http://2006.igem.org/wiki/index.php?title=Special:Contributions/MDolinar&feed=atom&limit=50&target=MDolinar&year=&month=2006.igem.org - User contributions [en]2024-03-28T13:34:44ZFrom 2006.igem.orgMediaWiki 1.16.5http://2006.igem.org/wiki/index.php/User:MDolinarUser:MDolinar2010-05-31T14:06:29Z<p>MDolinar: </p>
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<div>'''Marko Dolinar'''<br />
<br />
In 2006 and 2007 co-supervisor of the Slovenian iGEM team together with Roman Jerala. <br />
<br />
I am assistant professor of biochemistry and molecular biology at the University of Ljubljana Faculty of Chemistry and Chemical Technology. Currently I teach Recombinant DNA Technology, Molecular Biology, Molecular Fundamentals of Life Sciences and Biological Membranes. <br />
For details, please visit my [http://www.fkkt.uni-lj.si/en/?381 Faculty homepage].</div>MDolinarhttp://2006.igem.org/wiki/index.php/Research_Papers_in_Synthetic_Biology:YResearch Papers in Synthetic Biology:Y2007-09-24T08:05:11Z<p>MDolinar: link moved from entry under L (Laising Y.)</p>
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<div>* Yen, Laising et al. Exogenous Control of Mammalian Gene Expression through Modulation of RNA Self-cleavage. (mRNA-mediated regulation of translation.) Nature. Vol. 431: 471 - 476. [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15386015&query_hl=7&itool=pubmed_docsum Pubmed] [http://www.nature.com/nature/journal/v431/n7007/abs/nature02844.html Journal]</div>MDolinarhttp://2006.igem.org/wiki/index.php/Research_Papers_in_Synthetic_Biology:LResearch Papers in Synthetic Biology:L2007-09-24T08:01:03Z<p>MDolinar: </p>
<hr />
<div></div>MDolinarhttp://2006.igem.org/wiki/index.php/Research_Papers_in_Synthetic_Biology:CResearch Papers in Synthetic Biology:C2007-09-24T07:57:01Z<p>MDolinar: link repaired & poster link added</p>
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<div>*Carlson R. 2003. The pace and proliferation of biological technologies. Biosecur Bioterror. 1:203.<br />
*Che, Austin. 2004. BioBricks++: Simplifying Assembly of Standard DNA Components [http://austin.mit.edu/docs/bbpp.pdf Article] [http://austin.mit.edu/docs/bbpp_poster.pdf Poster]</div>MDolinarhttp://2006.igem.org/wiki/index.php/User:MDolinarUser:MDolinar2007-08-06T12:30:23Z<p>MDolinar: </p>
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<div>'''Marko Dolinar'''<br />
<br />
In 2006, co-supervisor of the (first-ever) [http://2006.igem.org/wiki/index.php/University_of_Ljubljana_%26_National_Institute_of_Chemistry%2C_Ljubljana%2C_Slovenia_2006 Slovenian iGEM team] together with Roman Jerala. In [http://2007.igem.org07/index.php/Ljubljana 2007], I am in the same role on a related topic: SB in cell medicine. <br />
<br />
I am assistant professor of biochemistry and molecular biology at the University of Ljubljana Faculty of Chemistry and Chemical Technology. I teach Recombinant DNA Technology, Biochemistry and Biological Membranes in Biochemistry and Chemistry undergraduate programmes. For details, please visit my [http://www.fkkt.uni-lj.si/en/?381 Faculty homepage].</div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Navodila2007Ljubljana, Slovenia 2006/Navodila20072007-02-16T16:14:16Z<p>MDolinar: </p>
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<div>Natančna navodila za pripravo projektov sva vsem zainteresiranim poslala po e-pošti 16. februarja popoldne. <br />
Če navodil niste dobili, sporočite kateremu od mentorjev. <br />
Rok za oddajo projektov in prilog je 20. marec.</div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Navodila2007Ljubljana, Slovenia 2006/Navodila20072007-02-13T15:51:19Z<p>MDolinar: </p>
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<div>Na tej strani boste najkasneje 19. februarja zvečer našli ključne podatke glede priprave projekta in okvirni časovni razpored raziskovalne naloge za letošnje tekmovanje iGEM.</div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Vabilo2007Ljubljana, Slovenia 2006/Vabilo20072007-01-20T21:29:47Z<p>MDolinar: </p>
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<div>'''Vabilo za prijavo v študentsko ekipo iGEM 2007'''<br />
<br />
Na [http://www.mit.edu/aboutmit/ MIT] (Massachussets Institute of Technology) že od leta 2003 prirejajo srečanja ekip študentov, ki sodelujejo z raziskovalnim projektom s področja<br />
gensko spremenjenih strojev (organizmov) oz. sintezne biologije - [http://2006.igem.org/Main_Page international Genetically Engineered Machines]. Lansko leto se je srečanja v Cambridgu (ZDA) udeležilo 33 ekip z vsega, sveta, med njimi veliko najuglednejših univerz (MIT, Harvard, Princeton, Berkeley, Cambridge, ETH...). Na lanskoletnem<br />
tekmovanju je slovenska ekipa osvojila prvo mesto, zato je prav, da se letos spet udeležimo srečanja in obranimo dober ugled slovenske znanosti in izobraževanja. <br />
<br />
Ekipo bo sestavljalo okrog 6 študentov, ki bodo do novembra pripravili projekt, s katerim bi se predstavili na srečanju. Najbrž je potrebno računati s 4-6 meseci eksperimentalnega dela. Vabljeni so študenti s širšega področja ved o življenju, naravoslovja in tehnike. Temo projekta bomo še izbrali, zagotovo pa bo povezana s sesalskimi sistemi v povezavi z medicinskim problemom. Za kandidate je zaželeno poznavanje metod rekombinantne DNA in izkušnje z laboratorijskim delom, nujno potrebno pa navdušenje in volja do dela. Eksperimentalno delo bo potekalo na [http://www.ki.si/ Kemijskem inštitutu] ([http://www.ki.si/index.php?id=192&no_cache=1 Laboratoriju za biotehnologijo]) in [http://bio.ijs.si/Katedra/katedra.html Katedri za biokemijo] [http://www.fkkt.uni-lj.si/si/?1 Fakultete za kemijo in kemijsko tehnologijo] [http://www.uni-lj.si/ Univerze v Ljubljani]. Izbor študentov bomo opravili na osnovi intervjujev. <br />
<br />
Prosim, da kandidati za ekipo do 10. februarja 2006 sporočite svoj interes na naslov: [mailto:roman.jerala@ki.si roman.jerala@ki.si].<br />
<br />
Sredi februarja načrtujemo sestanek z vsemi kandidati, ekipo pa bi izbrali do konca marca. Z delom bomo najbrž pričeli maja ali junija. Za dodatne informacije se lahko oglasite ali pa si informacijo o [http://www.igem2006.com/ tekmovanju] in [http://2006.igem.org/wiki/index.php/Ljubljana%2C_Slovenia_2006 lanskem projektu] naše ekipe ogledate na spletu.<br />
<br />
Roman Jerala <br><br />
Kemijski inštitut</div>MDolinarhttp://2006.igem.org/wiki/index.php/File:Lek3.jpgFile:Lek3.jpg2006-11-13T16:07:49Z<p>MDolinar: Logo of Lek pharmaceutical company.</p>
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<div>Logo of Lek pharmaceutical company.</div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006Ljubljana, Slovenia 20062006-11-13T16:04:52Z<p>MDolinar: </p>
<hr />
<div><center><br />
[[Image:Logo-si1.gif|120 px]] [[Image:fotka1b.jpg|475 px]] [[Image:Logo-si1.gif|120 px]]<br />
<br style="clear:both;"/> </center><br />
<br />
<br><br><br />
<center><font style="font size="9" face="Arial"><b>Engineered Human Cells: <br><br><br> SAY <font color="red">NO </font> TO SEPSIS<br />
<br><br></b></font><br />
<br />
<br><br />
[[Image:line-si4.jpg]]<br />
<table><br />
<tr><th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signaling Pathway]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Project & Model|Project & Model]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Results & Conclusions|Results & Conclusions]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr><br />
</table><br />
<br />
[[Image:line-si3.jpg]]<br />
</center><br />
<br />
{|<br />
|-valign="center" align="justify"<br />
|border="1" solid #affaaa" cellspacing="1" cellpadding="1" style="border:1px solid black; background:#cedff2" | <font style="font size="3" font face="Times New Roman""><p><b>Mammalian systems can be a subject of cellular engineering similarly to bacterial cells. We decided to tinker with the existing cell signaling network of the response to the bacterial infection. Binding of bacterial components ([[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]s – Pathogen associated molecular patterns) to a family of Toll-like receptors activates the cells of the immune system but the exaggerated response may lead to systemic inflammation and sepsis which is often fatal. We designed a feedback loop, which inhibits the signaling cascade at the »weak spot« - [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]], a consensus adaptor protein of the Toll-like receptors. A mathematical model of cell activation with engineered feedback loop was constructed, which predicts the decrease of the cellular activation after the repeated stimulation. Twenty-six new BrioBricks were constructed specially for the mammalian system. We have experimentally confirmed the function of the feedback device by detecting the inhibition of cellular activation after the repeated stimulation. Cell activation decreased without completely deleting the responsiveness to the bacterial infection, thus our engineered cell system represents a type of artificial immunotolerance.</b></p><br />
| [[Image:sepsa.png|450px|right|]]<br />
|}<br />
<br><br />
<br />
-----<br />
<br />
'''We would like to express our thanks to the Sponsors'''<br />
<br />
{| border="0" cellspacing="0" cellpadding="5" align="center"<br />
| [http://www.ki.si/ http://parts2.mit.edu/wiki/images/9/90/Logo_ki2.jpg]<br />
| [http://www.fkkt.uni-lj.si/en/ http://www.fkkt.uni-lj.si/img/menuleft_logo_unilj.gif]<br />
| [http://www.lek.si/ [[Image:Lek3.jpg]]]<br />
| [[Image:EU-flag.gif |thumb|120px|EU Synbiocomm (thank you Sven)]]<br />
|}<br />
<br />
<br />
'''and Donators'''<br />
<br>[http://www.ad-futura.si Ad Futura], [http://www.krka.si/si/ Krka pharmaceutical company], [http://www.mediline.si/ Mediline], Farmadent</div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006Ljubljana, Slovenia 20062006-11-01T17:10:46Z<p>MDolinar: </p>
<hr />
<div><center><br />
[[Image:Logo-si1.gif|120 px]] [[Image:fotka1b.jpg|475 px]] [[Image:Logo-si1.gif|120 px]]<br />
<br style="clear:both;"/> </center><br />
<br />
<br><br><br />
<center><font style="font size="9" face="Arial"><b>Engineered Human Cells: <br><br><br> SAY <font color="red">NO </font> TO SEPSIS<br />
<br><br></b></font><br />
<br />
<br><br />
[[Image:line-si4.jpg]]<br />
<table><br />
<tr><th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signaling Pathway]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Project & Model|Project & Model]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Results & Conclusions|Results & Conclusions]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr><br />
</table><br />
<br />
[[Image:line-si3.jpg]]<br />
</center><br />
<br />
{|<br />
|-valign="center" align="justify"<br />
|border="1" solid #affaaa" cellspacing="1" cellpadding="1" style="border:1px solid black; background:#cedff2" | <font style="font size="3" font face="Times New Roman""><p><b>Mammalian systems can be a subject of cellular engineering similarly to bacterial cells. We decided to tinker with the existing cell signaling network of the response to the bacterial infection. Binding of bacterial components ([[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]s – Pathogen associated molecular patterns) to a family of Toll-like receptors activates the cells of the immune system but the exaggerated response may lead to systemic inflammation and sepsis which is often fatal. We designed a feedback loop, which inhibits the signaling cascade at the »weak spot« - [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]], a consensus adaptor protein of the Toll-like receptors. A mathematical model of cell activation with engineered feedback loop was constructed, which predicts the decrease of the cellular activation after the repeated stimulation. Twenty-six new BrioBricks were constructed specially for the mammalian system. We have experimentally confirmed the function of the feedback device by detecting the inhibition of cellular activation after the repeated stimulation. Cell activation decreased without completely deleting the responsiveness to the bacterial infection, thus our engineered cell system represents a type of artificial immunotolerance.</b></p><br />
| [[Image:sepsa.png|450px|right|]]<br />
|}<br />
<br><br />
<br />
-----<br />
<br />
'''We would like to express our thanks to the Sponsors'''<br />
<br />
{| border="0" cellspacing="0" cellpadding="5" align="center"<br />
| [http://www.ki.si/ http://parts2.mit.edu/wiki/images/9/90/Logo_ki2.jpg]<br />
| [http://www.fkkt.uni-lj.si/en/ http://www.fkkt.uni-lj.si/img/menuleft_logo_unilj.gif]<br />
| [http://www.lek.si/ http://parts2.mit.edu/wiki/images/0/09/Lek2.jpg]<br />
| [[Image:EU-flag.gif |thumb|120px|EU Synbiocomm (thank you Sven)]]<br />
|}<br />
<br />
<br />
'''and Donators'''<br />
<br>[http://www.ad-futura.si Ad Futura], [http://www.krka.si/si/ Krka pharmaceutical company], [http://www.mediline.si/ Mediline], Farmadent</div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006Ljubljana, Slovenia 20062006-10-31T14:46:38Z<p>MDolinar: </p>
<hr />
<div><center><br />
[[Image:Logo-si1.gif|120 px]] [[Image:fotka1b.jpg|475 px]] [[Image:Logo-si1.gif|120 px]]<br />
<br style="clear:both;"/> </center><br />
<br />
<br><br><br />
<center><font style="font size="9" face="Arial"><b>Engineered Human Cells: <br><br><br> SAY STOP TO SEPSIS<br />
<br><br></b></font><br />
<br />
<br><br />
[[Image:line-si4.jpg]]<br />
<table><br />
<tr><th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signaling Pathway]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Project & Model|Project & Model]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Results & Conclusions|Results & Conclusions]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr><br />
</table><br />
<br />
[[Image:line-si3.jpg]]<br />
</center><br />
<br />
{|<br />
|-valign="center" align="justify"<br />
|border="1" solid #affaaa" cellspacing="1" cellpadding="1" style="border:1px solid black; background:#cedff2" | <font style="font size="3" font face="Times New Roman""><p><b>Mammalian systems can be a subject of cellular engineering similarly to bacterial cells. We decided to tinker with the existing cell signaling network of the response to the bacterial infection. Binding of bacterial components ([[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]s – Pathogen associated molecular patterns) to a family of Toll-like receptors activates the cells of the immune system but the exaggerated response may lead to systemic inflammation and sepsis which is often fatal. We designed a feedback loop, which inhibits the signaling cascade at the »weak spot« - [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]], a consensus adaptor protein of the Toll-like receptors. A mathematical model of cell activation with engineered feedback loop was constructed, which predicts the decrease of the cellular activation after the repeated stimulation. Twenty-six new BrioBricks were constructed specially for the mammalian system. We have experimentally confirmed the function of the feedback device by detecting the inhibition of cellular activation after the repeated stimulation. Cell activation decreased without completely deleting the responsiveness to the bacterial infection, thus our engineered cell system represents a type of artificial immunotolerance.</b></p><br />
| [[Image:sepsa.png|450px|right|]]<br />
|}<br />
<br><br />
<br />
-----<br />
<br />
'''We would like to express our thanks to the Sponsors'''<br />
<br />
{| border="0" cellspacing="0" cellpadding="5" align="center"<br />
| [http://www.ki.si/ http://parts2.mit.edu/wiki/images/9/90/Logo_ki2.jpg]<br />
| [http://www.fkkt.uni-lj.si/en/ http://www.fkkt.uni-lj.si/img/menuleft_logo_unilj.gif]<br />
| [http://www.lek.si/ http://parts2.mit.edu/wiki/images/0/09/Lek2.jpg]<br />
| [[Image:EU-flag.gif |thumb|120px|EU Synbiocomm (thank you Sven)]]<br />
|}<br />
<br />
<br />
'''and Donators'''<br />
<br>[http://www.ad-futura.si Ad Futura], [http://www.krka.si/si/ Krka pharmaceutical company], [http://www.mediline.si/ Mediline], Farmadent</div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Results_%26_ConclusionsLjubljana, Slovenia 2006/Results & Conclusions2006-10-31T14:45:39Z<p>MDolinar: </p>
<hr />
<div><center>[[Image:Logo-si1.gif|center|120 px]] <br><br />
<br />
[[Image:line-si4.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006|Home]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signaling Pathway]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Project & Model|Project & Model]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr></table>[[Image:line-si3.jpg]]<br />
<br><br />
</center><br />
<br><br><br />
<br />
<h1>Results</h1><br />
<br />
<h2>Construction of Biobricks</h2><br />
We prepared individual BioBricks that could be used in mammalian cells. The following BioBricks were used:<br />
*promoter (constitutive CMV and [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] inducible promoter), <br />
*terminator (which we included into plasmids to avoid repeated additional steps in construction of each functional Part) <br />
*protein coding sequences, which include: <br />
**two inhibitory (dominant negative) proteins of the signaling cascade ([[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] and dn[[Ljubljana, Slovenia 2006/Terms & References#Terms|TRAF6]]), <br />
**two reporters: ''Renilla'' luciferase (in order to allow simultaneous dual luciferase assay to normalize the luminescence for the efficiency of transfection and cell number) and GFP <br />
**[[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]] to decrease the lifetime of the inhibitor.<br />
<br />
Linkers between the protein coding sequences (e.g. [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]]+luciferase+[[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]]) were prepared by PCR ligation to avoid addition of unwanted aminoacids in the linker region between the protein domains.<br />
<br><br />
<br />
<br />
<br />
<center><table><br />
<caption>''Table 1: List of prepared Parts''</caption><br />
<tr><th>Registration number</th><br />
<th>Part's Name</th><br />
<th>Part/Device</th><br />
<th>Vector</th></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52008 BBa_J52008]</td><td>rluc</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52010 BBa_J52010]</td><td>NF&kappa;B</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52011 BBa_J52011]</td><td>dnMyD88-likn-rLuc</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52012 BBa_J52012]</td><td>rluc-link-PEST191</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52013 BBa_J52013]</td><td>dnMyD88-link-rluc-link-pest191</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52014 BBa_J52014]</td><td>NF&kappa;B+dnMyD88-link-rLuc</td><td>Device</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52016 BBa_J52016]</td><td>eukaryotic terminator</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52017 BBa_J52017]</td><td>eukaryotic terminator vector</td><td></td><td>pSB1AK3</td></tr></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52018 BBa_J52018]</td><td>NF&kappa;B+rLuc</td><td>Device</td><td>pSB1AC3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52019 BBa_J52019]</td><td>dnTRAF6</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52021 BBa_J52021]</td><td>dnTRAF6-link-GFP</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52022 BBa_J52022]</td><td>NF&kappa;B+dnTRAF6-link-GFP</td><td>Device</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52023 BBa_J52023]</td><td>NF&kappa;B+rLuc-link-PEST191</td><td>Device</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52024 BBa_J52024]</td><td>NF&kappa;B+dnMyD88-link-rLuc-link-PEST191</td><td>Device</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52026 BBa_J52026]</td><td>dnMyD88-link-GFP</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52027 BBa_J52027]</td><td>NF&kappa;B+dnMyD88-link-GFP</td><td>Device</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52028 BBa_J52028]</td><td>GFP-PEST191</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52029 BBa_J52029]</td><td>NF&kappa;B+GFP-PEST191</td><td>Device</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52034 BBa_J52034]</td><td>CMV</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52035 BBa_J52035]</td><td>dnMyD88</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52036 BBa_J52036]</td><td>NF&kappa;B+dnMyD88</td><td>Device</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52038 BBa_J52038]</td><td>CMV-rLuc</td><td>Device</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52039 BBa_J52039]</td><td>CMV+rLuc-link-PEST191</td><td>Device</td><td>pSB1A2</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52040 BBa_J52040]</td><td>CMV+GFP-PEST191</td><td>Device</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52642 BBa_J52642]</td><td>GFP</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52648 BBa_J52648]</td><td>CMV+GFP</td><td>Device</td><td>pSB1AK3+TER</td></tr><br />
</table></center><br />
<br><br />
<br />
<h2>Detection of phosphorylation of ERK kinases fy flow cytometry reflects the activation of the TLR signaling </h2><br />
<br />
<br style="clear:both;"/><br />
<br />
[[Image:cito1.gif|cito1.gif|center|thumb|400px|<b> Figure 19: Flow cytometric profile of phosphorylated ERK kinases in HEK293 cells stimulated by LPS for 0, 20 and 80 minutes.</b>]]<br />
<br />
<br style="clear:both;"/><br />
<br />
Flow Cytometry experiments showed that after 80 minute stimulation the increase in the level of phosphorylated ERK kinases was evident(Figure 19). The method was not as sensitive to the rapid response as we hoped. Further optimization of the method would be necessary in order to obtain the quantitative data to accurately model the cell signaling.<br />
<br />
<h2>Detection of transcriptionally competent[[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] in whole cell extracts dependent on the time after stimulation with [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]] determined with promoter ELISA method</h2><br />
<br />
<br style="clear:both;"/><br />
<br />
[[Image:Elisa_results.gif|thumb|center|800px|<b>Figure 20: Activity of peroxidase (conjugated on secondary antibodies) in luminescence units as a function of time after stimulation with [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]].</b> The amount of free [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] (and consequently luminescence) increases with time of stimulation (7 hours) and decreases after extended (23 hours) stimulation.]]<br />
<br />
<br style="clear:both;"/><br />
<br />
Cells were transfected with [[Ljubljana, Slovenia 2006/Terms & References#Terms|TLR4]] plasmid and [[Ljubljana, Slovenia 2006/Terms & References#Terms|MD-2]] plasmid in ordeer to confer the LPS responsiveness. We have shown that cells with TRL4 receptor respond to [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]] stimulation and that ELISA is apropriate and sensitive method for the detection of stimulation. However we did not get satisfying results when stimulating (with [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]] or flagellin) cells transfected neither with [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] nor [[Ljubljana, Slovenia 2006/Terms & References#Terms|TRAF6]]. The response of the ELISA method, as the most direct measurement of the free NF-&kappa;B was not sufficiently reproducible and linear in comparison to the method of luciferase reporter plasmids (see below).<br />
<br />
<br />
<h2>Inducible transcription by the stimulation of Toll-like receptor</h2><br />
<br />
<p>Stimulation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|HEK293]] cells transfected by TLR with their agonist (flagellin in case of TLR5) stimulates the translocation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] into the nucleus and activation of transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]]-responsive genes. We have used the firefly luciferase under the [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] dependent promoter to monitor the promotor activation. We tested the functionality of our part containing [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] dependent promotor in a construct with [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]]-rLuc. We detected the R-luciferase activity only in stimulated cells with significant activity appearing a few hours after the cell stimulation, which proves the functionality of this part (inducible promoter).</p><br />
<br />
<br style="clear:both;"/><br />
<br />
<br />
[[Image:graf_gabi.gif|left|thumb|400px|<b>Figure 21: Transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with rLuc reporter is induced by bacterial flagellin </b> Activity of ''Renilla'' luciferase stimulated by the addition of a pulse of [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]]. [[Ljubljana, Slovenia 2006/Terms & References#Terms|HEK293]] cells were transfected with device comprising [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]]-inducible fusion of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]]-rLuc.]]<br />
<br />
[[Image:rezultati7.gif|left|thumb|400px|<b>Figure 22: Transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with rLuc reporter in our device is inducible by bacterial flagellin.</b> Activity of the Firefly luciferase stimulated by the addition of two pulses of [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]]. ]]<br />
<br />
<br style="clear:both;"/><br />
<p>We also measured NF-kB-responsive F-luciferase activity of this device. We expected a decrease in the activity after the amount of time sufficient for the sysnthesis of inhibitory fusion protein in cells. To our dissapointment no decrease of the cell activation was observed. One of the possible causes for the lack of inhibition of the dnMyD88-rLuc protein fusion could be the sterical hindrance of the activity of dnMyD88. This domain interacts with TIR domain of TLRs in the direct proximity of the cellular membrane, which could prevent interaction with larger protein domains. Based on those results we also conducted our experiments with a device containing dnMyD88 without of any protein appendix.<br />
<br />
<h2>Inhibition of cell signaling by a dnMyD88 feedback device</h2><br />
<br />
An adapter protein [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]], consisting of a TIR and death domain, is at the crossroads of Toll-like receptors. Therefore activation of each of the surface expressed TLR recruits [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] to the cell membrane. We selected to use the receptor TLR5 and flagellin instead of the often used [[Ljubljana, Slovenia 2006/Terms & References#Terms|TLR4]] and its agonist [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]], because the [[Ljubljana, Slovenia 2006/Terms & References#Terms|TLR4]] system is extremely sensitive to the contamination with bacterial [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]], which often copurifies with the plasmid. In this way we have avoided the contamination issue, but our system is designed to work on any signaling receptor that has [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] in its signaling pathway (i.e. TLR1,2,4,5,6,7,8,9,11,IL-1).<br />
Cells were cotransfected with TLR5, reporter plasmid (NF&kappa;B-Fluc) and our feedback device ([[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]]-[[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]]).</p><br />
<br />
<p>We expect that after stimulation with flagellin cytoplasmic [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] is released from its inhibitor. It then migrates into the nucleus and induces the transcription of our construct. Accumulation of the dominant negative protein should compete with wild-type [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] and attenuate the downstream signaling pathway. We can detect the [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] translocation, transcriptional activation and phosphorylation by our detection systems - luminescence, free [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]], phosphorylation detected by flow cytometry and confocal microscopy. In case that our construct is followed by [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]] (degradation) sequence, it is expected that product should degrade rapidly and shorten the inhibition period. If the stimulus is still present, cells should again respond to it and the cycle with inhibition will repeat as well.</p><br />
<br />
<p>We have designed the experiment by two consecutive pulses of TLR stimulation separated by 4-6 hours, which is sufficient time to accumulate the inducible inhibitor. Normal cells should respond to both stimuli, while the cells with our device should respond to the same extent only to the first pulse -- this induces the transcription of the inhibitor, therefore the response to the second stimulus should be decreased. However if the stimuli are separated by a longer timespan, the system should respond normally, as the inhibitor has already degraded and the systemn has reset to the normal state.</p><br />
<br />
<center><br />
[[Image:rezultati1a.gif|center|thumb|400px|<b>Figure 23: [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] feedback loop decreases the cellular activation by repeated stimulation with bacterial [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]]. </b> Normalized luciferase activity (Fluc/Rluc) of cells stimulated by the addition of two pulses of flagellin separated by 6 hours, indicated time after the second pulse is shown. [[Ljubljana, Slovenia 2006/Terms & References#Terms|HEK293]] cells were transfected with device comprising NF&kappa;B-inducible [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] in comparison to wild type.]]<br />
</center><br />
<br style="clear:both;"/><br />
<br />
<p>Our results show that the system with inducible [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] indeed responds weaker to the second stimulus. The minimal delay between two stimuli should correspond to the time required for the synthesis of the inhibitor, which is exactly as planned, since it does not completely abolish the cellular response but decreases it after extended stimulation.</p><br />
<br />
<br style="clear:both;"/><br />
<br />
<h2>Decrease of the protein lifetime by the [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]]</h2><br />
<br />
<p>Rapid degradation signature motif - "[[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]]" was considered to tune the lifetime of the inhibitor in order to reset the cells to the normal responsiveness more rapidly. Fusion of the luciferase and [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]] under the control of CMV (Device [http://partsregistry.org/Part:BBa_J52039 BBa_J52039]) was used to test the effectiveness of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]]. We have used the cycloheximide to stop the protein synthesis and analyse the lifetime of the fusion protein with [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]] sequence. The steady state of the luciferase activity was 5-10 fold lower in the Rluc-[[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]] fusin than without of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]] signature, as evident from the chart below.</p> <br />
<br />
[[Image:graf1.gif|thumb|center|800px|<b>Figure 24: Addition of the PEST degradation tag decreases the steady state of the protein in cells due to its rapid degradation </b>. Cycloheximide was added to cells in order to stop the protein synthesis and monitor the protein degradation kinetics.]]<br />
<p>Constructs of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] domain and [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]] were not effective as inhibitors of signaling because of the interference of the C-terminal extension of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] inhibitory domain, as described above for the fusion constructs with the reporter luciferase or GFP.</p><br />
<br />
<br />
<br />
<div style="float:left; background-color:#cedff2; padding: .2em .6em; border: 1px solid black">'''<br />
<br />
<h1>Conclusions</h1><br />
<br />
*the same principles of BioBricks can be used in the mammalian cell system as in bacteria and eucaryotes<br />
*we have succesfully implemented a feedback loop that decreases the cellular activation with some delay<br />
*response of this feedback loop is transient and the cell responsiveness is restored after the synthesized inhibitor has degraded<br />
*[[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] inhibitor was not effective as a C-terminal fusion with luciferase or GFP, which may be due to the steric hindrance; it is likely that N-terminal fusion of reporter domains would be functional as signaling inhibitors <br />
*our constructed device mimicks the natural mechanism of tolerance only that it is activated faster, which may be a benefit for organism,<br />
*simplified model of the TLR signaling qualitatively captures most of the features of the natural system<br />
</div><br />
<br />
<br />
<br />
<br />
<br><br />
<h2>Suggestions</h2><br />
* for mammalian cell systems an additional set of vectors may be constructed based on the backbone, which includes the selection marker for stable transfection or retroviral integration<br />
<br />
<center><br />
[[Image:line-si4.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006|Home]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signaling Pathway]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Project & Model|Project & Model]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr></table>[[Image:line-si3.jpg]]<br />
<br><br />
</center></div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Project_%26_ModelLjubljana, Slovenia 2006/Project & Model2006-10-31T14:44:54Z<p>MDolinar: </p>
<hr />
<div><center>[[Image:Logo-si1.gif|center|120 px]] <br><br />
<br />
[[Image:line-si4.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006|Home]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signaling Pathway]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Results & Conclusions|Results & Conclusions]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr></table><br />
[[Image:line-si3.jpg]]<br />
<br><br />
</center><br />
<br />
<br><br><br />
<br />
<h1>Project ideas</h1><br />
Reasearch in the Laboratory of Biotechnology at the National institute of Chemisty, where most of our instructors come from and where most of the experimental work was conducted, is in the area of molecular immunology, specifically interactions in the Toll-like receptor signaling to microbial infections. This determined the selection of the eukaryotic system as our arena of synthetic biology "sandbox". besides, several contributions at the Synthetic biology meetings demonstrated that mammalian cell engineering <br />
<br />
In the beginning we have discussed following project ideas:<br />
<ol><br />
<li>Inserton of a logical device into the signaling pathway to limit the cellular activation by bacterial infection<br />
- use of dominant negative versions of proteins involved in signaling pathway, which will arrest the signaling pathway thus preventing [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] (transcription factor mentioned above)to translocate to the nucleus<br />
- addition of degradation tags to this dominant negative proteins ([[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]]), which would cause inhibition (negative feedback loop) to be temporal. </li><br />
<br />
<li>Engineering of the response to pathogens/their [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]s, which are otherwise not recognized by cells<br />
- for example a response to beta glucans of fungi.</li><br />
<br />
<li>Engineering of novel interconnections between signaling pathways. </li> </ol><br />
<br><br />
<br />
<h1>Selected project</h1><br />
<br />
[[Image:sepsa.png|250px|thumb|<b>Figure 4: Unlimited cell stimulation due to the infection can lead to sepsis, which is in many cases fatal</b>. Introduction of the negative feedback could decrease the immune response.]]<br />
<p>The basic idea of our project was to introduce a feedback loop in TLR signaling pathway, which would decrease the overwhelming response to the persistent or repeated stimulus with [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]. However, completly shutting down the response to bacterial stimulation is not recommendable from the view of a host battling the infection. Ideally the feedback loop should decrease the response when it is too high but recover the responsiveness of the system after some time.</p><br />
<br />
<p>Inhibition of the overwhelming response could be achieved if [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]] is simultaneously able to activate the immune response and the expression of a [[Ljubljana, Slovenia 2006/Terms & References#Terms|dominant-negative]] adapter protein, that would inactivate [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]`s signaling pathway. Decreasing the lifetime of the dominant-negative inhibitior by the addition of a rapid degradation tag ([[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]]) should inactivate the inhibition and restore the responsiveness of the immune system.</p><br />
<br />
<p>This idea is similar to the natural mechanism of tolerance, which is already present in living cells and which decreases the response to repeated bacterial stimulations. This natural tolerance is activated slowly, on the order of days and operates through several different mechanisms. Our feedback mechanism (i.e. artificial tolerance) should decrease the response within hours and thus "attack" the signaling pathway at the point, which has not been used in the natural system.</p><br />
<br><br />
<br />
<h2>Model of the TLR signaling triggered by bacterial infection</h2><br />
<br />
<h3>[[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] is the hub of the TLR signaling pathway</h3><br />
<p><br />
Figure 5 represents the complexity of cell signaling mediated by TLRs. which contains more than 700 molecules and complexes (Oda & Kitano, Molecular Systems Biology, 2006).</p><br />
<br />
<br style="clear:both;"/><br />
<br />
[[Image:model1.gif|thumb|center|800px|<b>Figure 5: Comprehensive scheme of the Toll-like receptor signaling of microbial infection.</b>]]<br />
<br />
<br />
<br style="clear:both;"/><br />
<br />
<p>[[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] adapter protein is involved in the signal transduction immediately after ligand-induced TLR oligomerization. This adapter protein is common to most TLRs before the signaling network branches into several phosphpryation cascades. We selected this molecule as the most appropriate target of TLR signaling network engineering (Figures 6 and 7).</p><br />
<br />
[[Image:model2.gif|thumb|left|650px|<b>Figure 6: [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] adapter protein connects the response of all TLRs and represents the »bottleneck« of the system (shaded area).</b>]]<br />
[[Image:model3.gif|thumb|right|200px|<b>Figure 7: Schematic representation of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] as the hub of TLR signaling shaped like a bow tie.</b>]]<br />
<br />
<br style="clear:both;"/><br />
<br />
<h3>Construction of the model</h3><br />
<p><br />
Model of the TLR signaling was prepared in CellDesigner. Signaling steps, where the response remains the same were assembled into one block (Signaling cascade), whose activation directly affects the [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] translocation. Our engineered feedback loop is inserted as a transcriptional activation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] protein domain (red block in Figure 8).</p><br />
<br />
[[Image:model12a.gif|left|thumb|400px|<b>Figure 8: Simplified model of Toll-like receptor signaling constructed in CellDesigner.</b>]]<br />
[[Image:model12.gif|right|thumb|400px|<b>Figure 9: Model of TLR signaling with additional feedback loop for the inhibition of [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]].</b>]]<br />
<br />
<br style="clear:both;"/><br />
<br />
<p>Simulation of cellular response to TLR stimulation by a pulse of [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]] (e.g. flagellin). When available parameters were taken from the literaturte (Selvarajoo, 2006). Signaling cascade results in the transient translocation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] into the cell nucleus (green) and in the production of inflammatory mediators. Repeated pulse after some time results in the repeated activation and accumulation of inflammatory mediators.</p><br />
<br />
<br style="clear:both;"/><br />
<br />
[[Image:model6.gif|thumb|left|400px|<b>Figure 10: Simulation of the cell response to the bacterial stimulus [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP</b>]] (grey) – transient translocation of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] into the nucleus (green), production of stimulatory mediators (orange).]]<br />
[[Image:model7.gif|thumb|right|400px|<b>Figure 11: Simulation of the repeated cellular stimulation by [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]</b> (grey) - the amount of inflammatory mediators increases (orange).]]<br />
<br />
<br style="clear:both;"/><br />
<br />
<p>Simulation of the TLR signaling response in the system with a feedback device results in the production of inhibitor ([[Ljubljana, Slovenia 2006/Terms & References|dnMyD88]], black) which inhibits cellular activation by a repeated stimulation. Cellular responsiveness resets to the normal response if the inhibitor is rapidly degraded, such as by the addition of a [[Ljubljana, Slovenia 2006/Terms & References|PEST]] motif.</p><br />
<br />
[[Image:model8.gif|thumb|left|400px|<b>Figure 12: Introduction of the feedback loop to initiate the production of inhibitory [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]]</b>( (black) – cell does not respond to the repeated stimulation.]]<br />
[[Image:model9.gif|thumb|right|400px|<b>Figure 13: Feedback loop with rapid degradation of the inhibitor ([[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with PEST sequence</b> - black) – responsiveness of the system is again restored to a full extent.]]<br />
<br style="clear:both;"/><br />
<br />
[[Image:model10.gif|thumb|left|400px|<b>Figure 14: Speed of the degradation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] could modulate the responsiveness of the system to repeated (or continuous) challenge by TLR agonists.</b>]]<br />
<br />
<br style="clear:both;"/><br />
<br />
<hr><br />
<center><br />
[[Image:line-si4.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006|Home]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signaling Pathway]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Results & Conclusions|Results & Conclusions]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr></table>[[Image:line-si3.jpg]]<br />
</center></div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Project_%26_ModelLjubljana, Slovenia 2006/Project & Model2006-10-31T14:44:19Z<p>MDolinar: </p>
<hr />
<div><center>[[Image:Logo-si1.gif|center|120 px]] <br><br />
<br />
[[Image:line-si4.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006|Home]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signaling Pathway]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Results & Conclusions|Results & Conclusions]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr></table><br />
[[Image:line-si3.jpg]]<br />
<br><br />
</center><br />
<br />
<br><br><br />
<br />
<h1>Project ideas</h1><br />
Reasearch in the Laboratory of Biotechnology at the National institute of Chemisty, where most of our instructors come from and where most of the experimental work was conducted, is in the area of molecular immunology, specifically interactions in the Toll-like receptor signaling to microbial infections. This determined the selection of the eukaryotic system as our arena of synthetic biology "sandbox". besides, several contributions at the Synthetic biology meetings demonstrated that mammalian cell engineering <br />
<br />
In the beginning we have discussed following project ideas:<br />
<ol><br />
<li>Inserton of a logical device into the signaling pathway to limit the cellular activation by bacterial infection<br />
- use of dominant negative versions of proteins involved in signaling pathway, which will arrest the signaling pathway thus preventing [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] (transcription factor mentioned above)to translocate to the nucleus<br />
- addition of degradation tags to this dominant negative proteins ([[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]]), which would cause inhibition (negative feedback loop) to be temporal. </li><br />
<br />
<li>Engineering of the response to pathogens/their [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]s, which are otherwise not recognized by cells<br />
- for example a response to beta glucans of fungi.</li><br />
<br />
<li>Engineering of novel interconnections between signaling pathways. </li> </ol><br />
<br><br />
<br />
<h1>Selected project</h1><br />
<br />
[[Image:sepsa.png|250px|thumb|<b>Figure 4: Unlimited cell stimulation due to the infection can lead to sepsis, which is in many cases fatal</b>. Introduction of the negative feedback could decrease the immune response.]]<br />
<p>The basic idea of our project was to introduce a feedback loop in TLR signaling pathway, which would decrease the overwhelming response to the persistent or repeated stimulus with [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]. However, completly shutting down the response to bacterial stimulation is not recommendable from the view of a host battling the infection. Ideally the feedback loop should decrease the response when it is too high but recover the responsiveness of the system after some time.</p><br />
<br />
<p>Inhibition of the overwhelming response could be achieved if [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]] is simultaneously able to activate the immune response and the expression of a [[Ljubljana, Slovenia 2006/Terms & References#Terms|dominant-negative]] adapter protein, that would inactivate [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]`s signaling pathway. Decreasing the lifetime of the dominant-negative inhibitior by the addition of a rapid degradation tag ([[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]]) should inactivate the inhibition and restore the responsiveness of the immune system.</p><br />
<br />
<p>This idea is similar to the natural mechanism of tolerance, which is already present in living cells and which decreases the response to repeated bacterial stimulations. This natural tolerance is activated slowly, on the order of days and operates through several different mechanisms. Our feedback mechanism (i.e. artificial tolerance) should decrease the response within hours and thus "attack" the signaling pathway at the point, which has not been used in the natural system.</p><br />
<br><br />
<br />
<h2>Model of the TLR signaling triggered by bacterial infection</h2><br />
<br />
<h3>[[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] is the hub of the TLR signaling pathway</h3><br />
<p><br />
Figure 5 represents the complexity of cell signaling mediated by TLRs. which contains more than 700 molecules and complexes (Oda & Kitano, Molecular Systems Biology, 2006).</p><br />
<br />
<br style="clear:both;"/><br />
<br />
[[Image:model1.gif|thumb|center|800px|<b>Figure 5: Comprehensive scheme of the Toll-like receptor signaling of microbial infection.</b>]]<br />
<br />
<br />
<br style="clear:both;"/><br />
<br />
<p>[[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] adapter protein is involved in the signal transduction immediately after ligand-induced TLR oligomerization. This adapter protein is common to most TLRs before the signaling network branches into several phosphpryation cascades. We selected this molecule as the most appropriate target of TLR signaling network engineering (Figures 6 and 7).</p><br />
<br />
[[Image:model2.gif|thumb|left|650px|<b>Figure 6: [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] adapter protein connects the response of all TLRs and represents the »bottleneck« of the system (shaded area).</b>]]<br />
[[Image:model3.gif|thumb|right|200px|<b>Figure 7: Schematic representation of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] as the hub of TLR signaling shaped like a bow tie.</b>]]<br />
<br />
<br style="clear:both;"/><br />
<br />
<h3>Construction of the model</h3><br />
<p><br />
Model of the TLR signaling was prepared in CellDesigner. Signaling steps, where the response remains the same were assembled into one block (Signaling cascade), whose activation directly affects the [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] translocation. Our engineered feedback loop is inserted as a transcriptional activation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] protein domain (red block in Figure 8).</p><br />
<br />
[[Image:model12a.gif|left|thumb|400px|<b>Figure 8: Simplified model of Toll-like receptor signaling constructed in CellDesigner.</b>]]<br />
[[Image:model12.gif|right|thumb|400px|<b>Figure 9: Model of TLR signaling with additional feedback loop for the inhibition of [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]].</b>]]<br />
<br />
<br style="clear:both;"/><br />
<br />
<p>Simulation of cellular response to TLR stimulation by a pulse of [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]] (e.g. flagellin). When available parameters were taken from the literaturte (Selvarajoo, 2006). Signaling cascade results in the transient translocation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] into the cell nucleus (green) and in the production of inflammatory mediators. Repeated pulse after some time results in the repeated activation and accumulation of inflammatory mediators.</p><br />
<br />
<br style="clear:both;"/><br />
<br />
[[Image:model6.gif|thumb|left|400px|<b>Figure 10: Simulation of the cell response to the bacterial stimulus [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP</b>]] (grey) – transient translocation of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] into the nucleus (green), production of stimulatory mediators (orange).]]<br />
[[Image:model7.gif|thumb|right|400px|<b>Figure 11: Simulation of the repeated cellular stimulation by [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]</b> (grey) - the amount of inflammatory mediators increases (orange).]]<br />
<br />
<br style="clear:both;"/><br />
<br />
<p>Simulation of the TLR signaling response in the system with a feedback device results in the production of inhibitor ([[Ljubljana, Slovenia 2006/Terms & References|dnMyD88]], black) which inhibits cellular activation by a repeated stimulation. Cellular responsiveness resets to the normal response if the inhibitor is rapidly degraded, such as by the addition of a [[Ljubljana, Slovenia 2006/Terms & References|PEST]] motif.</p><br />
<br />
[[Image:model8.gif|thumb|left|400px|<b>Figure 12: Introduction of the feedback loop to initiate the production of inhibitory [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]]</b>( (black) – cell does not respond to the repeated stimulation.]]<br />
[[Image:model9.gif|thumb|right|400px|<b>Figure 13: Feedback loop with rapid degradation of the inhibitor ([[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with PEST sequence</b> - black) – responsiveness of the system is again restored to a full extent.]]<br />
<br style="clear:both;"/><br />
<br />
[[Image:model10.gif|thumb|left|400px|<b>Figure 14: Speed of the degradation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] could modulate the responsiveness of the system to repeated (or continuous) challenge by TLR agonists.</b>]]<br />
<br />
<br style="clear:both;"/><br />
<br />
<hr><br />
<center><br />
[[Image:line-si4.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006|Home]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signalling Pathway]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Results & Conclusions|Results & Conclusions]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr></table>[[Image:line-si3.jpg]]<br />
</center></div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Project_%26_ModelLjubljana, Slovenia 2006/Project & Model2006-10-31T14:43:40Z<p>MDolinar: </p>
<hr />
<div><center>[[Image:Logo-si1.gif|center|120 px]] <br><br />
<br />
[[Image:line-si4.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006|Home]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signaling Pathway]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Results & Conclusions|Results & Conclusions]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr></table><br />
[[Image:line-si3.jpg]]<br />
<br><br />
</center><br />
<br />
<br><br><br />
<br />
<h1>Project ideas</h1><br />
Reasearch in the Laboratory of Biotechnology at the National institute of Chemisty, where most of our instructors come from and where most of the experimental work was conducted, is in the area of molecular immunology, specifically interactions in the Toll-like receptor signaling to microbial infections. This determined the selection of the eukaryotic system as our arena of synthetic biology "sandbox". besides, several contributions at the Synthetic biology meetings demonstrated that mammalian cell engineering <br />
<br />
In the beginning we have discussed following project ideas:<br />
<ol><br />
<li>Inserton of a logical device into the signaling pathway to limit the cellular activation by bacterial infection<br />
- use of dominant negative versions of proteins involved in signaling pathway, which will arrest the signaling pathway thus preventing [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] (transcription factor mentioned above)to translocate to the nucleus<br />
- addition of degradation tags to this dominant negative proteins ([[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]]), which would cause inhibition (negative feedback loop) to be temporal. </li><br />
<br />
<li>Engineering of the response to pathogens/their [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]s, which are otherwise not recognized by cells<br />
- for example a response to beta glucans of fungi.</li><br />
<br />
<li>Engineering of novel interconnections between signaling pathways. </li> </ol><br />
<br><br />
<br />
<h1>Selected project</h1><br />
<br />
[[Image:sepsa.png|250px|thumb|<b>Figure 4: Unlimited cell stimulation due to the infection can lead to sepsis, which is in many cases fatal</b>. Introduction of the negative feedback could decrease the immune response.]]<br />
<p>The basic idea of our project was to introduce a feedback loop in TLR signaling pathway, which would decrease the overwhelming response to the persistent or repeated stimulus with [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]. However, completly shutting down the response to bacterial stimulation is not recommendable from the view of a host battling the infection. Ideally the feedback loop should decrease the response when it is too high but recover the responsiveness of the system after some time.</p><br />
<br />
<p>Inhibition of the overwhelming response could be achieved if [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]] is simultaneously able to activate the immune response and the expression of a [[Ljubljana, Slovenia 2006/Terms & References#Terms|dominant-negative]] adapter protein, that would inactivate [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]`s signaling pathway. Decreasing the lifetime of the dominant-negative inhibitior by the addition of a rapid degradation tag ([[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]]) should inactivate the inhibition and restore the responsiveness of the immune system.</p><br />
<br />
<p>This idea is similar to the natural mechanism of tolerance, which is already present in living cells and which decreases the response to repeated bacterial stimulations. This natural tolerance is activated slowly, on the order of days and operates through several different mechanisms. Our feedback mechanism (i.e. artificial tolerance) should decrease the response within hours and thus "attack" the signaling pathway at the point, which has not been used in the natural system.</p><br />
<br><br />
<br />
<h2>Model of the TLR signaling triggered by bacterial infection</h2><br />
<br />
<h3>[[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] is the hub of the TLR signaling pathway</h3><br />
<p><br />
Figure 5 represents the complexity of cell signalling mediated by TLRs. which contains more than 700 molecules and complexes (Oda & Kitano, Molecular Systems Biology, 2006).</p><br />
<br />
<br style="clear:both;"/><br />
<br />
[[Image:model1.gif|thumb|center|800px|<b>Figure 5: Comprehensive scheme of the Toll-like receptor signaling of microbial infection.</b>]]<br />
<br />
<br />
<br style="clear:both;"/><br />
<br />
<p>[[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] adapter protein is involved in the signal transduction immediately after ligand-induced TLR oligomerization. This adapter protein is common to most TLRs before the signaling network branches into several phosphpryation cascades. We selected this molecule as the most appropriate target of TLR signaling network engineering (Figures 6 and 7).</p><br />
<br />
[[Image:model2.gif|thumb|left|650px|<b>Figure 6: [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] adapter protein connects the response of all TLRs and represents the »bottleneck« of the system (shaded area).</b>]]<br />
[[Image:model3.gif|thumb|right|200px|<b>Figure 7: Schematic representation of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] as the hub of TLR signaling shaped like a bow tie.</b>]]<br />
<br />
<br style="clear:both;"/><br />
<br />
<h3>Construction of the model</h3><br />
<p><br />
Model of the TLR signaling was prepared in CellDesigner. Signaling steps, where the response remains the same were assembled into one block (Signaling cascade), whose activation directly affects the [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] translocation. Our engineered feedback loop is inserted as a transcriptional activation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] protein domain (red block in Figure 8).</p><br />
<br />
[[Image:model12a.gif|left|thumb|400px|<b>Figure 8: Simplified model of Toll-like receptor signaling constructed in CellDesigner.</b>]]<br />
[[Image:model12.gif|right|thumb|400px|<b>Figure 9: Model of TLR signaling with additional feedback loop for the inhibition of [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]].</b>]]<br />
<br />
<br style="clear:both;"/><br />
<br />
<p>Simulation of cellular response to TLR stimulation by a pulse of [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]] (e.g. flagellin). When available parameters were taken from the literaturte (Selvarajoo, 2006). Signaling cascade results in the transient translocation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] into the cell nucleus (green) and in the production of inflammatory mediators. Repeated pulse after some time results in the repeated activation and accumulation of inflammatory mediators.</p><br />
<br />
<br style="clear:both;"/><br />
<br />
[[Image:model6.gif|thumb|left|400px|<b>Figure 10: Simulation of the cell response to the bacterial stimulus [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP</b>]] (grey) – transient translocation of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] into the nucleus (green), production of stimulatory mediators (orange).]]<br />
[[Image:model7.gif|thumb|right|400px|<b>Figure 11: Simulation of the repeated cellular stimulation by [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]</b> (grey) - the amount of inflammatory mediators increases (orange).]]<br />
<br />
<br style="clear:both;"/><br />
<br />
<p>Simulation of the TLR signaling response in the system with a feedback device results in the production of inhibitor ([[Ljubljana, Slovenia 2006/Terms & References|dnMyD88]], black) which inhibits cellular activation by a repeated stimulation. Cellular responsiveness resets to the normal response if the inhibitor is rapidly degraded, such as by the addition of a [[Ljubljana, Slovenia 2006/Terms & References|PEST]] motif.</p><br />
<br />
[[Image:model8.gif|thumb|left|400px|<b>Figure 12: Introduction of the feedback loop to initiate the production of inhibitory [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]]</b>( (black) – cell does not respond to the repeated stimulation.]]<br />
[[Image:model9.gif|thumb|right|400px|<b>Figure 13: Feedback loop with rapid degradation of the inhibitor ([[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with PEST sequence</b> - black) – responsiveness of the system is again restored to a full extent.]]<br />
<br style="clear:both;"/><br />
<br />
[[Image:model10.gif|thumb|left|400px|<b>Figure 14: Speed of the degradation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] could modulate the responsiveness of the system to repeated (or continuous) challenge by TLR agonists.</b>]]<br />
<br />
<br style="clear:both;"/><br />
<br />
<hr><br />
<center><br />
[[Image:line-si4.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006|Home]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signalling Pathway]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Results & Conclusions|Results & Conclusions]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr></table>[[Image:line-si3.jpg]]<br />
</center></div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Project_%26_ModelLjubljana, Slovenia 2006/Project & Model2006-10-31T14:43:04Z<p>MDolinar: </p>
<hr />
<div><center>[[Image:Logo-si1.gif|center|120 px]] <br><br />
<br />
[[Image:line-si4.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006|Home]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signaling Pathway]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Results & Conclusions|Results & Conclusions]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr></table><br />
[[Image:line-si3.jpg]]<br />
<br><br />
</center><br />
<br />
<br><br><br />
<br />
<h1>Project ideas</h1><br />
Reasearch in the Laboratory of Biotechnology at the National institute of Chemisty, where most of our instructors come from and where most of the experimental work was conducted, is in the area of molecular immunology, specifically interactions in the Toll-like receptor signaling to microbial infections. This determined the selection of the eukaryotic system as our arena of synthetic biology "sandbox". besides, several contributions at the Synthetic biology meetings demonstrated that mammalian cell engineering <br />
<br />
In the beginning we have discussed following project ideas:<br />
<ol><br />
<li>Inserton of a logical device into the signaling pathway to limit the cellular activation by bacterial infection<br />
- use of dominant negative versions of proteins involved in signaling pathway, which will arrest the signaling pathway thus preventing [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] (transcription factor mentioned above)to translocate to the nucleus<br />
- addition of degradation tags to this dominant negative proteins ([[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]]), which would cause inhibition (negative feedback loop) to be temporal. </li><br />
<br />
<li>Engineering of the response to pathogens/their [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]s, which are otherwise not recognized by cells<br />
- for example a response to beta glucans of fungi.</li><br />
<br />
<li>Engineering of novel interconnections between signaling pathways. </li> </ol><br />
<br><br />
<br />
<h1>Selected project</h1><br />
<br />
[[Image:sepsa.png|250px|thumb|<b>Figure 4: Unlimited cell stimulation due to the infection can lead to sepsis, which is in many cases fatal</b>. Introduction of the negative feedback could decrease the immune response.]]<br />
<p>The basic idea of our project was to introduce a feedback loop in TLR signaling pathway, which would decrease the overwhelming response to the persistent or repeated stimulus with [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]. However, completly shutting down the response to bacterial stimulation is not recommendable from the view of a host battling the infection. Ideally the feedback loop should decrease the response when it is too high but recover the responsiveness of the system after some time.</p><br />
<br />
<p>Inhibition of the overwhelming response could be achieved if [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]] is simultaneously able to activate the immune response and the expression of a [[Ljubljana, Slovenia 2006/Terms & References#Terms|dominant-negative]] adapter protein, that would inactivate [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]`s signaling pathway. Decreasing the lifetime of the dominant-negative inhibitior by the addition of a rapid degradation tag ([[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]]) should inactivate the inhibition and restore the responsiveness of the immune system.</p><br />
<br />
<p>This idea is similar to the natural mechanism of tolerance, which is already present in living cells and which decreases the response to repeated bacterial stimulations. This natural tolerance is activated slowly, on the order of days and operates through several different mechanisms. Our feedback mechanism (i.e. artificial tolerance) should decrease the response within hours and thus "attack" the signaling pathway at the point, which has not been used in the natural system.</p><br />
<br><br />
<br />
<h2>Model of the TLR signaling triggered by bacterial infection</h2><br />
<br />
<h3>[[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] is the hub of the TLR signalling pathway</h3><br />
<p><br />
Figure 5 represents the complexity of cell signalling mediated by TLRs. which contains more than 700 molecules and complexes (Oda, Kitano, Molecular Systems Biology 2006).</p><br />
<br />
<br style="clear:both;"/><br />
<br />
[[Image:model1.gif|thumb|center|800px|<b>Figure 5: Comprehensive scheme of the Toll-like receptor signaling of microbial infection.</b>]]<br />
<br />
<br />
<br style="clear:both;"/><br />
<br />
<p>[[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] adapter protein is involved in the signal transduction immediately after ligand-induced TLR oligomerization. This adapter protein is common to most TLRs before the signaling network branches into several phosphpryation cascades. We selected this molecule as the most appropriate target of TLR signaling network engineering (Figures 6 and 7).</p><br />
<br />
[[Image:model2.gif|thumb|left|650px|<b>Figure 6: [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] adapter protein connects the response of all TLRs and represents the »bottleneck« of the system (shaded area).</b>]]<br />
[[Image:model3.gif|thumb|right|200px|<b>Figure 7: Schematic representation of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] as the hub of TLR signaling shaped like a bow tie.</b>]]<br />
<br />
<br style="clear:both;"/><br />
<br />
<h3>Construction of the model</h3><br />
<p><br />
Model of the TLR signaling was prepared in CellDesigner. Signaling steps, where the response remains the same were assembled into one block (Signaling cascade), whose activation directly affects the [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] translocation. Our engineered feedback loop is inserted as a transcriptional activation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] protein domain (red block in Figure 8).</p><br />
<br />
[[Image:model12a.gif|left|thumb|400px|<b>Figure 8: Simplified model of Toll-like receptor signaling constructed in CellDesigner.</b>]]<br />
[[Image:model12.gif|right|thumb|400px|<b>Figure 9: Model of TLR signaling with additional feedback loop for the inhibition of [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]].</b>]]<br />
<br />
<br style="clear:both;"/><br />
<br />
<p>Simulation of cellular response to TLR stimulation by a pulse of [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]] (e.g. flagellin). When available parameters were taken from the literaturte (Selvarajoo, 2006). Signaling cascade results in the transient translocation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] into the cell nucleus (green) and in the production of inflammatory mediators. Repeated pulse after some time results in the repeated activation and accumulation of inflammatory mediators.</p><br />
<br />
<br style="clear:both;"/><br />
<br />
[[Image:model6.gif|thumb|left|400px|<b>Figure 10: Simulation of the cell response to the bacterial stimulus [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP</b>]] (grey) – transient translocation of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] into the nucleus (green), production of stimulatory mediators (orange).]]<br />
[[Image:model7.gif|thumb|right|400px|<b>Figure 11: Simulation of the repeated cellular stimulation by [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]</b> (grey) - the amount of inflammatory mediators increases (orange).]]<br />
<br />
<br style="clear:both;"/><br />
<br />
<p>Simulation of the TLR signaling response in the system with a feedback device results in the production of inhibitor ([[Ljubljana, Slovenia 2006/Terms & References|dnMyD88]], black) which inhibits cellular activation by a repeated stimulation. Cellular responsiveness resets to the normal response if the inhibitor is rapidly degraded, such as by the addition of a [[Ljubljana, Slovenia 2006/Terms & References|PEST]] motif.</p><br />
<br />
[[Image:model8.gif|thumb|left|400px|<b>Figure 12: Introduction of the feedback loop to initiate the production of inhibitory [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]]</b>( (black) – cell does not respond to the repeated stimulation.]]<br />
[[Image:model9.gif|thumb|right|400px|<b>Figure 13: Feedback loop with rapid degradation of the inhibitor ([[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with PEST sequence</b> - black) – responsiveness of the system is again restored to a full extent.]]<br />
<br style="clear:both;"/><br />
<br />
[[Image:model10.gif|thumb|left|400px|<b>Figure 14: Speed of the degradation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] could modulate the responsiveness of the system to repeated (or continuous) challenge by TLR agonists.</b>]]<br />
<br />
<br style="clear:both;"/><br />
<br />
<hr><br />
<center><br />
[[Image:line-si4.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006|Home]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signalling Pathway]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Results & Conclusions|Results & Conclusions]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr></table>[[Image:line-si3.jpg]]<br />
</center></div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/MethodsLjubljana, Slovenia 2006/Methods2006-10-31T14:42:23Z<p>MDolinar: </p>
<hr />
<div><center>[[Image:Logo-si1.gif|center|120 px]] <br><br />
<br />
[[Image:line-si4.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006|Home]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signaling Pathway]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Project & Model|Project & Model]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Results & Conclusions|Results & Conclusions]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr></table>[[Image:line-si3.jpg]]<br />
<br><br />
</center><br />
<br><br><br />
<br />
<h2>Parts design</h2><br />
<br />
<p>At first we had to design primers to replicate a desired DNA fragments. In primers we included restriction sites - on left site ''XbaI'' and on the right site ''SpeI'', ''NcoI'' and ''PstI''. We cloned that part into BioBrick plasmids with ''ccdB'' domain to get all restriction sites needed for BioBrick assembly. We had to design all parts ''de novo'', since no parts like promoters, terminators, desired proteins for signaling pathway modification, degradation flags and reporters had been designed so far - neither to work in mammalian cells. List of desired constructs is shown below. For our use we designed a special vector ([http://partsregistry.org/Part:BBa_J52017 BBa_J52017]) with terminator to simplify constructs assembly. All our composite parts (promoter plus part) were then cloned in this vector.</p><br />
<br />
<p>We also needed fusion proteins e.g. [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]]-rLuc-PEST ([http://partsregistry.org/Part:BBa_J52013 BBa_J52013]) - that is our dominant negative protein linked with reporter and degradation flag. This parts are designed like basic parts - not composite, although they are fusion proteins. Between proteins there is a six aminoacid long linker because only two aminoacid long linker formed during biobrick assembly could affect protein folding. We introduced a six amino acids long linker inbetween protein - reporter and reporter - degradation flag with primers using PCR Overlap Extension method. These parts were then combined with promoter ([[Ljubljana, Slovenia 2006/Terms & References|NF-&kappa;B]]) in BioBrick assembly technique. The part was then inserted into the vector with terminator and ready to use in human cells ([[Ljubljana, Slovenia 2006/Terms & References#Terms|HEK293]]).</p><br />
<br><br />
<br />
<h2>Experiments on mammalian cell cultures</h2><br />
<br />
<p>In September and October we were transfecting our constructs into human embrional kidney cells ([[Ljubljana, Slovenia 2006/Terms & References#Terms|HEK293]]). At the same time we were optimizing three detection systems mentioned below. At first we had to optimize the methods (read articles, test negative and positive controls) and learn how to work with human cells.</p><br />
<br />
<p>Cells were transfected not only with our constructs but also with TLRs which are not natively present on the surface of [[Ljubljana, Slovenia 2006/Terms & References#Terms|HEK293]] cells (only TLR3 and TLR6 are present).</p><br />
<br />
<p>First we transfected all [[Ljubljana, Slovenia 2006/Terms & References#Terms|HEK293]] cells with our constructs and with two additional plasmids coding for [[Ljubljana, Slovenia 2006/Terms & References#Terms|TLR4]] and the accessory [[Ljubljana, Slovenia 2006/Terms & References#Terms|MD-2]] protein. Very soon, we found out that our plasmid preparations were contaminated with [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]] from ''E. coli'' (strain DH5&alpha; used for plasmid preparation), since there was no difference in the response between stimulated (with [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]]) and unstimulated cells. To overcome this problem we decided to focus on TLR5 receptor which reacts upon contact with bacterial [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]]. Signal transfer through TLR5 does not depend on the presence of [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]].</p><br />
<br><br />
<br />
<h2>Detection systems</h2><br />
<br />
<p>In order to monitor the activbation of the signaling pathway and function of our Parts we considered using several detection systems. We expected as the result the synthesis of dominant negative protein ([[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] or [[Ljubljana, Slovenia 2006/Terms & References#Terms|TRAF6]]), blocking of signaling pathway and consequently transcription termination of dominant negative protein. The constructs (parts) we prepared were designed for each detection system respectively.</p><br />
<br />
<p>Requests for the optimal detection system were:<br />
<br />
<ul type="circle"> <li>velocity;</li><br />
<li>sensitivity;</li><br />
<li>paralelization;</li><br />
<li>optical signal would be preferred for the ease of data collection;</li><br />
<li>''in vivo'' and real time detection as a bonus;</li><br />
<li>low price per assay.</li></ul></p><br />
<br />
<p>On the basis of those requests we have decided for the following detection systems: flow cytometry, luminometry and ELISA. Members of the team in charge for each detection system are :<br />
<li>for flow cytometry:Jernej and Ota, (mentor Mateja)</li><br />
<li>for luminometry:Alja, Moni and Rok, (mentor Mojca)</li> <br />
<li>for ELISA:Jelka and Matej (mentor Gabi).</li></p><br />
<br />
<br />
<p>Constucts used in individual detection system and short description of detection systems:</p><br />
<br />
<br><br />
<h4>Flow cytometry</h4><br />
<br />
Used composite parts: NF&kappa;B+dnMyD88-link-rLuc ([http://partsregistry.org/Part:BBa_J52014 BBa_J52014]).<br />
<br />
[[Image:Flow cytometer.jpg|right|thumb|200px|Figure 15: Ota working on the flow cytometer.]]<br />
<br />
<p>Flow cytometry is a sophisticated method, which gives very precise and highly valuable results. The main advantage of this method is that measurements are done on individual cells, so the results are not the average value of the population. In our project we could detect cellular activation based on the phosphorylation cascade activation and anti-phosphoprotein antibodies. In our experiments we used primary antibodies (Ab) from mouse, which specifically detect only phosphorylated form of [[Ljubljana, Slovenia 2006/Terms & References#Terms|ERK kinase]] (pERK). The secondary antibodies used were anti-mouse Abs labelled with a fluorescence marker phycoerithrin (PE), which we can detect on flow cytometer. Cells were transfected with TLR5 or TLR4/MD-2 and with or without [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]], which is under [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] promoter. On the second day the cells were stimulated with flagellin or [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]] (depends on the selected TLR receptor). According to the literature phosphorylation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|ERK]] can already be detected after 15 min. The amount of p[[Ljubljana, Slovenia 2006/Terms & References#Terms|ERK kinase]] increases for additional 20 min and then it starts to decline. After 40 min no p[[Ljubljana, Slovenia 2006/Terms & References#Terms|ERK]] is detected.</p><br />
<br />
<p>We used pulsed stimulation of cells. First we stimulated cells with 5 ng/ml of flagellin to activate the expression of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]]. We repeated the stimulation with the second, stronger stimulus (50 ng/ml of flagellin) at different time intervals (2, 4, 6 and 8 hours after the first stimulation. 30 min after the second stimulus cells were fixed in 2% paraformaldehid, permeabilized with ice cold methanol and stained for p[[Ljubljana, Slovenia 2006/Terms & References#Terms|ERK]] and secondary Ab labelled with PE.</p> <br />
<br />
<p>Expected results: The activation of the signaling pathway also activates the expression of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dominant negative protein MyD88]], which should block the signaling, so we expect to detect decreased amount of p[[Ljubljana, Slovenia 2006/Terms & References#Terms|ERK kinase]] after second stimulation in comparison to cells without of inducible [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]].</p><br />
<br><br />
<br />
<h4>Detection of transcriptional activation by luminometry</h4><br />
<br />
Used composite parts: NF&kappa;B+dnMyD88 ([http://partsregistry.org/Part:BBa_J52036 BBa_J52036]), NF&kappa;B+dnMyD88-link-rLuc ([http://partsregistry.org/Part:BBa_J52014 BBa_J52014]), NF&kappa;B+dnMyD88-link-rLuc-link-PEST191([http://partsregistry.org/Part:BBa_J52024 BBa_J52024]), NF&kappa;B+rLuc-link-PEST191([http://partsregistry.org/Part:BBa_J52023 BBa_J52023]), NF&kappa;B ([http://partsregistry.org/Part:BBa_J52010 BBa_J52010]), CMV-rLuc([http://partsregistry.org/Part:BBa_J52038 BBa_J52038]) and CMV+rLuc-link-PEST191([http://partsregistry.org/Part:BBa_J52039 BBa_J52039]).<br />
<br />
[[Image:luminometer.jpg|200px|right|thumb|Mithras luminometer]]<br />
<p>Luminometry is a very used method to measure activation of signaling pathways. In many cases it replaced ELISA because it is much more sensitive. The basis of the method is the measurment of light, which is emited after substrate cleavage. We used two different enzymes; the firefly luciferase and the Renilla (sea pansy) luciferase can discriminate between their respective bioluminescent substrates and do not cross-activate, that is why we can measure the activity of both in one sample.</p> <br />
<br />
<p>Dual luciferase assay</p><br />
<p>Dual luciferase assay includes two different luciferase reporter enzymes that are expressed simultaneously in each cell. Typically, the experimental reporter (in our case NFkB-fLuc) is correlated with the effect of specific experimental conditions, while the activity of the co-transfected "control" reporter gene (in our case CMV-rLuc) provides an internal control, which serves as the baseline response. Normalizing the experimental reporter gene to the activity of an internal control minimizes the variability caused by differences in cell viability and transfection efficiency. Thus, dual reporter assays allow more reliable interpretation of the experimental data by reducing extraneous influences.</p><br />
<br />
<br />
<p>Different experiments were conducted:</p><br />
<br />
<p>1. To be able to measure the activation of dnMyD88 expression after stimulation, we prepared dnMyD88 construct under [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] inducible promoter in the fusion with rLuc (construct NFkB+dnMyD88-link-rLuc). We transfected cells with TLR5 receptor plasmid and our biobrick construct. Cells were stimulated with [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]]. The expression of NFkB+dnMyD88-link-rLuc was measured in time intervals. We expect that amount of rLuc will increase in time only if cells are stimulated with a ligand.</p><br />
<br />
<p>2. CMV-rLuc-link-PEST191 biobrick was constructed to measure the kinetics of degradation of proteins in fusion with PEST. Cells were transfected with the construct. We found out that it takes four hours after the transfection to detect luciferase activity. To calculate half-life of rLuc and rLuc-PEST we transfected cells with either of those two constructs and incubated them to allow proteins to sythesize. New protein synthesis was inhibited by adding cycloheximide (inhibitor of protein synthesis in higher eucaryotes) and the activity of rLuc was measured in time intervals . Because the addition of cycloheximide stops the sythesis of new proteins there should be a decrease in rLuc activity due to protein degradation. A construct with [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]] should decrease more rapidly as rLuc alone.</p><br />
<br />
<p>3. Cells were transfected with TLR5, NFkB-fLuc, CMV-rLuc and +/- NFkB+dnMyD88. To have the amount of NF-kB binding sites equal in all cells we added the same amount of plasmid containing only [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] inducible promotor to cells without NFkB+dnMyD88. The next day after the transfection cells were first stimulated with 5 ng/ml of flagellin to activate the signaling pathway and thus dnMyD88 expression. After 6 hours (time, which was measured as an optimal time for detection of the expression of dnMyD88) cells were restimulated with 50 ng/ml of flagellin. Activation of fLuc was measured in time intervals (0, 2, 4, and 6 hours after the second stimulation). The data were normalized for transfection efficiency by measuring rLuc. We expect that there will be a lower activation after the second stimulation in cells, which express dnMyD88.</p><br />
<br />
<h4>ELISA for detection of transcription factors</h4> <br />
<br />
[[Image:NF-kB-ELISA.jpg|200px|right|thumb|Figure 17: Schematic representation of the ELISA for detection of the transcriptionally-active NF&kappa;B.]]<br />
<br />
Used composite parts: NF&kappa;B+dnMyD88 ([http://partsregistry.org/Part:BBa_J52036 BBa_J52036]) and NF&kappa;B+dnTRAF6-link–GFP ([http://partsregistry.org/Part:BBa_J52022 BBa_J52022]).<br />
<br />
<p>With ELISA detection system we are detecting free [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] transcription factor, which it was already shown that it is a good measurment of pathway activation. Active form of this protein is present after the activation of signaling pathway when protein is released from a complex with IkB inhibitor. Free NF-kB migrates into the nucleus where it acts as transcription activator. We are using high bind 96-well plates coated with [http://en.wikipedia.org/wiki/Streptavidin streptavidin](we prepare it ourselves). The probe for free [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] is biotinilated double stranded [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] binding sequence. The free [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] protein from whole cell lysate is captured by a probe, which biotin group binds to streptavidin coated on a plate. We detect [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] with primary mouse antibodies and secondary peroxidase-conjugated rabbit anti-mouse antibodies. Peroxidase is an enzyme, which cleaves a substrate that emits light, which we measure on luminometer. </p><br />
<br><br />
<br />
<h4>Confocal microscopy</h4><br />
[[Image:celica2.gif|right|thumb|200px|Figure 18: Fluorescence of MyD88-GFP in transfected HEK293 cells.]]<br />
<p>Microscope allows localization of signaling molecules within each individual cell, such as translocation of transcription factors, trafficking of proteins fused with reporter fluorescent proteins etc. Therefore this technique should be very usefull particularly for monitoring the expression of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] inhibitory domain fused with fluorescent protein and the timecourse of its degradation. Therefore we designed Parts and several Devices, which included fusion with fluorescent protein:</p> <br />
<br><br><br />
[http://partsregistry.org/Part:BBa_J52021 BBa_J52021]<br />
<br>[http://partsregistry.org/Part:BBa_J52021 BBa_J52022]<br />
<br>[http://partsregistry.org/Part:BBa_J52021 BBa_J52026]<br />
<br>[http://partsregistry.org/Part:BBa_J52021 BBa_J52027]<br />
<br>[http://partsregistry.org/Part:BBa_J52021 BBa_J52040]<br />
<br>[http://partsregistry.org/Part:BBa_J52021 BBa_J52048].<br />
<br><br />
<br />
<h1>Problems and Troubleshooting</h1><br />
<br />
''Construction of Parts using bad oligos''<br> <br />
<p>We can only suggest to the other teams in the future to order oligos from a reliable and well established company (not necessarily the onw with the lowest price). When we repeatedly failed to produce PCR products we thought it could have been the beginner's bad luck, but when we determined a huge number of point mutations in the primer region even the mentors decided that we better avoid this company in the future (although they did offer us the replacement of oligos for free). There'll be no next time for this company in our lab.</p><br />
<br />
''Theory versus practise''<br><br />
<p>We spent a lot of time trying to find an answer to the following question: "Why didn't our transformants grow on the LB-Tc plates?". First we accused our our tetracycline, than to the proper concentration, but at the end we found out that one of the previous year's parts was incorrectly registered. Now we know the importance of checking everything twice... </p><br />
<br />
<br />
''Real-time luminescence''<br> <br />
<p>We have attempted to monitor "real time" cell activation using luminescence and invested considerable amount of time and effort into this. We could not detectluminescence ''in vivo'' since coelentrazine (substrate of Renilla luciferaze) decays too quickly, while the luciferin could not penetrate the cells. We have also used the caged luciferin as well as its methy esther but in neither case did we suceed in detecting the luciferase in the living cells (several authors of published protocols were unavailable for comments :-( ).</p><br />
<br />
<br />
''Optimization of activation intervals''<br><br />
<p>Much od lab work (more than initially planned) was devoted to the design of experiments to monitor cell activation. We had to find out the optimal concentration of inducers, the timespan between the first and second activation which gave the best difference, lenght of the experiment in which difference between genetically engineered and wild-type cells would be sufficiently prominent etc. Methods from the literature could not simply be adopted into our experimental procedure.</p><br />
<br />
<center><br />
<br><br />
<br />
[[Image:line-si4.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006|Home]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signaling Pathway]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Project & Model|Project & Model]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Results & Conclusions|Results & Conclusions]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr></table><br />
[[Image:line-si3.jpg]]<br />
<br><br />
</center></div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Background_and_Signalling_PathwayLjubljana, Slovenia 2006/Background and Signalling Pathway2006-10-31T14:41:40Z<p>MDolinar: </p>
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[[Image:line-si4.jpg]]<br />
<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006|Home]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Project & Model|Project & Model]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Results & Conclusions|Results & Conclusions]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr></table><br />
[[Image:line-si3.jpg]]<br />
</center><br />
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<br />
<h1>Background</h1><br />
<br />
[[Image:scheme2.jpg|right|thumb|360px|Figure 1: Toll-like receptors sense the presence of microbial infection and initiate the innate immune resonse.]]<br />
<br />
<p>When bacteria invade the human body, it has to respond quickly by activation of the innate branch of the immune system, which recognizes the broad spectrum of molecules, specific for pathogenic microorganisms. Immune response cells have on their surface a collection of receptors (Toll-like receptors - TLR), which recognize those "Pathogen associated molecular patterns" or [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]s. [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]s include various components of microorganisms (e.g. lipopolysaccharides, which are part of outer cell membrane of Gram-negative bacteria, flagellin, lipopeptides but also double-stranded RNA from viruses etc., which are each recognized by one of the 11 human TLRs) (Akira et al., 2004). This kind of cell signaling is the core of our efficient innate immune response (i.e. the fast-acting, not mediated by antibodies) against microbes. In some instances, however, extensive signaling mediated by TLR is not beneficial. If the cellular response gets out ouf control and becomes exaggerated, excessive inflammatory response may lead to sepsis, which can lead to severe organ failure and often the consequences are fatal (Cohen, 2002). Sepsis claims '''each year more than 150,000 casualties''' within the USA and a similar number within the EU. In the absence of an effective treatment of sepsis, the analysis of the signaling pathway may indicate the potential therapeutic targets. </p><br />
<br />
<p>Response to bacterial stimulus is a double-edged sword - too strong response may lead to sepsis, while the too weak response does not overcome the bacterial infection, therefore both extremes have to be in the balance. '''The feedback mechanism that would limit the excessive cell stimulation, but would nevertheless maintain the responsiveness to bacterial infection could provide the path to an effective antiseptic strategy.'''</p><br />
<br />
<br />
<br />
[[Image:balance.jpg|center|thumb|360px|Figure 2: Innate immune response has to maintain the delicate balance between the infection in case of low response and excessive inflammation in case of the exaggerated response.]]<br />
<br />
<h1>Signaling pathway of bacterial recognition in a nutshell</h1><br />
<br />
<p>Toll-like receptors (TLRs) are the key gatekeepers of innate immunity, sensing and responding to invading microorganisms. So far in humans eleven different receptors are konwn that recognize specific molecular patterns which are specific for microbes, e.g. lipopolysaccharide ([[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]]), lipopeptides, double and single stranded RNA of viruses, nonmethylated [[Ljubljana, Slovenia 2006/Terms & References#Terms|CpG]] containing DNA, glycolipids, and several others.</p><br />
<br />
<p>Most of the receptors and transducers in the signaling cascade involved in the initial stages of cell transduction have been identified, however the complete response is highly complex as for example [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]] alone can affect transcription of more than 1000 different genes (Beutler, 2004).</p><br />
<br />
<p>Binding of bacterial constituents ([[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]s) to TLR triggers the association of its intracellular TIR domains, which recruit from the cytoplasm to the membrane an adapter molecule [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]], which in turn transmits the signal to other components of the signaling cascade, particularly kinases, as shown in Figure 2. </p><br />
<br />
[[Image:signalling1b.jpg|left|thumb|450px|Figure 3: TLR5 signaling pathway. The main step in the flagellin activation of TLR5 is the release of a transcription factor [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] and its translocation to the nucleus, where it is responsible for the activation of its target genes. Adapted from Liaudet et al. (2002) Drug News Perspect. 15, 1-13]]<br />
<br />
<p>When receptors sense the presence of microbial components, stimulation of TLRs occurs and triggers the association of MyD88 (myeloid differentiation primary-response protein 88), which in turn recruits IRAK4, thereby allowing its association with IRAK1. IRAK4 phosphorylates IRAK1 and with phosphorylated IRAK1 associates [[Ljubljana, Slovenia 2006/Terms & References#Terms|TRAF6]] (tumour necrosis factor receptor- associated factor 6). The complex of phosphorylated IRAK1 and [[Ljubljana, Slovenia 2006/Terms & References#Terms|TRAF6]] dissociates from the receptor and forms a complex with TAK1, TAB1 and TAB2, which induces the phosphorylation of TAB2 and TAK1. This leads to the ubiquitylation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|TRAF6]], which induces the activation of TAK1. TAK1 phosphorylates IKK complex (inhibitor of nuclear factor &kappa;B (I&kappa;B)-kinase complex), which consists of IKK&alpha;, IKK&beta; and IKK&gamma;. The IKK complex then phosphorylates I&kappa;B, which is associated with [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] thus enabling its translocation to the nucleus. Phosphorylation of I&kappa;B leads to its ubiquitylation and subsequent degradation. This allows the important inflammatory transcription factor [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] to release and to translocate to the nucleus and to induce the transcription of a range of its target genes, such as cytokines and chemokines.</p><br />
<br />
<p>Inflammatory cytokines, chemokines and interferons attract cells of the immune system (lymphocytes, macrophages) to the infected area. This is beneficial to organism, but if overactivated it can also lead to severe inflammation and sepsis (Cohen,2002; Beutler, 2004).</p><br />
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<center><br />
[[Image:line-si4.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006|Home]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Project & Model|Project & Model]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Results & Conclusions|Results & Conclusions]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr></table><br />
[[Image:line-si3.jpg]]<br />
</center></div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Team_membersLjubljana, Slovenia 2006/Team members2006-10-31T14:40:14Z<p>MDolinar: </p>
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[[Image:line-si4.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006|Home]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signaling Pathway]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Project & Model|Project & Model]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Results & Conclusions|Results & Conclusions]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th></tr></table>[[Image:line-si3.jpg]]<br />
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<center>[[Image:SLOteam06a.jpg|center|From left to right: Alja, Ota, Jelka, Matej, Monika, Jernej, Rok, Simon, Marko, Mojca M., Roman]]<br />
From left to right: Alja, Ota, Jelka, Matej, Monika, Jernej, Rok, Simon, Marko, Mojca M., Roman</center><br />
<br />
----<br />
'''STUDENTS:'''<br />
<br />
*[[User:Moni|Monika Ciglic (Microbiology)]]<br />
*[[User:Ota|Ota Fekonja]](Microbiology)<br />
*[[User:Jernej|Jernej Kovac]] (Biochemistry)<br />
*[[User:Mojca Miklavec|Mojca Miklavec]] (Physics & Bioinformatics)<br />
*[[User:Alja|Alja Oblak]] (Microbiology)<br />
*[[User:Jelka.pohar|Jelka Pohar]] (Microbiology)<br />
*[[User:Matej|Matej Skocaj]] (Microbiology)<br />
*[[User:rtkavc|Rok Tkavc (Microbiology)]]<br />
<br />
'''SUPERVISORS:'''<br />
<br />
*Roman Jerala (National Institute of Chemistry = [http://www.ki.si/index.php?id=218&L=1 NIC])<br />
*[[User:MDolinar|Marko Dolinar]] ([http://www.fkkt.uni-lj.si/en/ Faculty of Chemistry & Chemical Technology])<br />
<br />
<br />
'''INSTRUCTORS:'''<br />
*Mojca Bencina (NIC) <br />
*Mateja Mancek Keber (NIC) <br />
*Gabriela Panter (NIC) <br />
<br />
{|<br />
|-<br />
|[[Image:Mojca.jpg|thumb|150px|Mojca at work]]<br />
|[[Image:Mateja.jpg|thumb|150px|Mateja]]<br />
|[[Image:Gabi.jpg|thumb|150px|Gabi and Moni having a discussion]]<br />
|-<br />
|}<br />
<br />
<br />
To see more pics, click [[Ljubljana, Slovenia 2006/Gallery|HERE]]<br />
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<center>[[Image:SLOteam06b.jpg|center|From left to right: Gabriela, Roman, Jelka, Jernej, Monika, Matej, Alja, Rok, Mojca M., Marko]] <br />
From left to right: Gabriela, Roman, Jelka, Jernej, Monika, Matej, Alja, Rok, Mojca M., Marko.</center><br />
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<table cellpadding="10"><br />
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<th>[[Ljubljana, Slovenia 2006/Project & Model|Project & Model]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Results & Conclusions|Results & Conclusions]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th></tr></table><br />
[[Image:line-si3.jpg]]</div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Terms_%26_ReferencesLjubljana, Slovenia 2006/Terms & References2006-10-31T14:39:17Z<p>MDolinar: </p>
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<th>[[Ljubljana, Slovenia 2006/Project & Model|Project & Model]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Results & Conclusions|Results & Conclusions]]</th><br />
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<br />
<h1>Terms</h1><br />
<br />
<dl><dt>''CpG''</dt><br />
<dd>Unmethylated CpG motifs usually present in bacterial DNA. They are recognised by TLR9 receptor.</dd><br />
<br />
<dt>''dnMyD88''</dt><br />
<dd>Dominant negative form of MyD88, which has only TIR-domain.</dd><br />
<br />
<dt>''Dominant negative protein''</dt> <br />
<dd>A protein that is mutated in a way to compete with wild type protein, but does not support the cascade of additional interactions downstream. This results in a blocking of the pathway.</dd><br />
<br />
<dt>''ERK kinases''</dt><br />
<dd>Extracellular signal-regulated kinases (ERKs) are regulatory proteins that mediate cell survival, proliferation and differentiation. Activation of ERK is coupled to stimulation of cell-surface proteins via several different upstream signaling pathways, and contributes to the regulation of diverse cellular processes - from cell excitability to gene expression.</dd><br />
<br />
<dt>''Flagellin''</dt><br />
<dd>This protein can be found in bacterial flagellae. It arranges itself in a hollow cylinder to form the filament in bacterial flagellum.</dd><br />
<br />
<dt>''HEK293''</dt><br />
<dd>Human embryonic kidney epithelial cell line, that was generated by transformation of human embryonic kidney cell cultures (hence HEK) with sheared adenovirus 5 DNA. They are very easy to work with, and so are a widely-used cell line in cell biology research.</dd><br />
<br />
<dt>''LPS''</dt><br />
<dd>LPS stands for lipopolysaccharide. It is a characteristic component of the cell membrane of Gram-negative bacteria. It is also an endotoxin, and induces a strong response from normal animal/human immune systems. It binds to the TLR4/MD2/CD14 receptor complex, which promotes the secretion of pro-inflammatory cytokines in many cell types.</dd><br />
<br />
<dt>''MD2''</dt><br />
<dd>MD2 is an essential protein component that stabilizes TLR4. It has a critical role LPS linking to the LPS receptor.</dd><br />
<br />
<dt>''MyD88''</dt><br />
<dd>A central protein of the TLR signaling pathway that transfers signal from TLR receptor to downstream proteins (IRAK4) resulting in the NF&kappa;B activation. MyD88 is composed of TIR domain, with which it interacts with TIR domains of TLRs and of a death domain, which interacts with heteromeric death domains of IRAK kinases. Truncated MyD88, comprising only of TIR domain has a dominant-negative phenotype (dnMyD88), because it interacts with cytoplasmic domain of TLRs and competes with wild type MyD88. Because of the lack of a death domain dnMyD88 does not support the cell signaling.</dd><br />
<br />
<dt>''NF-&kappa;B''</dt><br />
<dd>A transcription factor that functions as heterodimer. NF-&kappa;B dimers are usually sequestered in the cytoplasm in an inactive form. Activation of NF-&kappa;B involves phosphorylation and proteolysis of the inhibitory I&kappa;B proteins, release and nuclear translocation of the NF-&kappa;B, which results in activation of NF&kappa;B-dependent transcription.</dd><br />
<br />
<dt>''PAMP''</dt><br />
<dd>Abbreviation of Pathogen Associated Molecular Pattern. Those molecules (flagellin, LPS, CpG etc.) are characteristic of pathogenic microorganisms and recognition of which triggers imune response.<br />
<br />
<dt>''PEST sequence''</dt><br />
<dd>A sequence that has been associated with rapidly degraded proteins. The short life-time of a protein is signaled by a region rich in the amino acids proline (P); glutamic acid (E); serine (S); or threonine (T).</dd><br />
<br />
<dt>''TLR 4''</dt><br />
<dd>Member of TLR receptor familiy that is predominantly activated by lipopolysaccharide. It can activate MyD88 dependent and independent way. For activation also MD2 and CD14 accessory molecules are needed. </dd><br />
<br />
<dt>''TRAF6''</dt><br />
<dd>A protein that functions as a signaling mediator. It binds to IRAK1 and transfers signal downstream, also resulting in NF-&kappa;B activation.</dd></dl><br />
<br />
<h1>References</h1><br />
<p>Akira, S. and K. Takeda (2004). "Toll-like receptor signalling." Nat Rev Immunol 4(7): 499-511.</p><br />
<br />
<p>Akira, S., M. Yamamoto, et al. (2003). "Role of adapters in Toll-like receptor signalling." Biochem Soc Trans 31(Pt 3): 637-42.</p><br />
<br />
<p>Beutler, B. (2004). "Inferences, questions and possibilities in Toll-like receptor signalling." Nature 430(6996): 257-63.</p><br />
<br />
<p>Cohen, J. (2002). "The immunopathogenesis of sepsis." Nature 420(6917): 885-91.<br />
Dueber, J. E., B. J. Yeh, et al. (2004). "Rewiring cell signaling: the logic and plasticity of eukaryotic protein circuitry." Curr Opin Struct Biol 14(6): 690-9.</p><br />
<br />
<p>Dupraz, P., S. Cottet, et al. (2000). "Dominant negative MyD88 proteins inhibit interleukin-1beta /interferon-gamma -mediated induction of nuclear factor kappa B-dependent nitrite production and apoptosis in beta cells." J Biol Chem 275(48): 37672-8.</p><br />
<br />
<p>Li, X., X. Zhao, et al. (1998). "Generation of destabilized green fluorescent protein as a transcription reporter." J Biol Chem 273(52): 34970-5.</p><br />
<br />
<p>Oda, K. and H. Kitano (2006). "A comprehensive map of the toll-like receptor signaling network." Mol Syst Biol 2: 2006 0015.</p><br />
<br />
<p>Selvarajoo, K. (2006). "Discovering differential activation machinery of the Toll-like receptor 4 signaling pathways in MyD88 knockouts." FEBS Lett 580(5): 1457-64.</p><br />
<br />
<p>Smith, K. D. and A. Ozinsky (2002). "Toll-like receptor-5 and the innate immune response to bacterial flagellin." Curr Top Microbiol Immunol 270: 93-108.</p><br />
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[[Image:line-si4.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006|Home]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signaling Pathway]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Project & Model|Project & Model]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Results & Conclusions|Results & Conclusions]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr></table>[[Image:line-si3.jpg]]</div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Results_%26_ConclusionsLjubljana, Slovenia 2006/Results & Conclusions2006-10-31T14:38:10Z<p>MDolinar: </p>
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<div><center>[[Image:Logo-si1.gif|center|120 px]] <br><br />
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[[Image:line-si4.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006|Home]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signaling Pathway]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Project & Model|Project & Model]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr></table>[[Image:line-si3.jpg]]<br />
<br><br />
</center><br />
<br><br><br />
<br />
<h1>Results</h1><br />
<br />
<h2>Construction of Biobricks</h2><br />
We prepared individual BioBricks that could be used in mammalian cells. The following BioBricks were used:<br />
*promoter (constitutive CMV and [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] inducible promoter), <br />
*terminator (which we included into plasmids to avoid repeated additional steps in construction of each functional Part) <br />
*protein coding sequences, which include: <br />
**two inhibitory (dominant negative) proteins of the signaling cascade ([[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] and dn[[Ljubljana, Slovenia 2006/Terms & References#Terms|TRAF6]]), <br />
**two reporters: ''Renilla'' luciferase (in order to allow simultaneous dual luciferase assay to normalize the luminescence for the efficiency of transfection and cell number) and GFP <br />
**[[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]] to decrease the lifetime of the inhibitor.<br />
<br />
Linkers between the protein coding sequences (e.g. [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]]+luciferase+[[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]]) were prepared by PCR ligation to avoid addition of unwanted aminoacids in the linker region between the protein domains.<br />
<br><br />
<br />
<br />
<br />
<center><table><br />
<caption>''Table 1: List of prepared Parts''</caption><br />
<tr><th>Registration number</th><br />
<th>Part's Name</th><br />
<th>Part/Device</th><br />
<th>Vector</th></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52008 BBa_J52008]</td><td>rluc</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52010 BBa_J52010]</td><td>NF&kappa;B</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52011 BBa_J52011]</td><td>dnMyD88-likn-rLuc</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52012 BBa_J52012]</td><td>rluc-link-PEST191</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52013 BBa_J52013]</td><td>dnMyD88-link-rluc-link-pest191</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52014 BBa_J52014]</td><td>NF&kappa;B+dnMyD88-link-rLuc</td><td>Device</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52016 BBa_J52016]</td><td>eukaryotic terminator</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52017 BBa_J52017]</td><td>eukaryotic terminator vector</td><td></td><td>pSB1AK3</td></tr></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52018 BBa_J52018]</td><td>NF&kappa;B+rLuc</td><td>Device</td><td>pSB1AC3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52019 BBa_J52019]</td><td>dnTRAF6</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52021 BBa_J52021]</td><td>dnTRAF6-link-GFP</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52022 BBa_J52022]</td><td>NF&kappa;B+dnTRAF6-link-GFP</td><td>Device</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52023 BBa_J52023]</td><td>NF&kappa;B+rLuc-link-PEST191</td><td>Device</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52024 BBa_J52024]</td><td>NF&kappa;B+dnMyD88-link-rLuc-link-PEST191</td><td>Device</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52026 BBa_J52026]</td><td>dnMyD88-link-GFP</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52027 BBa_J52027]</td><td>NF&kappa;B+dnMyD88-link-GFP</td><td>Device</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52028 BBa_J52028]</td><td>GFP-PEST191</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52029 BBa_J52029]</td><td>NF&kappa;B+GFP-PEST191</td><td>Device</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52034 BBa_J52034]</td><td>CMV</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52035 BBa_J52035]</td><td>dnMyD88</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52036 BBa_J52036]</td><td>NF&kappa;B+dnMyD88</td><td>Device</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52038 BBa_J52038]</td><td>CMV-rLuc</td><td>Device</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52039 BBa_J52039]</td><td>CMV+rLuc-link-PEST191</td><td>Device</td><td>pSB1A2</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52040 BBa_J52040]</td><td>CMV+GFP-PEST191</td><td>Device</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52642 BBa_J52642]</td><td>GFP</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52648 BBa_J52648]</td><td>CMV+GFP</td><td>Device</td><td>pSB1AK3+TER</td></tr><br />
</table></center><br />
<br><br />
<br />
<h2>Detection of phosphorylation of ERK kinases fy flow cytometry reflects the activation of the TLR signaling </h2><br />
<br />
<br style="clear:both;"/><br />
<br />
[[Image:cito1.gif|cito1.gif|center|thumb|400px|<b> Figure 19: Flow cytometric profile of phosphorylated ERK kinases in HEK293 cells stimulated by LPS for 0, 20 and 80 minutes.</b>]]<br />
<br />
<br style="clear:both;"/><br />
<br />
Flow Cytometry experiments showed that after 80 minute stimulation the increase in the level of phosphorylated ERK kinases was evident(Figure 19). The method was not as sensitive to the rapid response as we hoped. Further optimization of the method would be necessary in order to obtain the quantitative data to accurately model the cell signaling.<br />
<br />
<h2>Detection of transcriptionally competent[[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] in whole cell extracts dependent on the time after stimulation with [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]] determined with promoter ELISA method</h2><br />
<br />
<br style="clear:both;"/><br />
<br />
[[Image:Elisa_results.gif|thumb|center|800px|<b>Figure 20: Activity of peroxidase (conjugated on secondary antibodies) in luminescence units as a function of time after stimulation with [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]].</b> The amount of free [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] (and consequently luminescence) increases with time of stimulation (7 hours) and decreases after extended (23 hours) stimulation.]]<br />
<br />
<br style="clear:both;"/><br />
<br />
Cells were transfected with [[Ljubljana, Slovenia 2006/Terms & References#Terms|TLR4]] plasmid and [[Ljubljana, Slovenia 2006/Terms & References#Terms|MD-2]] plasmid in ordeer to confer the LPS responsiveness. We have shown that cells with TRL4 receptor respond to [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]] stimulation and that ELISA is apropriate and sensitive method for the detection of stimulation. However we did not get satisfying results when stimulating (with [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]] or flagellin) cells transfected neither with [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] nor [[Ljubljana, Slovenia 2006/Terms & References#Terms|TRAF6]]. The response of the ELISA method, as the most direct measurement of the free NF-&kappa;B was not sufficiently reproducible and linear in comparison to the method of luciferase reporter plasmids (see below).<br />
<br />
<br />
<h2>Inducible transcription by the stimulation of Toll-like receptor</h2><br />
<br />
<p>Stimulation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|HEK293]] cells transfected by TLR with their agonist (flagellin in case of TLR5) stimulates the translocation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] into the nucleus and activation of transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]]-responsive genes. We have used the firefly luciferase under the [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] dependent promoter to monitor the promotor activation. We tested the functionality of our part containing [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] dependent promotor in a construct with [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]]-rLuc. We detected the R-luciferase activity only in stimulated cells with significant activity appearing a few hours after the cell stimulation, which proves the functionality of this part (inducible promoter).</p><br />
<br />
<br style="clear:both;"/><br />
<br />
<br />
[[Image:graf_gabi.gif|left|thumb|400px|<b>Figure 21: Transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with rLuc reporter is induced by bacterial flagellin </b> Activity of ''Renilla'' luciferase stimulated by the addition of a pulse of [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]]. [[Ljubljana, Slovenia 2006/Terms & References#Terms|HEK293]] cells were transfected with device comprising [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]]-inducible fusion of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]]-rLuc.]]<br />
<br />
[[Image:rezultati7.gif|left|thumb|400px|<b>Figure 22: Transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with rLuc reporter in our device is inducible by bacterial flagellin.</b> Activity of the Firefly luciferase stimulated by the addition of two pulses of [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]]. ]]<br />
<br />
<br style="clear:both;"/><br />
<p>We also measured NF-kB-responsive F-luciferase activity of this device. We expected a decrease in the activity after the amount of time sufficient for the sysnthesis of inhibitory fusion protein in cells. To our dissapointment no decrease of the cell activation was observed. One of the possible causes for the lack of inhibition of the dnMyD88-rLuc protein fusion could be the sterical hindrance of the activity of dnMyD88. This domain interacts with TIR domain of TLRs in the direct proximity of the cellular membrane, which could prevent interaction with larger protein domains. Based on those results we also conducted our experiments with a device containing dnMyD88 without of any protein appendix.<br />
<br />
<h2>Inhibition of cell signaling by a dnMyD88 feedback device</h2><br />
<br />
An adapter protein [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]], consisting of a TIR and death domain, is at the crossroads of Toll-like receptors. Therefore activation of each of the surface expressed TLR recruits [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] to the cell membrane. We selected to use the receptor TLR5 and flagellin instead of the often used [[Ljubljana, Slovenia 2006/Terms & References#Terms|TLR4]] and its agonist [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]], because the [[Ljubljana, Slovenia 2006/Terms & References#Terms|TLR4]] system is extremely sensitive to the contamination with bacterial [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]], which often copurifies with the plasmid. In this way we have avoided the contamination issue, but our system is designed to work on any signalling receptor that has [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] in its signaling pathway (i.e. TLR1,2,4,5,6,7,8,9,11,IL-1).<br />
Cells were cotransfected with TLR5, reporter plasmid (NF&kappa;B-Fluc) and our feedback device ([[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]]-[[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]]).</p><br />
<br />
<p>We expect that after stimulation with flagellin cytoplasmic [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] is released from its inhibitor. It then migrates into the nucleus and induces the transcription of our construct. Accumulation of the dominant negative protein should compete with wild-type [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] and attenuate the downstream signaling pathway. We can detect the [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] translocation, transcriptional activation and phosphorylation by our detection systems - luminescence, free [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]], phosphorylation detected by flow cytometry and confocal microscopy. In case that our construct is followed by [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]] (degradation) sequence, it is expected that product should degrade rapidly and shorten the inhibition period. If the stimulus is still present, cells should again respond to it and the cycle with inhibition will repeat as well.</p><br />
<br />
<p>We have designed the experiment by two consecutive pulses of TLR stimulation separated by 4-6 hours, which is sufficient time to accumulate the inducible inhibitor. Normal cells should respond to both stimuli, while the cells with our device should respond to the same extent only to the first pulse -- this induces the transcription of the inhibitor, therefore the response to the second stimulus should be decreased. However if the stimuli are separated by a longer timespan, the system should respond normally, as the inhibitor has already degraded and the systemn has reset to the normal state.</p><br />
<br />
<center><br />
[[Image:rezultati1a.gif|center|thumb|400px|<b>Figure 23: [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] feedback loop decreases the cellular activation by repeated stimulation with bacterial [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]]. </b> Normalized luciferase activity (Fluc/Rluc) of cells stimulated by the addition of two pulses of flagellin separated by 6 hours, indicated time after the second pulse is shown. [[Ljubljana, Slovenia 2006/Terms & References#Terms|HEK293]] cells were transfected with device comprising NF&kappa;B-inducible [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] in comparison to wild type.]]<br />
</center><br />
<br style="clear:both;"/><br />
<br />
<p>Our results show that the system with inducible [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] indeed responds weaker to the second stimulus. The minimal delay between two stimuli should correspond to the time required for the synthesis of the inhibitor, which is exactly as planned, since it does not completely abolish the cellular response but decreases it after extended stimulation.</p><br />
<br />
<br style="clear:both;"/><br />
<br />
<h2>Decrease of the protein lifetime by the [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]]</h2><br />
<br />
<p>Rapid degradation signature motif - "[[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]]" was considered to tune the lifetime of the inhibitor in order to reset the cells to the normal responsiveness more rapidly. Fusion of the luciferase and [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]] under the control of CMV (Device [http://partsregistry.org/Part:BBa_J52039 BBa_J52039]) was used to test the effectiveness of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]]. We have used the cycloheximide to stop the protein synthesis and analyse the lifetime of the fusion protein with [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]] sequence. The steady state of the luciferase activity was 5-10 fold lower in the Rluc-[[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]] fusin than without of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]] signature, as evident from the chart below.</p> <br />
<br />
[[Image:graf1.gif|thumb|center|800px|<b>Figure 24: Addition of the PEST degradation tag decreases the steady state of the protein in cells due to its rapid degradation </b>. Cycloheximide was added to cells in order to stop the protein synthesis and monitor the protein degradation kinetics.]]<br />
<p>Constructs of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] domain and [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]] were not effective as inhibitors of signaling because of the interference of the C-terminal extension of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] inhibitory domain, as described above for the fusion constructs with the reporter luciferase or GFP.</p><br />
<br />
<br />
<br />
<div style="float:left; background-color:#cedff2; padding: .2em .6em; border: 1px solid black">'''<br />
<br />
<h1>Conclusions</h1><br />
<br />
*the same principles of BioBricks can be used in the mammalian cell system as in bacteria and eucaryotes<br />
*we have succesfully implemented a feedback loop that decreases the cellular activation with some delay<br />
*response of this feedback loop is transient and the cell responsiveness is restored after the synthesized inhibitor has degraded<br />
*[[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] inhibitor was not effective as a C-terminal fusion with luciferase or GFP, which may be due to the steric hindrance; it is likely that N-terminal fusion of reporter domains would be functional as signaling inhibitors <br />
*our constructed device mimicks the natural mechanism of tolerance only that it is activated faster, which may be a benefit for organism,<br />
*simplified model of the TLR signaling qualitatively captures most of the features of the natural system<br />
</div><br />
<br />
<br />
<br />
<br />
<br><br />
<h2>Suggestions</h2><br />
* for mammalian cell systems an additional set of vectors may be constructed based on the backbone, which includes the selection marker for stable transfection or retroviral integration<br />
<br />
<center><br />
[[Image:line-si4.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006|Home]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signaling Pathway]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Project & Model|Project & Model]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr></table>[[Image:line-si3.jpg]]<br />
<br><br />
</center></div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Results_%26_ConclusionsLjubljana, Slovenia 2006/Results & Conclusions2006-10-31T14:37:43Z<p>MDolinar: </p>
<hr />
<div><center>[[Image:Logo-si1.gif|center|120 px]] <br><br />
<br />
[[Image:line-si4.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006|Home]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signalling Pathway]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Project & Model|Project & Model]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr></table>[[Image:line-si3.jpg]]<br />
<br><br />
</center><br />
<br><br><br />
<br />
<h1>Results</h1><br />
<br />
<h2>Construction of Biobricks</h2><br />
We prepared individual BioBricks that could be used in mammalian cells. The following BioBricks were used:<br />
*promoter (constitutive CMV and [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] inducible promoter), <br />
*terminator (which we included into plasmids to avoid repeated additional steps in construction of each functional Part) <br />
*protein coding sequences, which include: <br />
**two inhibitory (dominant negative) proteins of the signaling cascade ([[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] and dn[[Ljubljana, Slovenia 2006/Terms & References#Terms|TRAF6]]), <br />
**two reporters: ''Renilla'' luciferase (in order to allow simultaneous dual luciferase assay to normalize the luminescence for the efficiency of transfection and cell number) and GFP <br />
**[[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]] to decrease the lifetime of the inhibitor.<br />
<br />
Linkers between the protein coding sequences (e.g. [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]]+luciferase+[[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]]) were prepared by PCR ligation to avoid addition of unwanted aminoacids in the linker region between the protein domains.<br />
<br><br />
<br />
<br />
<br />
<center><table><br />
<caption>''Table 1: List of prepared Parts''</caption><br />
<tr><th>Registration number</th><br />
<th>Part's Name</th><br />
<th>Part/Device</th><br />
<th>Vector</th></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52008 BBa_J52008]</td><td>rluc</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52010 BBa_J52010]</td><td>NF&kappa;B</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52011 BBa_J52011]</td><td>dnMyD88-likn-rLuc</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52012 BBa_J52012]</td><td>rluc-link-PEST191</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52013 BBa_J52013]</td><td>dnMyD88-link-rluc-link-pest191</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52014 BBa_J52014]</td><td>NF&kappa;B+dnMyD88-link-rLuc</td><td>Device</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52016 BBa_J52016]</td><td>eukaryotic terminator</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52017 BBa_J52017]</td><td>eukaryotic terminator vector</td><td></td><td>pSB1AK3</td></tr></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52018 BBa_J52018]</td><td>NF&kappa;B+rLuc</td><td>Device</td><td>pSB1AC3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52019 BBa_J52019]</td><td>dnTRAF6</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52021 BBa_J52021]</td><td>dnTRAF6-link-GFP</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52022 BBa_J52022]</td><td>NF&kappa;B+dnTRAF6-link-GFP</td><td>Device</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52023 BBa_J52023]</td><td>NF&kappa;B+rLuc-link-PEST191</td><td>Device</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52024 BBa_J52024]</td><td>NF&kappa;B+dnMyD88-link-rLuc-link-PEST191</td><td>Device</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52026 BBa_J52026]</td><td>dnMyD88-link-GFP</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52027 BBa_J52027]</td><td>NF&kappa;B+dnMyD88-link-GFP</td><td>Device</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52028 BBa_J52028]</td><td>GFP-PEST191</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52029 BBa_J52029]</td><td>NF&kappa;B+GFP-PEST191</td><td>Device</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52034 BBa_J52034]</td><td>CMV</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52035 BBa_J52035]</td><td>dnMyD88</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52036 BBa_J52036]</td><td>NF&kappa;B+dnMyD88</td><td>Device</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52038 BBa_J52038]</td><td>CMV-rLuc</td><td>Device</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52039 BBa_J52039]</td><td>CMV+rLuc-link-PEST191</td><td>Device</td><td>pSB1A2</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52040 BBa_J52040]</td><td>CMV+GFP-PEST191</td><td>Device</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52642 BBa_J52642]</td><td>GFP</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52648 BBa_J52648]</td><td>CMV+GFP</td><td>Device</td><td>pSB1AK3+TER</td></tr><br />
</table></center><br />
<br><br />
<br />
<h2>Detection of phosphorylation of ERK kinases fy flow cytometry reflects the activation of the TLR signaling </h2><br />
<br />
<br style="clear:both;"/><br />
<br />
[[Image:cito1.gif|cito1.gif|center|thumb|400px|<b> Figure 19: Flow cytometric profile of phosphorylated ERK kinases in HEK293 cells stimulated by LPS for 0, 20 and 80 minutes.</b>]]<br />
<br />
<br style="clear:both;"/><br />
<br />
Flow Cytometry experiments showed that after 80 minute stimulation the increase in the level of phosphorylated ERK kinases was evident(Figure 19). The method was not as sensitive to the rapid response as we hoped. Further optimization of the method would be necessary in order to obtain the quantitative data to accurately model the cell signaling.<br />
<br />
<h2>Detection of transcriptionally competent[[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] in whole cell extracts dependent on the time after stimulation with [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]] determined with promoter ELISA method</h2><br />
<br />
<br style="clear:both;"/><br />
<br />
[[Image:Elisa_results.gif|thumb|center|800px|<b>Figure 20: Activity of peroxidase (conjugated on secondary antibodies) in luminescence units as a function of time after stimulation with [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]].</b> The amount of free [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] (and consequently luminescence) increases with time of stimulation (7 hours) and decreases after extended (23 hours) stimulation.]]<br />
<br />
<br style="clear:both;"/><br />
<br />
Cells were transfected with [[Ljubljana, Slovenia 2006/Terms & References#Terms|TLR4]] plasmid and [[Ljubljana, Slovenia 2006/Terms & References#Terms|MD-2]] plasmid in ordeer to confer the LPS responsiveness. We have shown that cells with TRL4 receptor respond to [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]] stimulation and that ELISA is apropriate and sensitive method for the detection of stimulation. However we did not get satisfying results when stimulating (with [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]] or flagellin) cells transfected neither with [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] nor [[Ljubljana, Slovenia 2006/Terms & References#Terms|TRAF6]]. The response of the ELISA method, as the most direct measurement of the free NF-&kappa;B was not sufficiently reproducible and linear in comparison to the method of luciferase reporter plasmids (see below).<br />
<br />
<br />
<h2>Inducible transcription by the stimulation of Toll-like receptor</h2><br />
<br />
<p>Stimulation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|HEK293]] cells transfected by TLR with their agonist (flagellin in case of TLR5) stimulates the translocation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] into the nucleus and activation of transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]]-responsive genes. We have used the firefly luciferase under the [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] dependent promoter to monitor the promotor activation. We tested the functionality of our part containing [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] dependent promotor in a construct with [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]]-rLuc. We detected the R-luciferase activity only in stimulated cells with significant activity appearing a few hours after the cell stimulation, which proves the functionality of this part (inducible promoter).</p><br />
<br />
<br style="clear:both;"/><br />
<br />
<br />
[[Image:graf_gabi.gif|left|thumb|400px|<b>Figure 21: Transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with rLuc reporter is induced by bacterial flagellin </b> Activity of ''Renilla'' luciferase stimulated by the addition of a pulse of [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]]. [[Ljubljana, Slovenia 2006/Terms & References#Terms|HEK293]] cells were transfected with device comprising [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]]-inducible fusion of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]]-rLuc.]]<br />
<br />
[[Image:rezultati7.gif|left|thumb|400px|<b>Figure 22: Transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with rLuc reporter in our device is inducible by bacterial flagellin.</b> Activity of the Firefly luciferase stimulated by the addition of two pulses of [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]]. ]]<br />
<br />
<br style="clear:both;"/><br />
<p>We also measured NF-kB-responsive F-luciferase activity of this device. We expected a decrease in the activity after the amount of time sufficient for the sysnthesis of inhibitory fusion protein in cells. To our dissapointment no decrease of the cell activation was observed. One of the possible causes for the lack of inhibition of the dnMyD88-rLuc protein fusion could be the sterical hindrance of the activity of dnMyD88. This domain interacts with TIR domain of TLRs in the direct proximity of the cellular membrane, which could prevent interaction with larger protein domains. Based on those results we also conducted our experiments with a device containing dnMyD88 without of any protein appendix.<br />
<br />
<h2>Inhibition of cell signaling by a dnMyD88 feedback device</h2><br />
<br />
An adapter protein [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]], consisting of a TIR and death domain, is at the crossroads of Toll-like receptors. Therefore activation of each of the surface expressed TLR recruits [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] to the cell membrane. We selected to use the receptor TLR5 and flagellin instead of the often used [[Ljubljana, Slovenia 2006/Terms & References#Terms|TLR4]] and its agonist [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]], because the [[Ljubljana, Slovenia 2006/Terms & References#Terms|TLR4]] system is extremely sensitive to the contamination with bacterial [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]], which often copurifies with the plasmid. In this way we have avoided the contamination issue, but our system is designed to work on any signalling receptor that has [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] in its signaling pathway (i.e. TLR1,2,4,5,6,7,8,9,11,IL-1).<br />
Cells were cotransfected with TLR5, reporter plasmid (NF&kappa;B-Fluc) and our feedback device ([[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]]-[[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]]).</p><br />
<br />
<p>We expect that after stimulation with flagellin cytoplasmic [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] is released from its inhibitor. It then migrates into the nucleus and induces the transcription of our construct. Accumulation of the dominant negative protein should compete with wild-type [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] and attenuate the downstream signaling pathway. We can detect the [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] translocation, transcriptional activation and phosphorylation by our detection systems - luminescence, free [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]], phosphorylation detected by flow cytometry and confocal microscopy. In case that our construct is followed by [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]] (degradation) sequence, it is expected that product should degrade rapidly and shorten the inhibition period. If the stimulus is still present, cells should again respond to it and the cycle with inhibition will repeat as well.</p><br />
<br />
<p>We have designed the experiment by two consecutive pulses of TLR stimulation separated by 4-6 hours, which is sufficient time to accumulate the inducible inhibitor. Normal cells should respond to both stimuli, while the cells with our device should respond to the same extent only to the first pulse -- this induces the transcription of the inhibitor, therefore the response to the second stimulus should be decreased. However if the stimuli are separated by a longer timespan, the system should respond normally, as the inhibitor has already degraded and the systemn has reset to the normal state.</p><br />
<br />
<center><br />
[[Image:rezultati1a.gif|center|thumb|400px|<b>Figure 23: [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] feedback loop decreases the cellular activation by repeated stimulation with bacterial [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]]. </b> Normalized luciferase activity (Fluc/Rluc) of cells stimulated by the addition of two pulses of flagellin separated by 6 hours, indicated time after the second pulse is shown. [[Ljubljana, Slovenia 2006/Terms & References#Terms|HEK293]] cells were transfected with device comprising NF&kappa;B-inducible [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] in comparison to wild type.]]<br />
</center><br />
<br style="clear:both;"/><br />
<br />
<p>Our results show that the system with inducible [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] indeed responds weaker to the second stimulus. The minimal delay between two stimuli should correspond to the time required for the synthesis of the inhibitor, which is exactly as planned, since it does not completely abolish the cellular response but decreases it after extended stimulation.</p><br />
<br />
<br style="clear:both;"/><br />
<br />
<h2>Decrease of the protein lifetime by the [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]]</h2><br />
<br />
<p>Rapid degradation signature motif - "[[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]]" was considered to tune the lifetime of the inhibitor in order to reset the cells to the normal responsiveness more rapidly. Fusion of the luciferase and [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]] under the control of CMV (Device [http://partsregistry.org/Part:BBa_J52039 BBa_J52039]) was used to test the effectiveness of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]]. We have used the cycloheximide to stop the protein synthesis and analyse the lifetime of the fusion protein with [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]] sequence. The steady state of the luciferase activity was 5-10 fold lower in the Rluc-[[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]] fusin than without of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]] signature, as evident from the chart below.</p> <br />
<br />
[[Image:graf1.gif|thumb|center|800px|<b>Figure 24: Addition of the PEST degradation tag decreases the steady state of the protein in cells due to its rapid degradation </b>. Cycloheximide was added to cells in order to stop the protein synthesis and monitor the protein degradation kinetics.]]<br />
<p>Constructs of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] domain and [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]] were not effective as inhibitors of signaling because of the interference of the C-terminal extension of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] inhibitory domain, as described above for the fusion constructs with the reporter luciferase or GFP.</p><br />
<br />
<br />
<br />
<div style="float:left; background-color:#cedff2; padding: .2em .6em; border: 1px solid black">'''<br />
<br />
<h1>Conclusions</h1><br />
<br />
*the same principles of BioBricks can be used in the mammalian cell system as in bacteria and eucaryotes<br />
*we have succesfully implemented a feedback loop that decreases the cellular activation with some delay<br />
*response of this feedback loop is transient and the cell responsiveness is restored after the synthesized inhibitor has degraded<br />
*[[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] inhibitor was not effective as a C-terminal fusion with luciferase or GFP, which may be due to the steric hindrance; it is likely that N-terminal fusion of reporter domains would be functional as signaling inhibitors <br />
*our constructed device mimicks the natural mechanism of tolerance only that it is activated faster, which may be a benefit for organism,<br />
*simplified model of the TLR signaling qualitatively captures most of the features of the natural system<br />
</div><br />
<br />
<br />
<br />
<br />
<br><br />
<h2>Suggestions</h2><br />
* for mammalian cell systems an additional set of vectors may be constructed based on the backbone, which includes the selection marker for stable transfection or retroviral integration<br />
<br />
<center><br />
[[Image:line-si4.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006|Home]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signaling Pathway]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Project & Model|Project & Model]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr></table>[[Image:line-si3.jpg]]<br />
<br><br />
</center></div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Results_%26_ConclusionsLjubljana, Slovenia 2006/Results & Conclusions2006-10-31T14:36:56Z<p>MDolinar: </p>
<hr />
<div><center>[[Image:Logo-si1.gif|center|120 px]] <br><br />
<br />
[[Image:line-si4.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006|Home]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signalling Pathway]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Project & Model|Project & Model]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr></table>[[Image:line-si3.jpg]]<br />
<br><br />
</center><br />
<br><br><br />
<br />
<h1>Results</h1><br />
<br />
<h2>Construction of Biobricks</h2><br />
We prepared individual BioBricks that could be used in mammalian cells. The following BioBricks were used:<br />
*promoter (constitutive CMV and [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] inducible promoter), <br />
*terminator (which we included into plasmids to avoid repeated additional steps in construction of each functional Part) <br />
*protein coding sequences, which include: <br />
**two inhibitory (dominant negative) proteins of the signaling cascade ([[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] and dn[[Ljubljana, Slovenia 2006/Terms & References#Terms|TRAF6]]), <br />
**two reporters: ''Renilla'' luciferase (in order to allow simultaneous dual luciferase assay to normalize the luminescence for the efficiency of transfection and cell number) and GFP <br />
**[[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]] to decrease the lifetime of the inhibitor.<br />
<br />
Linkers between the protein coding sequences (e.g. [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]]+luciferase+[[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]]) were prepared by PCR ligation to avoid addition of unwanted aminoacids in the linker region between the protein domains.<br />
<br><br />
<br />
<br />
<br />
<center><table><br />
<caption>''Table 1: List of prepared Parts''</caption><br />
<tr><th>Registration number</th><br />
<th>Part's Name</th><br />
<th>Part/Device</th><br />
<th>Vector</th></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52008 BBa_J52008]</td><td>rluc</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52010 BBa_J52010]</td><td>NF&kappa;B</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52011 BBa_J52011]</td><td>dnMyD88-likn-rLuc</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52012 BBa_J52012]</td><td>rluc-link-PEST191</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52013 BBa_J52013]</td><td>dnMyD88-link-rluc-link-pest191</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52014 BBa_J52014]</td><td>NF&kappa;B+dnMyD88-link-rLuc</td><td>Device</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52016 BBa_J52016]</td><td>eukaryotic terminator</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52017 BBa_J52017]</td><td>eukaryotic terminator vector</td><td></td><td>pSB1AK3</td></tr></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52018 BBa_J52018]</td><td>NF&kappa;B+rLuc</td><td>Device</td><td>pSB1AC3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52019 BBa_J52019]</td><td>dnTRAF6</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52021 BBa_J52021]</td><td>dnTRAF6-link-GFP</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52022 BBa_J52022]</td><td>NF&kappa;B+dnTRAF6-link-GFP</td><td>Device</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52023 BBa_J52023]</td><td>NF&kappa;B+rLuc-link-PEST191</td><td>Device</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52024 BBa_J52024]</td><td>NF&kappa;B+dnMyD88-link-rLuc-link-PEST191</td><td>Device</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52026 BBa_J52026]</td><td>dnMyD88-link-GFP</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52027 BBa_J52027]</td><td>NF&kappa;B+dnMyD88-link-GFP</td><td>Device</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52028 BBa_J52028]</td><td>GFP-PEST191</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52029 BBa_J52029]</td><td>NF&kappa;B+GFP-PEST191</td><td>Device</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52034 BBa_J52034]</td><td>CMV</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52035 BBa_J52035]</td><td>dnMyD88</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52036 BBa_J52036]</td><td>NF&kappa;B+dnMyD88</td><td>Device</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52038 BBa_J52038]</td><td>CMV-rLuc</td><td>Device</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52039 BBa_J52039]</td><td>CMV+rLuc-link-PEST191</td><td>Device</td><td>pSB1A2</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52040 BBa_J52040]</td><td>CMV+GFP-PEST191</td><td>Device</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52642 BBa_J52642]</td><td>GFP</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52648 BBa_J52648]</td><td>CMV+GFP</td><td>Device</td><td>pSB1AK3+TER</td></tr><br />
</table></center><br />
<br><br />
<br />
<h2>Detection of phosphorylation of ERK kinases fy flow cytometry reflects the activation of the TLR signaling </h2><br />
<br />
<br style="clear:both;"/><br />
<br />
[[Image:cito1.gif|cito1.gif|center|thumb|400px|<b> Figure 19: Flow cytometric profile of phosphorylated ERK kinases in HEK293 cells stimulated by LPS for 0, 20 and 80 minutes.</b>]]<br />
<br />
<br style="clear:both;"/><br />
<br />
Flow Cytometry experiments showed that after 80 minute stimulation the increase in the level of phosphorylated ERK kinases was evident(Figure 19). The method was not as sensitive to the rapid response as we hoped. Further optimization of the method would be necessary in order to obtain the quantitative data to accurately model the cell signaling.<br />
<br />
<h2>Detection of transcriptionally competent[[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] in whole cell extracts dependent on the time after stimulation with [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]] determined with promoter ELISA method</h2><br />
<br />
<br style="clear:both;"/><br />
<br />
[[Image:Elisa_results.gif|thumb|center|800px|<b>Figure 20: Activity of peroxidase (conjugated on secondary antibodies) in luminescence units as a function of time after stimulation with [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]].</b> The amount of free [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] (and consequently luminescence) increases with time of stimulation (7 hours) and decreases after extended (23 hours) stimulation.]]<br />
<br />
<br style="clear:both;"/><br />
<br />
Cells were transfected with [[Ljubljana, Slovenia 2006/Terms & References#Terms|TLR4]] plasmid and [[Ljubljana, Slovenia 2006/Terms & References#Terms|MD-2]] plasmid in ordeer to confer the LPS responsiveness. We have shown that cells with TRL4 receptor respond to [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]] stimulation and that ELISA is apropriate and sensitive method for the detection of stimulation. However we did not get satisfying results when stimulating (with [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]] or flagellin) cells transfected neither with [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] nor [[Ljubljana, Slovenia 2006/Terms & References#Terms|TRAF6]]. The response of the ELISA method, as the most direct measurement of the free NF-&kappa;B was not sufficiently reproducible and linear in comparison to the method of luciferase reporter plasmids (see below).<br />
<br />
<br />
<h2>Inducible transcription by the stimulation of Toll-like receptor</h2><br />
<br />
<p>Stimulation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|HEK293]] cells transfected by TLR with their agonist (flagellin in case of TLR5) stimulates the translocation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] into the nucleus and activation of transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]]-responsive genes. We have used the firefly luciferase under the [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] dependent promoter to monitor the promotor activation. We tested the functionality of our part containing [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] dependent promotor in a construct with [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]]-rLuc. We detected the R-luciferase activity only in stimulated cells with significant activity appearing a few hours after the cell stimulation, which proves the functionality of this part (inducible promoter).</p><br />
<br />
<br style="clear:both;"/><br />
<br />
<br />
[[Image:graf_gabi.gif|left|thumb|400px|<b>Figure 21: Transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with rLuc reporter is induced by bacterial flagellin </b> Activity of ''Renilla'' luciferase stimulated by the addition of a pulse of [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]]. [[Ljubljana, Slovenia 2006/Terms & References#Terms|HEK293]] cells were transfected with device comprising [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]]-inducible fusion of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]]-rLuc.]]<br />
<br />
[[Image:rezultati7.gif|left|thumb|400px|<b>Figure 22: Transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with rLuc reporter in our device is inducible by bacterial flagellin.</b> Activity of the Firefly luciferase stimulated by the addition of two pulses of [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]]. ]]<br />
<br />
<br style="clear:both;"/><br />
<p>We also measured NF-kB-responsive F-luciferase activity of this device. We expected a decrease in the activity after the amount of time sufficient for the sysnthesis of inhibitory fusion protein in cells. To our dissapointment no decrease of the cell activation was observed. One of the possible causes for the lack of inhibition of the dnMyD88-rLuc protein fusion could be the sterical hindrance of the activity of dnMyD88. This domain interacts with TIR domain of TLRs in the direct proximity of the cellular membrane, which could prevent interaction with larger protein domains. Based on those results we also conducted our experiments with a device containing dnMyD88 without of any protein appendix.<br />
<br />
<h2>Inhibition of cell signaling by a dnMyD88 feedback device</h2><br />
<br />
An adapter protein [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]], consisting of a TIR and death domain, is at the crossroads of Toll-like receptors. Therefore activation of each of the surface expressed TLR recruits [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] to the cell membrane. We selected to use the receptor TLR5 and flagellin instead of the often used [[Ljubljana, Slovenia 2006/Terms & References#Terms|TLR4]] and its agonist [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]], because the [[Ljubljana, Slovenia 2006/Terms & References#Terms|TLR4]] system is extremely sensitive to the contamination with bacterial [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]], which often copurifies with the plasmid. In this way we have avoided the contamination issue, but our system is designed to work on any signalling receptor that has [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] in its signaling pathway (i.e. TLR1,2,4,5,6,7,8,9,11,IL-1).<br />
Cells were cotransfected with TLR5, reporter plasmid (NF&kappa;B-Fluc) and our feedback device ([[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]]-[[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]]).</p><br />
<br />
<p>We expect that after stimulation with flagellin cytoplasmic [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] is released from its inhibitor. It then migrates into the nucleus and induces the transcription of our construct. Accumulation of the dominant negative protein should compete with wild-type [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] and attenuate the downstream signaling pathway. We can detect the [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] translocation, transcriptional activation and phosphorylation by our detection systems - luminescence, free [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]], phosphorylation detected by flow cytometry and confocal microscopy. In case that our construct is followed by [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]] (degradation) sequence, it is expected that product should degrade rapidly and shorten the inhibition period. If the stimulus is still present, cells should again respond to it and the cycle with inhibition will repeat as well.</p><br />
<br />
<p>We have designed the experiment by two consecutive pulses of TLR stimulation separated by 4-6 hours, which is sufficient time to accumulate the inducible inhibitor. Normal cells should respond to both stimuli, while the cells with our device should respond to the same extent only to the first pulse -- this induces the transcription of the inhibitor, therefore the response to the second stimulus should be decreased. However if the stimuli are separated by a longer timespan, the system should respond normally, as the inhibitor has already degraded and the systemn has reset to the normal state.</p><br />
<br />
<center><br />
[[Image:rezultati1a.gif|center|thumb|400px|<b>Figure 23: [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] feedback loop decreases the cellular activation by repeated stimulation with bacterial [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]]. </b> Normalized luciferase activity (Fluc/Rluc) of cells stimulated by the addition of two pulses of flagellin separated by 6 hours, indicated time after the second pulse is shown. [[Ljubljana, Slovenia 2006/Terms & References#Terms|HEK293]] cells were transfected with device comprising NF&kappa;B-inducible [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] in comparison to wild type.]]<br />
</center><br />
<br style="clear:both;"/><br />
<br />
<p>Our results show that the system with inducible [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] indeed responds weaker to the second stimulus. The minimal delay between two stimuli should correspond to the time required for the synthesis of the inhibitor, which is exactly as planned, since it does not completely abolish the cellular response but decreases it after extended stimulation.</p><br />
<br />
<br style="clear:both;"/><br />
<br />
<h2>Decrease of the protein lifetime by the [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]]</h2><br />
<br />
<p>Rapid degradation signature motif - "[[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]]" was considered to tune the lifetime of the inhibitor in order to reset the cells to the normal responsiveness more rapidly. Fusion of the luciferase and [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]] under the control of CMV (Device [http://partsregistry.org/Part:BBa_J52039 BBa_J52039]) was used to test the effectiveness of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]]. We have used the cycloheximide to stop the protein synthesis and analyse the lifetime of the fusion protein with [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]] sequence. The steady state of the luciferase activity was 5-10 fold lower in the Rluc-[[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]] fusin than without of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]] signature, as evident from the chart below.</p> <br />
<br />
[[Image:graf1.gif|thumb|center|800px|<b>Figure 24: Addition of the PEST degradation tag decreases the steady state of the protein in cells due to its rapid degradation </b>. Cycloheximide was added to cells in order to stop the protein synthesis and monitor the protein degradation kinetics.]]<br />
<p>Constructs of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] domain and [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]] were not effective as inhibitors of signaling because of the interference of the C-terminal extension of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] inhibitory domain, as described above for the fusion constructs with the reporter luciferase or GFP.</p><br />
<br />
<br />
<br />
<div style="float:left; background-color:#cedff2; padding: .2em .6em; border: 1px solid black">'''<br />
<br />
<h1>Conclusions</h1><br />
<br />
*the same principles of BioBricks can be used in the mammalian cell system as in bacteria and eucaryotes<br />
*we have succesfully implemented a feedback loop that decreases the cellular activation with some delay<br />
*response of this feedback loop is transient and the cell responsiveness is restored after the synthesized inhibitor has degraded<br />
*[[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] inhibitor was not effective as a C-terminal fusion with luciferase or GFP, which may be due to the steric hindrance; it is likely that N-terminal fusion of reporter domains would be functional as signaling inhibitors <br />
*our constructed device mimicks the natural mechanism of tolerance only that it is activated faster, which may be a benefit for organism,<br />
*simplified model of the TLR signaling qualitatively captures most of the features of the natural system<br />
</div><br />
<br />
<br />
<br />
<br />
<h2>Suggestions</h2><br />
* for mammalian cell systems an additional set of vectors may be constructed based on the backbone, which includes the selection marker for stable transfection or retroviral integration<br />
<br />
<center><br />
[[Image:line-si4.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006|Home]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signalling Pathway]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Project & Model|Project & Model]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr></table>[[Image:line-si3.jpg]]<br />
<br><br />
</center></div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Results_%26_ConclusionsLjubljana, Slovenia 2006/Results & Conclusions2006-10-31T14:35:57Z<p>MDolinar: </p>
<hr />
<div><center>[[Image:Logo-si1.gif|center|120 px]] <br><br />
<br />
[[Image:line-si4.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006|Home]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signalling Pathway]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Project & Model|Project & Model]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr></table>[[Image:line-si3.jpg]]<br />
<br><br />
</center><br />
<br><br><br />
<br />
<h1>Results</h1><br />
<br />
<h2>Construction of Biobricks</h2><br />
We prepared individual BioBricks that could be used in mammalian cells. The following BioBricks were used:<br />
*promoter (constitutive CMV and [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] inducible promoter), <br />
*terminator (which we included into plasmids to avoid repeated additional steps in construction of each functional Part) <br />
*protein coding sequences, which include: <br />
**two inhibitory (dominant negative) proteins of the signaling cascade ([[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] and dn[[Ljubljana, Slovenia 2006/Terms & References#Terms|TRAF6]]), <br />
**two reporters: ''Renilla'' luciferase (in order to allow simultaneous dual luciferase assay to normalize the luminescence for the efficiency of transfection and cell number) and GFP <br />
**[[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]] to decrease the lifetime of the inhibitor.<br />
<br />
Linkers between the protein coding sequences (e.g. [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]]+luciferase+[[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]]) were prepared by PCR ligation to avoid addition of unwanted aminoacids in the linker region between the protein domains.<br />
<br><br />
<br />
<br />
<br />
<center><table><br />
<caption>''Table 1: List of prepared Parts''</caption><br />
<tr><th>Registration number</th><br />
<th>Part's Name</th><br />
<th>Part/Device</th><br />
<th>Vector</th></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52008 BBa_J52008]</td><td>rluc</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52010 BBa_J52010]</td><td>NF&kappa;B</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52011 BBa_J52011]</td><td>dnMyD88-likn-rLuc</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52012 BBa_J52012]</td><td>rluc-link-PEST191</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52013 BBa_J52013]</td><td>dnMyD88-link-rluc-link-pest191</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52014 BBa_J52014]</td><td>NF&kappa;B+dnMyD88-link-rLuc</td><td>Device</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52016 BBa_J52016]</td><td>eukaryotic terminator</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52017 BBa_J52017]</td><td>eukaryotic terminator vector</td><td></td><td>pSB1AK3</td></tr></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52018 BBa_J52018]</td><td>NF&kappa;B+rLuc</td><td>Device</td><td>pSB1AC3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52019 BBa_J52019]</td><td>dnTRAF6</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52021 BBa_J52021]</td><td>dnTRAF6-link-GFP</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52022 BBa_J52022]</td><td>NF&kappa;B+dnTRAF6-link-GFP</td><td>Device</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52023 BBa_J52023]</td><td>NF&kappa;B+rLuc-link-PEST191</td><td>Device</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52024 BBa_J52024]</td><td>NF&kappa;B+dnMyD88-link-rLuc-link-PEST191</td><td>Device</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52026 BBa_J52026]</td><td>dnMyD88-link-GFP</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52027 BBa_J52027]</td><td>NF&kappa;B+dnMyD88-link-GFP</td><td>Device</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52028 BBa_J52028]</td><td>GFP-PEST191</td><td>Part</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52029 BBa_J52029]</td><td>NF&kappa;B+GFP-PEST191</td><td>Device</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52034 BBa_J52034]</td><td>CMV</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52035 BBa_J52035]</td><td>dnMyD88</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52036 BBa_J52036]</td><td>NF&kappa;B+dnMyD88</td><td>Device</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52038 BBa_J52038]</td><td>CMV-rLuc</td><td>Device</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52039 BBa_J52039]</td><td>CMV+rLuc-link-PEST191</td><td>Device</td><td>pSB1A2</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52040 BBa_J52040]</td><td>CMV+GFP-PEST191</td><td>Device</td><td>pSB1AK3</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52642 BBa_J52642]</td><td>GFP</td><td>Part</td><td>pSB1AK3+TER</td></tr><br />
<br />
<tr><td>[http://partsregistry.org/Part:BBa_J52648 BBa_J52648]</td><td>CMV+GFP</td><td>Device</td><td>pSB1AK3+TER</td></tr><br />
</table></center><br />
<br><br />
<br />
<h2>Detection of phosphorylation of ERK kinases fy flow cytometry reflects the activation of the TLR signaling </h2><br />
<br />
<br style="clear:both;"/><br />
<br />
[[Image:cito1.gif|cito1.gif|center|thumb|400px|<b> Figure 19: Flow cytometric profile of phosphorylated ERK kinases in HEK293 cells stimulated by LPS for 0, 20 and 80 minutes.</b>]]<br />
<br />
<br style="clear:both;"/><br />
<br />
Flow Cytometry experiments showed that after 80 minute stimulation the increase in the level of phosphorylated ERK kinases was evident(Figure 19). The method was not as sensitive to the rapid response as we hoped. Further optimization of the method would be necessary in order to obtain the quantitative data to accurately model the cell signaling.<br />
<br />
<h2>Detection of transcriptionally competent[[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] in whole cell extracts dependent on the time after stimulation with [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]] determined with promoter ELISA method</h2><br />
<br />
<br style="clear:both;"/><br />
<br />
[[Image:Elisa_results.gif|thumb|center|800px|<b>Figure 20: Activity of peroxidase (conjugated on secondary antibodies) in luminescence units as a function of time after stimulation with [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]].</b> The amount of free [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] (and consequently luminescence) increases with time of stimulation (7 hours) and decreases after extended (23 hours) stimulation.]]<br />
<br />
<br style="clear:both;"/><br />
<br />
Cells were transfected with [[Ljubljana, Slovenia 2006/Terms & References#Terms|TLR4]] plasmid and [[Ljubljana, Slovenia 2006/Terms & References#Terms|MD-2]] plasmid in ordeer to confer the LPS responsiveness. We have shown that cells with TRL4 receptor respond to [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]] stimulation and that ELISA is apropriate and sensitive method for the detection of stimulation. However we did not get satisfying results when stimulating (with [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]] or flagellin) cells transfected neither with [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] nor [[Ljubljana, Slovenia 2006/Terms & References#Terms|TRAF6]]. The response of the ELISA method, as the most direct measurement of the free NF-&kappa;B was not sufficiently reproducible and linear in comparison to the method of luciferase reporter plasmids (see below).<br />
<br />
<br />
<h2>Inducible transcription by the stimulation of Toll-like receptor</h2><br />
<br />
<p>Stimulation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|HEK293]] cells transfected by TLR with their agonist (flagellin in case of TLR5) stimulates the translocation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] into the nucleus and activation of transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]]-responsive genes. We have used the firefly luciferase under the [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] dependent promoter to monitor the promotor activation. We tested the functionality of our part containing [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] dependent promotor in a construct with [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]]-rLuc. We detected the R-luciferase activity only in stimulated cells with significant activity appearing a few hours after the cell stimulation, which proves the functionality of this part (inducible promoter).</p><br />
<br />
<br style="clear:both;"/><br />
<br />
<br />
[[Image:graf_gabi.gif|left|thumb|400px|<b>Figure 21: Transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with rLuc reporter is induced by bacterial flagellin </b> Activity of ''Renilla'' luciferase stimulated by the addition of a pulse of [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]]. [[Ljubljana, Slovenia 2006/Terms & References#Terms|HEK293]] cells were transfected with device comprising [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]]-inducible fusion of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]]-rLuc.]]<br />
<br />
[[Image:rezultati7.gif|left|thumb|400px|<b>Figure 22: Transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with rLuc reporter in our device is inducible by bacterial flagellin.</b> Activity of the Firefly luciferase stimulated by the addition of two pulses of [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]]. ]]<br />
<br />
<br style="clear:both;"/><br />
<p>We also measured NF-kB-responsive F-luciferase activity of this device. We expected a decrease in the activity after the amount of time sufficient for the sysnthesis of inhibitory fusion protein in cells. To our dissapointment no decrease of the cell activation was observed. One of the possible causes for the lack of inhibition of the dnMyD88-rLuc protein fusion could be the sterical hindrance of the activity of dnMyD88. This domain interacts with TIR domain of TLRs in the direct proximity of the cellular membrane, which could prevent interaction with larger protein domains. Based on those results we also conducted our experiments with a device containing dnMyD88 without of any protein appendix.<br />
<br />
<h2>Inhibition of cell signaling by a dnMyD88 feedback device</h2><br />
<br />
An adapter protein [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]], consisting of a TIR and death domain, is at the crossroads of Toll-like receptors. Therefore activation of each of the surface expressed TLR recruits [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] to the cell membrane. We selected to use the receptor TLR5 and flagellin instead of the often used [[Ljubljana, Slovenia 2006/Terms & References#Terms|TLR4]] and its agonist [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]], because the [[Ljubljana, Slovenia 2006/Terms & References#Terms|TLR4]] system is extremely sensitive to the contamination with bacterial [[Ljubljana, Slovenia 2006/Terms & References#Terms|LPS]], which often copurifies with the plasmid. In this way we have avoided the contamination issue, but our system is designed to work on any signalling receptor that has [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] in its signaling pathway (i.e. TLR1,2,4,5,6,7,8,9,11,IL-1).<br />
Cells were cotransfected with TLR5, reporter plasmid (NF&kappa;B-Fluc) and our feedback device ([[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]]-[[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]]).</p><br />
<br />
<p>We expect that after stimulation with flagellin cytoplasmic [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] is released from its inhibitor. It then migrates into the nucleus and induces the transcription of our construct. Accumulation of the dominant negative protein should compete with wild-type [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] and attenuate the downstream signalling pathway. We can detect the [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] translocation, transcriptional activation and phosphorylation by our detection systems - luminescence, free [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]], phosphorylation detected by flow cytometry and confocal microscopy. In case that our construct is followed by [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]] (degradation) sequence, it is expected that product should degrade rapidly and shorten the inhibition period. If the stimulus is still present, cells should again respond to it and the cycle with inhibition will repeat as well.</p><br />
<br />
<p>We have designed the experiment by two consecutive pulses of TLR stimulation separated by 4-6 hours, which is sufficient time to accumulate the inducible inhibitor. Normal cells should respond to both stimuli, while the cells with our device should respond to the same extent only to the first pulse -- this induces the transcription of the inhibitor, therefore the response to the second stimulus should be decreased. However if the stimuli are separated by a longer timespan, the system should respond normally, as the inhibitor has already degraded and the systemn has reset to the normal state.</p><br />
<br />
<center><br />
[[Image:rezultati1a.gif|center|thumb|400px|<b>Figure 23: [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] feedback loop decreases the cellular activation by repeated stimulation with bacterial [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]]. </b> Normalized luciferase activity (Fluc/Rluc) of cells stimulated by the addition of two pulses of flagellin separated by 6 hours, indicated time after the second pulse is shown. [[Ljubljana, Slovenia 2006/Terms & References#Terms|HEK293]] cells were transfected with device comprising NF&kappa;B-inducible [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] in comparison to wild type.]]<br />
</center><br />
<br style="clear:both;"/><br />
<br />
<p>Our results show that the system with inducible [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] indeed responds weaker to the second stimulus. The minimal delay between two stimuli should correspond to the time required for the synthesis of the inhibitor, which is exactly as planned, since it does not completely abolish the cellular response but decreases it after extended stimulation.</p><br />
<br />
<br style="clear:both;"/><br />
<br />
<h2>Decrease of the protein lifetime by the [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]]</h2><br />
<br />
<p>Rapid degradation signature motif - "[[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]]" was considered to tune the lifetime of the inhibitor in order to reset the cells to the normal responsiveness more rapidly. Fusion of the luciferase and [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]] under the control of CMV (Device [http://partsregistry.org/Part:BBa_J52039 BBa_J52039]) was used to test the effectiveness of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]]. We have used the cycloheximide to stop the protein synthesis and analyse the lifetime of the fusion protein with [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]] sequence. The steady state of the luciferase activity was 5-10 fold lower in the Rluc-[[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]] fusin than without of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST]] signature, as evident from the chart below.</p> <br />
<br />
[[Image:graf1.gif|thumb|center|800px|<b>Figure 24: Addition of the PEST degradation tag decreases the steady state of the protein in cells due to its rapid degradation </b>. Cycloheximide was added to cells in order to stop the protein synthesis and monitor the protein degradation kinetics.]]<br />
<p>Constructs of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] domain and [[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]] were not effective as inhibitors of signaling because of the interference of the C-terminal extension of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] inhibitory domain, as described above for the fusion constructs with the reporter luciferase or GFP.</p><br />
<br />
<br />
<br />
<div style="float:left; background-color:#cedff2; padding: .2em .6em; border: 1px solid black">'''<br />
<br />
<h1>Conclusions</h1><br />
<br />
*the same principles of BioBricks can be used in the mammalian cell system as in bacteria and eucaryotes<br />
*we have succesfully implemented a feedback loop that decreases the cellular activation with some delay<br />
*response of this feedback loop is transient and the cell responsiveness is restored after the synthesized inhibitor has degraded<br />
*[[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] inhibitor was not effective as a C-terminal fusion with luciferase or GFP, which may be due to the steric hindrance; it is likely that N-terminal fusion of reporter domains would be functional as signaling inhibitors <br />
*our constructed device mimicks the natural mechanism of tolerance only that it is activated faster, which may be a benefit for organism,<br />
*simplified model of the TLR signaling qualitatively captures most of the features of the natural system<br />
</div><br />
<br />
<br />
<br />
<br />
<h2>Suggestions</h2><br />
* for mammalian cell systems an additional set of vectors may be constructed based on the backbone, which includes the selection marker for stable transfection or retroviral integration<br />
<br />
<center><br />
[[Image:line-si4.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006|Home]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signalling Pathway]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Project & Model|Project & Model]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr></table>[[Image:line-si3.jpg]]<br />
<br><br />
</center></div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006Ljubljana, Slovenia 20062006-10-31T14:33:26Z<p>MDolinar: </p>
<hr />
<div><center><br />
[[Image:Logo-si1.gif|120 px]] [[Image:fotka1b.jpg|475 px]] [[Image:Logo-si1.gif|120 px]]<br />
<br style="clear:both;"/> </center><br />
<br />
<br><br><br />
<center><font style="font size="9" face="Arial"><b>Engineered Human Cells: <br><br><br> SAY STOP TO SEPSIS<br />
<br><br></b></font><br />
<br />
<br><br />
[[Image:line-si4.jpg]]<br />
<table><br />
<tr><th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signaling Pathway]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Project & Model|Project & Model]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Results & Conclusions|Results & Conclusions]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr><br />
</table><br />
<br />
[[Image:line-si3.jpg]]<br />
</center><br />
<br />
{|<br />
|-valign="center" align="justify"<br />
|border="1" solid #affaaa" cellspacing="1" cellpadding="1" style="border:1px solid black; background:#cedff2" | <font style="font size="3" font face="Times New Roman""><p><b>Mammalian systems can be a subject of cellular engineering similarly to bacterial cells. We decided to tinker with the existing cell signaling network of the response to the bacterial infection. Binding of bacterial components ([[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]s – Pathogen associated molecular patterns) to a family of Toll-like receptors activates the cells of the immune system but the exaggerated response may lead to systemic inflammation and sepsis which is often fatal. We designed a feedback loop, which inhibits the signalling cascade at the »weak spot« - [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]], a consensus adaptor protein of the Toll-like receptors. A mathematical model of cell activation with engineered feedback loop was constructed, which predicts the decrease of the cellular activation after the repeated stimulation. Twenty-six new BrioBricks were constructed specially for the mammalian system. We have experimentally confirmed the function of the feedback device by detecting the inhibition of cellular activation after the repeated stimulation. Cell activation decreased without completely deleting the responsiveness to the bacterial infection, thus our engineered cell system represents a type of artificial immunotolerance.</b></p><br />
| [[Image:sepsa.png|450px|right|]]<br />
|}<br />
<br><br />
<br />
-----<br />
<br />
'''We would like to express our thanks to the Sponsors'''<br />
<br />
{| border="0" cellspacing="0" cellpadding="5" align="center"<br />
| [http://www.ki.si/ http://parts2.mit.edu/wiki/images/9/90/Logo_ki2.jpg]<br />
| [http://www.fkkt.uni-lj.si/en/ http://www.fkkt.uni-lj.si/img/menuleft_logo_unilj.gif]<br />
| [http://www.lek.si/ http://parts2.mit.edu/wiki/images/0/09/Lek2.jpg]<br />
| [[Image:EU-flag.gif |thumb|120px|EU Synbiocomm (thank you Sven)]]<br />
|}<br />
<br />
<br />
'''and Donators'''<br />
<br>[http://www.ad-futura.si Ad Futura], [http://www.krka.si/si/ Krka pharmaceutical company], [http://www.mediline.si/ Mediline], Farmadent</div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006Ljubljana, Slovenia 20062006-10-31T14:32:52Z<p>MDolinar: </p>
<hr />
<div><center><br />
[[Image:Logo-si1.gif|120 px]] [[Image:fotka1b.jpg|475 px]] [[Image:Logo-si1.gif|120 px]]<br />
<br style="clear:both;"/> </center><br />
<br />
<br><br><br />
<center><font style="font size="9" face="Arial"><b>Engineered Human Cells: <br><br><br> SAY STOP TO SEPSIS<br />
<br><br></b></font><br />
<br />
<br><br />
[[Image:line-si4.jpg]]<br />
<table><br />
<tr><th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signaling Pathway]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Project & Model|Project & Model]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Results & Conclusions|Results & Conclusions]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr><br />
</table><br />
<br />
[[Image:line-si3.jpg]]<br />
</center><br />
<br />
{|<br />
|-valign="center" align="justify"<br />
|border="1" solid #affaaa" cellspacing="1" cellpadding="1" style="border:1px solid black; background:#cedff2" | <font style="font size="3" font face="Times New Roman""><p><b>Mammalian systems can be a subject of cellular engineering similarly to bacterial cells. We decided to tinker with the existing cell signalling network of the response to the bacterial infection. Binding of bacterial components ([[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]s – Pathogen associated molecular patterns) to a family of Toll-like receptors activates the cells of the immune system but the exaggerated response may lead to systemic inflammation and sepsis which is often fatal. We designed a feedback loop, which inhibits the signalling cascade at the »weak spot« - [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]], a consensus adaptor protein of the Toll-like receptors. A mathematical model of cell activation with engineered feedback loop was constructed, which predicts the decrease of the cellular activation after the repeated stimulation. Twenty-six new BrioBricks were constructed specially for the mammalian system. We have experimentally confirmed the function of the feedback device by detecting the inhibition of cellular activation after the repeated stimulation. Cell activation decreased without completely deleting the responsiveness to the bacterial infection, thus our engineered cell system represents a type of artificial immunotolerance.</b></p><br />
| [[Image:sepsa.png|450px|right|]]<br />
|}<br />
<br><br />
<br />
-----<br />
<br />
'''We would like to express our thanks to the Sponsors'''<br />
<br />
{| border="0" cellspacing="0" cellpadding="5" align="center"<br />
| [http://www.ki.si/ http://parts2.mit.edu/wiki/images/9/90/Logo_ki2.jpg]<br />
| [http://www.fkkt.uni-lj.si/en/ http://www.fkkt.uni-lj.si/img/menuleft_logo_unilj.gif]<br />
| [http://www.lek.si/ http://parts2.mit.edu/wiki/images/0/09/Lek2.jpg]<br />
| [[Image:EU-flag.gif |thumb|120px|EU Synbiocomm (thank you Sven)]]<br />
|}<br />
<br />
<br />
'''and Donators'''<br />
<br>[http://www.ad-futura.si Ad Futura], [http://www.krka.si/si/ Krka pharmaceutical company], [http://www.mediline.si/ Mediline], Farmadent</div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Project_%26_ModelLjubljana, Slovenia 2006/Project & Model2006-10-31T14:32:08Z<p>MDolinar: </p>
<hr />
<div><center>[[Image:Logo-si1.gif|center|120 px]] <br><br />
<br />
[[Image:line-si4.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006|Home]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signaling Pathway]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Results & Conclusions|Results & Conclusions]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr></table><br />
[[Image:line-si3.jpg]]<br />
<br><br />
</center><br />
<br />
<br><br><br />
<br />
<h1>Project ideas</h1><br />
Reasearch in the Laboratory of Biotechnology at the National institute of Chemisty, where most of our instructors come from and where most of the experimental work was conducted, is in the area of molecular immunology, specifically interactions in the Toll-like receptor signaling to microbial infections. This determined the selection of the eukaryotic system as our arena of synthetic biology "sandbox". besides, several contributions at the Synthetic biology meetings demonstrated that mammalian cell engineering <br />
<br />
In the beginning we have discussed following project ideas:<br />
<ol><br />
<li>Inserton of a logical device into the signaling pathway to limit the cellular activation by bacterial infection<br />
- use of dominant negative versions of proteins involved in signaling pathway, which will arrest the signaling pathway thus preventing [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] (transcription factor mentioned above)to translocate to the nucleus<br />
- addition of degradation tags to this dominant negative proteins ([[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]]), which would cause inhibition (negative feedback loop) to be temporal. </li><br />
<br />
<li>Engineering of the response to pathogens/their [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]s, which are otherwise not recognized by cells<br />
- for example a response to beta glucans of fungi.</li><br />
<br />
<li>Engineering of novel interconnections between signaling pathways. </li> </ol><br />
<br><br />
<br />
<h1>Selected project</h1><br />
<br />
[[Image:sepsa.png|250px|thumb|<b>Figure 4: Unlimited cell stimulation due to the infection can lead to sepsis, which is in many cases fatal</b>. Introduction of the negative feedback could decrease the immune response.]]<br />
<p>The basic idea of our project was to introduce a feedback loop in TLR signaling pathway, which would decrease the overwhelming response to the persistent or repeated stimulus with [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]. However, completly shutting down the response to bacterial stimulation is not recommendable from the view of a host battling the infection. Ideally the feedback loop should decrease the response when it is too high but recover the responsiveness of the system after some time.</p><br />
<br />
<p>Inhibition of the overwhelming response could be achieved if [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]] is simultaneously able to activate the immune response and the expression of a [[Ljubljana, Slovenia 2006/Terms & References#Terms|dominant-negative]] adapter protein, that would inactivate [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]`s signaling pathway. Decreasing the lifetime of the dominant-negative inhibitior by the addition of a rapid degradation tag ([[Ljubljana, Slovenia 2006/Terms & References#Terms|PEST sequence]]) should inactivate the inhibition and restore the responsiveness of the immune system.</p><br />
<br />
<p>This idea is similar to the natural mechanism of tolerance, which is already present in living cells and which decreases the response to repeated bacterial stimulations. This natural tolerance is activated slowly, on the order of days and operates through several different mechanisms. Our feedback mechanism (i.e. artificial tolerance) should decrease the response within hours and thus "attack" the signaling pathway at the point, which has not been used in the natural system.</p><br />
<br><br />
<br />
<h2>Model of the TLR signalling triggered by bacterial infection</h2><br />
<h3>[[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] is the hub of the TLR signalling pathway</h3><br />
<p><br />
Figure 5 represents the complexity of cell signalling mediated by TLRs. which contains more than 700 molecules and complexes (Oda, Kitano, Molecular Systems Biology 2006).</p><br />
<br />
<br style="clear:both;"/><br />
<br />
[[Image:model1.gif|thumb|center|800px|<b>Figure 5: Comprehensive scheme of the Toll-like receptor signaling of microbial infection.</b>]]<br />
<br />
<br />
<br style="clear:both;"/><br />
<br />
<p>[[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] adapter protein is involved in the signal transduction immediately after ligand-induced TLR oligomerization. This adapter protein is common to most TLRs before the signaling network branches into several phosphpryation cascades. We selected this molecule as the most appropriate target of TLR signaling network engineering (Figures 6 and 7).</p><br />
<br />
[[Image:model2.gif|thumb|left|650px|<b>Figure 6: [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] adapter protein connects the response of all TLRs and represents the »bottleneck« of the system (shaded area).</b>]]<br />
[[Image:model3.gif|thumb|right|200px|<b>Figure 7: Schematic representation of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]] as the hub of TLR signaling shaped like a bow tie.</b>]]<br />
<br />
<br style="clear:both;"/><br />
<br />
<h3>Construction of the model</h3><br />
<p><br />
Model of the TLR signaling was prepared in CellDesigner. Signaling steps, where the response remains the same were assembled into one block (Signaling cascade), whose activation directly affects the [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] translocation. Our engineered feedback loop is inserted as a transcriptional activation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] protein domain (red block in Figure 8).</p><br />
<br />
[[Image:model12a.gif|left|thumb|400px|<b>Figure 8: Simplified model of Toll-like receptor signaling constructed in CellDesigner.</b>]]<br />
[[Image:model12.gif|right|thumb|400px|<b>Figure 9: Model of TLR signaling with additional feedback loop for the inhibition of [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]].</b>]]<br />
<br />
<br style="clear:both;"/><br />
<br />
<p>Simulation of cellular response to TLR stimulation by a pulse of [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]] (e.g. flagellin). When available parameters were taken from the literaturte (Selvarajoo, 2006). Signaling cascade results in the transient translocation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] into the cell nucleus (green) and in the production of inflammatory mediators. Repeated pulse after some time results in the repeated activation and accumulation of inflammatory mediators.</p><br />
<br />
<br style="clear:both;"/><br />
<br />
[[Image:model6.gif|thumb|left|400px|<b>Figure 10: Simulation of the cell response to the bacterial stimulus [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP</b>]] (grey) – transient translocation of the [[Ljubljana, Slovenia 2006/Terms & References#Terms|NF-&kappa;B]] into the nucleus (green), production of stimulatory mediators (orange).]]<br />
[[Image:model7.gif|thumb|right|400px|<b>Figure 11: Simulation of the repeated cellular stimulation by [[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]</b> (grey) - the amount of inflammatory mediators increases (orange).]]<br />
<br />
<br style="clear:both;"/><br />
<br />
<p>Simulation of the TLR signaling response in the system with a feedback device results in the production of inhibitor ([[Ljubljana, Slovenia 2006/Terms & References|dnMyD88]], black) which inhibits cellular activation by a repeated stimulation. Cellular responsiveness resets to the normal response if the inhibitor is rapidly degraded, such as by the addition of a [[Ljubljana, Slovenia 2006/Terms & References|PEST]] motif.</p><br />
<br />
[[Image:model8.gif|thumb|left|400px|<b>Figure 12: Introduction of the feedback loop to initiate the production of inhibitory [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]]</b>( (black) – cell does not respond to the repeated stimulation.]]<br />
[[Image:model9.gif|thumb|right|400px|<b>Figure 13: Feedback loop with rapid degradation of the inhibitor ([[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with PEST sequence</b> - black) – responsiveness of the system is again restored to a full extent.]]<br />
<br style="clear:both;"/><br />
<br />
[[Image:model10.gif|thumb|left|400px|<b>Figure 14: Speed of the degradation of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] could modulate the responsiveness of the system to repeated (or continuous) challenge by TLR agonists.</b>]]<br />
<br />
<br style="clear:both;"/><br />
<br />
<hr><br />
<center><br />
[[Image:line-si4.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006|Home]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signalling Pathway]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Results & Conclusions|Results & Conclusions]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th><br />
<th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr></table>[[Image:line-si3.jpg]]<br />
</center></div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006Ljubljana, Slovenia 20062006-10-30T12:40:37Z<p>MDolinar: </p>
<hr />
<div><center><br />
[[Image:Logo-si1.gif|120 px]] [[Image:fotka1b.jpg|475 px]] [[Image:Logo-si1.gif|120 px]]<br />
<br style="clear:both;"/> </center><br />
<br />
<br><br><br />
<center><font style="font size="9" face="Arial"><b>Engineered Human Cells: <br><br><br> SAY NO TO SEPSIS<br />
<br><br></b></font><br />
<br />
<br><br />
[[Image:line-si4.jpg]]<br />
<table><br />
<tr><th>[[Ljubljana, Slovenia 2006/Background and Signalling Pathway|Background and Signalling Pathway]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Project & Model|Project & Model]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Methods|Methods]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Results & Conclusions|Results & Conclusions]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Terms & References|Terms & References]]</th></tr><br />
<tr><th>[[Ljubljana, Slovenia 2006/Team members|Team members]]</th></tr><br />
</table><br />
<br />
[[Image:line-si3.jpg]]<br />
</center><br />
<br />
{|<br />
|-valign="center" align="justify"<br />
|border="1" solid #affaaa" cellspacing="1" cellpadding="1" style="border:1px solid black; background:#cedff2" | <font style="font size="3" font face="Times New Roman""><p><b>Mammalian systems can be a subject of cellular engineering similarly to bacterial cells. We decided to tinker with the existing cell signalling network of the response to the bacterial infection. Binding of bacterial components ([[Ljubljana, Slovenia 2006/Terms & References#Terms|PAMP]]s – Pathogen associated molecular patterns) to a family of Toll-like receptors activates the cells of the immune system but the exaggerated response may lead to systemic inflammation and sepsis which is often fatal. We designed a feedback loop, which inhibits the signalling cascade at the »weak spot« - [[Ljubljana, Slovenia 2006/Terms & References#Terms|MyD88]], a consensus adaptor protein of the Toll-like receptors. A mathematical model of cell activation with engineered feedback loop was constructed, which predicts the decrease of the cellular activation after the repeated stimulation. Twenty-six new BrioBricks were constructed specially for the mammalian system. We have experimentally confirmed the function of the feedback device by detecting the inhibition of cellular activation after the repeated stimulation. Cell activation decreased without completely deleting the responsiveness to the bacterial infection, thus our engineered cell system represents a type of artificial immunotolerance.</b></p><br />
| [[Image:sepsa.png|450px|right|]]<br />
|}<br />
<br><br />
<br />
-----<br />
<br />
'''We would like to express our thanks to the Sponsors'''<br />
<br />
{| border="0" cellspacing="0" cellpadding="5" align="center"<br />
| [http://www.ki.si/ http://parts2.mit.edu/wiki/images/9/90/Logo_ki2.jpg]<br />
| [http://www.fkkt.uni-lj.si/en/ http://www.fkkt.uni-lj.si/img/menuleft_logo_unilj.gif]<br />
| [http://www.lek.si/ http://parts2.mit.edu/wiki/images/0/09/Lek2.jpg]<br />
| [[Image:EU-flag.gif |thumb|120px|EU Synbiocomm (thank you Sven)]]<br />
|}<br />
<br />
<br />
'''and Donators'''<br />
<br>[http://www.ad-futura.si Ad Futura], [http://www.krka.si/si/ Krka pharmaceutical company], [http://www.mediline.si/ Mediline], Farmadent</div>MDolinarhttp://2006.igem.org/wiki/index.php/JamboreeJamboree2006-10-25T08:36:45Z<p>MDolinar: </p>
<hr />
<div>[[Image:Statagraphic.jpg]]<br />
<br />
== 2006 Jamboree Location and Directions ==<br />
[[Image:IGEM 2006 Map.jpg|thumb|MIT campus in vicinity of Stata Center]]<br />
<br />
The Jamboree will be held at MIT's [http://web.mit.edu/evolving/buildings/stata/index.html Stata Center], also known as Building 32. Click [http://whereis.mit.edu/map-jpg?mapterms=32&mapsearch=go here] for a map of campus and directions to MIT.<br />
<br />
== Accommodations ==<br />
<br />
Hotels around MIT are expensive ($250-$350), but lower rates (about $120) can be found nearby, within a short cab or subway ride.<br />
<br />
== Hostels ==<br />
[http://www.hostelsweb.com/hostelsweb.com/hostel.php?HostelNumber=1852 Hostelling International] has a youth hostel about 10 mins walk from the MIT campus. Beds are about $37 per night. <br />
<br />
=== Hotel@MIT ===<br />
*The [http://www.hotelatmit.com Hotel@MIT] (20 Sidney St, Cambridge,(617) 577-0200) is excellent and close to campus (see map above). iGEM discounted rates expired on October 3rd.<br />
<br />
=== Other Hotels ===<br />
<br />
*The Boston Marriott Cambridge (2 Cambridge Ctr, Cambridge, (617) 494-6600) is also a good choice for nearby accommodation.<br />
*The [http://www.kendallhotel.com/ Kendall Hotel] is a block away from the Sata Center<br />
*[http://www.radisson.com/cambridgema The Radisson Hotel] (777 Memorial Drive) is about a mile from the Stata, and has less expensive rooms.<br />
*A list of '''temporary housing''' near MIT can be found [http://web.mit.edu/housing/och/temporary_housing.html here], or check major travel web sites (Travelocity, Expedia, or Hotels.com) for other accommodation options.''<br><br />
<br />
== Schedule ==<br />
<br />
<TABLE width='100% style='padding-left:100px'><TR valign='top'><br />
<TD width='50%'><br />
<TABLE cellspacing='0' cellpadding='5' style='border: 1px solid gray;line-height:75%'> <br />
<TR><TH colspan='9'> Saturday November 4th - Stata Center <br />
<TR><TD width='70' align='right'> 8:30 AM <TD width='40' align='right'> 0:30 <TD> Registration and Coffee<br />
<TR><TD width='70' align='right'> 9:00 AM <TD width='40' align='right'> 0:15 <TD> Introduction<br />
<TR><TD width='70' align='right'> 9:15 AM <TD width='40' align='right'> 0:30 <TD> Talk 1<br />
<TR><TD width='70' align='right'> 9:45 AM <TD width='40' align='right'> 0:30 <TD> Talk 2<br />
<TR><TD width='70' align='right'> 10:15 AM <TD width='40' align='right'> 0:30 <TD> Talk 3<br />
<TR><TD width='70' align='right'> 10:45 AM <TD width='40' align='right'> 0:30 <TD> Break<br />
<TR><TD width='70' align='right'> 10:45 AM <TD width='40' align='right'> 0:30 <TD> Talk 4<br />
<TR><TD width='70' align='right'> 11:15 AM <TD width='40' align='right'> 0:30 <TD> Talk 5<br />
<TR><TD width='70' align='right'> 11:45 AM <TD width='40' align='right'> 0:30 <TD> Talk 6<br />
<TR><TD width='70' align='right'> 12:15 PM <TD width='40' align='right'> 0:20 <TD> Group Picture<br />
<TR><TD width='70' align='right'> 12:35 PM <TD width='40' align='right'> 0:55 <TD> Lunch<br />
<TR><TD width='70' align='right'> 1:30 PM <TD width='40' align='right'> 0:30 <TD> Talk 7<br />
<TR><TD width='70' align='right'> 2:00 PM <TD width='40' align='right'> 0:30 <TD> Talk 8<br />
<TR><TD width='70' align='right'> 2:30 PM <TD width='40' align='right'> 0:30 <TD> Talk 9<br />
<TR><TD width='70' align='right'> 3:00 PM <TD width='40' align='right'> 0:30 <TD> Break<br />
<TR><TD width='70' align='right'> 3:30 PM <TD width='40' align='right'> 0:30 <TD> Ambassadors and Wrap-up<br />
<TR><TD width='70' align='right'> 4:00 PM <TD width='40' align='right'> 0:30 <TD> Talk 10<br />
<TR><TD width='70' align='right'> 4:30 PM <TD width='40' align='right'> 0:30 <TD> Talk 11<br />
<TR><TD width='70' align='right'> 5:00 PM <TD width='40' align='right'> 0:30 <TD> Talk 12<br />
<TR><TD width='70' align='right'> 5:30 PM <TD width='40' align='right'> 2:30 <TD> Posters and Reception<br />
<TR><TD width='70' align='right'> 8:00 PM <TD width='40' align='right'> 4:00 <TD> Social Event<br />
<TR><TD width='70' align='right'> 12:00 AM <TD width='40' align='right'><br />
</TABLE><br />
<br />
<TD width='50%'> <br />
<TABLE cellspacing='0' cellpadding='5' style='border: 1px solid gray;line-height:75%'><br />
<TR><TH colspan='9'> Sunday November 5th - Kressge Auditorium <br />
<TR><TD width='70' align='right'> 8:00 AM <TD width='40' align='right'> 0:30 <TD> Coffee<br />
<TR><TD width='70' align='right'> 8:30 AM <TD width='40' align='right'> 0:15 <TD> Award Session Introduction<br />
<TR><TD width='70' align='right'> 8:45 AM <TD width='40' align='right'> 0:45 <TD> Drew Endy<br />
<TR><TD width='70' align='right'> 9:30 AM <TD width='40' align='right'> 1:00 <TD> Awards<br />
<TR><TD width='70' align='right'> 10:30 AM <TD width='40' align='right'> 0:30 <TD> Break<br />
<TR><TD width='70' align='right'> 11:00 AM <TD width='40' align='right'> 1:00 <TD> 3 Finalists<br />
<TR><TD width='70' align='right'> 12:00 PM <TD width='40' align='right'> 0:15 <TD> Grand Prize Award<br />
<TR><TD width='70' align='right'> 12:15 PM <TD width='40' align='right'> 0:30 <TD> Wrap-up<br />
<TR><TD width='70' align='right'> 12:45 PM <TD width='40' align='right'> <TD> Lunch or Leave<br />
</TABLE> <br />
</TABLE> <br />
<br />
Notes:<br />
* Presentations will be 20 minutes per team plus an additional 5 minutes for questions and 5 minutes to set up the next team.<br />
* On Saturday, there will be a group photo taken.<br />
* On Sunday, the three finalists for the grand prize will repeat their presentations for everyone.<br />
* Box lunches will be provided Sunday and teams are urged to stay and compare notes, but are otherwise free to depart.<br />
<br />
== Posters ==<br />
<br />
Posters shall be 48 inches on a side (Randy's E-mail on Oct. 12) -- this equals to 121,9 cm. Closest ISO A-series format is A0 (841 × 1189 mm).<br />
<br />
== Planned Awards ==<br />
<br />
Best Part<br />
<br />
Best Device<br />
<br />
Best System<br />
<br />
Best Presentation<br />
<br />
Best Poster<br />
<br />
Best Documentation<br />
<br />
Best Measurement and Part Characterization<br />
<br />
Best Cooperation and Collaboration<br />
<br />
Best Conquest of Adversity<br />
<br />
Best Real World Application <br />
<br />
And, finally, the '''Grand Prize''' as determined by the judges...<br />
<br />
== Please Send Photos ==<br />
Got fantastic iGEM-related photos? Send them to ahessel@gmail.com so they can be included in a iGEM video montage! <br />
<br />
== Attendance Roster ==<br />
<br />
Prelimary headcounts are available at the [[Jamboree Attendance]] page<br />
<br />
== External Site == <br />
<br />
The iGEM 2006 external web site can be found [http://igem2006.com here]. (Some tweaks are still being made.)<br />
<br />
== 2005 Jamboree == <br />
Archived information about the 2005 Jamboree can be found [[Jamboree 2005|here]].<br />
<br />
<hr><br />
<font size=1; color="LightSlateGray">iGEM Competition c/o 32 Vassar Street, Room 314, Cambridge, MA 02139 (617) 258 5244 | <font size=1; color="Green"> Join iGEM </font> | Support iGEM </font></div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/ProjectLjubljana, Slovenia 2006/Project2006-10-23T11:46:42Z<p>MDolinar: </p>
<hr />
<div>Although in the past years all participating iGEM teams focused on bacteria and yeast, we decided to modify signalling pathways in higher eukaryotes. This is a far more complicated system to understand but we feel that the recently accumulated knowledge enables rational planning of cells with novel properties.<br />
<br />
== Background ==<br />
<br />
When bacteria attack a human body, immune response is the normal reaction of the organism. Cells which represent the first line of defence are macrophages and the way they act is rather complicated. On the surface of these cells (actually inserted into cell membrane) are several types of receptor molecules, commonly abbreviated as TLR (toll-like receptors). These can specifically bind various components of bacteria (e.g. lipopolysaccharides which are part of bacterial outer membranes) and upon binding, they interact with other molecules inside the cell, forming a cell signalling pathway that, after several relays, ends in the cell nucleus and induces production of immune response proteins, such as cytokines and chemokines. The complete pathway is depicted [http://www.komabiotech.co.kr/technical/review/toll_like_receptor.gif here] and is rather complicated. Stages of the signal transfer were elucidated recently. Cell signalling like this is the basis of our efficient innate immune response (i.e. the fast-acting, not mediated by antibodies) against microbes. In some instances, however, signalling mediated by TLR is not beneficial. Under some not yet fully understood circumstances, bacteria can trigger this same signalling pathway just that it results in excessive inflammatory response. As result, sepsis occurs, which can lead to severe organ failure and in about 20% cases cosequences are fatal.<br />
<br />
== Signalling pathway ==<br />
After binding of bacterial constituents to TLR, its intracellular TIR-domain interacts with either MyD88 or TRIF, two signalling molecules in the cytoplasm that relay signal to other components of the pathway as shown [http://www.biken.osaka-u.ac.jp/act/images/akiraB.png here].<br />
<br />
<br />
== Idea ==<br />
<br />
<br />
<br />
== Approach ==<br />
<br />
<br />
== Anticipated results ==<br />
<br />
----<br />
<br />
<br />
''(expect to see the full story here on October 30)''<br />
<br />
[[Ljubljana, Slovenia 2006|Back home]]</div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/NewsLjubljana, Slovenia 2006/News2006-10-23T11:45:29Z<p>MDolinar: </p>
<hr />
<div>(4th week of October)<br />
Testing cell lines continues with full speed. Preparations for poster and PowerPoint presentations.<br />
<br />
(3rd week of October)<br />
BioBricks are ready to be shipped to MIT.<br />
Finishing the content of the wiki (on our internal wiki site). Still needs some polishing...<br />
<br />
(2nd week of October)<br />
Travel arrangements were fixed. We will travel via Frankfurt and hope to be in Boston on 12:15. We will stay in a suburbs hotel and commute to the jamboree by two vans.<br />
<br />
(First week of October)<br />
Last-minute improvements of the layout delayed printing of the brochure for another week.<br />
Jobs for final presentation (wiki, PowerPoint, English brochure, poster) were taken by pairs of team members.<br />
<br />
(September 22)<br />
Deadline for presentation brochure layout - printing due September 25.<br />
<br />
(September 19)<br />
Transfection of human cell line with first 3 vectors.<br />
<br />
(September)<br />
Rok started working with cell lines. Introduction to fluorescence microscopy. Optimizing ELISAs as detection system. Correcting mutations in some of the constructs.<br />
<br />
(late August)<br />
First completed vectors containing all the BioBricks & correctly ligated. Continue working on other constructs.<br />
<br />
(August 2-4)<br />
iGEM ambassador Robin Kuenzler was visiting. Presentations of iGEM concept, ETH 2005 project, work with wikis and introduction to Registry of biological parts were scheduled as was a short presentation of our project and progress in the lab. Click [[Robin's visit|here]] to see some photos.<br />
<br />
(August 1)<br />
Work intensified. First constructs are due August 15 and Jelka, Matej and Jernej moved their samples to the [http://openwetware.org/wiki/FCCT_Biochemistry_Lab FCCT Biochemistry Lab] and continue preparing constructs they are in charge of.<br />
<br />
(July 19) <br />
Supervisors are back from vacation - time for a brief overview of the lab work...<br />
Work actually started on June 7 with basic cloning, vector preparations, PCRs etc. Instructors from Roman's group were great help all the time. First experiments with ELISA detection system were already performed (looks promising). Next team meeting is in one week. iGEM ambassador expected to visit in early August.<br />
<br />
[[Ljubljana, Slovenia 2006|Back home]]</div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Team_MembersLjubljana, Slovenia 2006/Team Members2006-10-23T11:40:10Z<p>MDolinar: </p>
<hr />
<div>[[Image:SLOteam06a.jpg]]<br />
<br />
''From left to right: Alja, Ota, Jelka, Matej, Monika, Jernej, Rok, Simon, Marko, Mojca M., Roman.<br />
''<br />
<br />
----<br />
'''STUDENTS:'''<br />
<br />
*Simon Belak (Physics) ''Simon unfortunately quit the team in summer.''<br />
*[[User:Moni|Monika Ciglič (Microbiology)]]<br />
*[[User:Ota|Ota Fekonja]](Microbiology)<br />
*[[User:Jernej|Jernej Kovač]] (Biochemistry)<br />
*[[User:Mojca Miklavec|Mojca Miklavec]] (Physics & Bioinformatics)<br />
*[[User:Alja|Alja Oblak]] (Microbiology)<br />
*[[User:Jelka.pohar|Jelka Pohar]] (Microbiology)<br />
*[[User:Matej|Matej Skočaj]] (Microbiology)<br />
*[[User:rtkavc|Rok Tkavc (Microbiology)]]<br />
<br />
'''SUPERVISORS:'''<br />
<br />
*[[User:MDolinar|Marko Dolinar]] (Faculty of Chemistry & Chemical Technology)<br />
*Roman Jerala (National Institute of Chemistry = NIC)<br />
<br />
'''INSTRUCTORS:'''<br />
*Mojca Benčina (NIC)<br />
*Mateja Manček Keber (NIC)<br />
*Gabriela Panter (NIC)<br />
<br />
----<br />
<br><br />
[[Image:SLOteam06b.jpg]] <br />
<br />
''From left to right: Gabriela, Roman, Jelka, Jernej, Monika, Matej, Alja, Rok, Mojca M., Marko.''<br />
<br />
[[Ljubljana, Slovenia 2006|Back home]]</div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006Ljubljana, Slovenia 20062006-10-23T11:37:53Z<p>MDolinar: </p>
<hr />
<div><center>[[Image:LJU2.jpg]]<br />
<div style="padding: 7px; color: #ffffff; background-color: #3674C2; width: 626px"><br />
<br />
[[Ljubljana, Slovenia 2006 | <font face="trebuchet ms" style="color:#ffffff"> '''Home''' </font>]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
[[Ljubljana, Slovenia 2006/News | <font face="trebuchet ms" style="color:#ffffff"> '''News''' </font>]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
[[Ljubljana, Slovenia 2006/Team Members | <font face="trebuchet ms" style="color:#ffffff"> '''Team Members''' </font>]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
[[Ljubljana, Slovenia 2006/Project | <font face="trebuchet ms" style="color:#ffffff"> '''Project''' </font>]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
[[Ljubljana, Slovenia 2006/Progress | <font face="trebuchet ms" style="color:#ffffff"> '''Progress''' </font>]] <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
[[Ljubljana, Slovenia 2006/Back Door | <font face="trebuchet ms" style="color:#ffffff"> '''Internal''' </font>]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
[[Ljubljana, Slovenia 2006/Slovenia | <font face="trebuchet ms" style="color:#ffffff"> '''Slovenia''' </font>]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<br />
</div><br />
</center><br />
<br />
Our team is composed of 8 students of different undergraduate programs within the [http://www.uni-lj.si/English/description.asp University of Ljubljana], 3 instructors from the National Institute of Chemistry and 2 supervisors. Project research will be conducted at the [http://www.ki.si/eng/ki/Introduction.html National Institute of Chemistry] (in the group of Prof. Roman Jerala) and at the [http://www.fkkt.uni-lj.si/en/ Faculty of Chemistry and Chemical Technology], [http://openwetware.org/wiki/FCCT_Biochemistry_Lab Biochemistry Chair] (under supervision of Asst. Prof. Marko Dolinar).<br />
<br />
34 students initially applied for the team. Project proposals were due March 10. We received 13 different project ideas. Both supervisors evaluated the proposed projects and had interviews in the week from April 3 to 7. The 'lab' team members were selected on April 12 and two physics students one week later. In May, we were mainly discussing possible projects and soon focused on a cellular signalisation issue. Laboratory work started on June 7. We were first preparing vector constructs and soon thereafter started testing detection systems (including enzyme-linked immune assays, cell sorter analyses and luminiscence measurements). The model (CellDesigner) of the signalling pathway was developed as well. We work hard to get the anticipated response in genetically modified cells. In parallel, we are preparing the final version of the wiki on another Web server, so expect to have the almost complete version here by the deadline.<br />
----<br />
<small>''In the center of the photograph is the rectorate building of the University of Ljubljana. Right behind, in the white building the first [http://www.fkkt.uni-lj.si/attachments/521/realka.jpg laboratories] of the Chemistry Department were located since 1919.''</small></div>MDolinarhttp://2006.igem.org/wiki/index.php/Jamboree_AttendanceJamboree Attendance2006-10-23T07:51:50Z<p>MDolinar: </p>
<hr />
<div>This page is the place to put information related to the [[Jamboree]] 2006 planning.<br />
<br />
=Attendance planning=<br />
{|| style="width:100%; height:10px" border="1"<br />
!style="background:silver"| school<br />
!style="background:silver"|number of students<br />
!style="background:silver"|number of advisors/staff<br />
!style="background:silver"|total number<br />
!style="background:silver"|accuracy of guess (according to the team)<br />
!style="background:silver"|status of travel planning (visa,...)<br />
!style="background:silver"| source of information<br />
|- <br />
| [[Cambridge_University_2006 | Cambridge]]<br />
| 6<br />
| 2-5<br />
| 8-13<br />
| <br />
| visa applications, as soon as the team is assembled<br />
| mail from Gos Micklem, [[User:Jonas|jonas]] 09:26, 7 July 2006 (EDT)<br />
|- <br />
| [[Imperial_College_2006 | Imperial College]]<br />
| 8<br />
| 2-3<br />
| 10-11<br />
| <br />
| <br />
| mail from Vincent Rouilly, [[User:Jonas|jonas]] 8 July 2006 (EDT)<br />
|- <br />
| [[University_of_Edinburgh_2006 | Edinburgh]]<br />
| 8<br />
| 2<br />
| 10<br />
| <br />
| <br />
| updated by C. French, 20 October 2006<br />
|- <br />
| [[Freiburg_University_2006 | Freiburg]]<br />
| 5<br />
| 1<br />
| <br />
| <br />
| probably webcast<br />
| mail from Andrei Kouznetsov, [[User:Tamara|Tamara]] 18 July 2006<br />
|- <br />
| [[ETH_Zurich_2006 | ETH Zurich]]<br />
| 9<br />
| 2-3<br />
| 12<br />
| 11-12<br />
| <br />
| mail from Sven Panke, [[User:Robin|robin]] 12:16, 12 July 2006 (EDT)<br />
|- <br />
| [[UPV-UV_Valencia%2C_Spain_2006 | Valencia]]<br />
| 10<br />
| 5<br />
| 13-17<br />
| <br />
| <br />
| talked to Alfonso Jaramillo, [[User:Robin|robin]] 12:00, 12 July 2006 (EDT)<br />
|- <br />
| [[Ljubljana, Slovenia 2006 | Ljubljana]]<br />
| 7<br />
| 3<br />
| 10<br />
| final number<br />
| <br />
| [[User:MDolinar|Marko]], 23 October 2006<br />
|-<br />
| [[Turkey_2006 | EGE Turkey]]<br />
| <br />
|<br />
| 3<br />
| <br />
| <br />
| mail from Bahattin, [[User:Robin|robin]] 13:37, 16 July 2006 (EDT)<br />
|-<br />
| [[Duke_2006 | Duke University]]<br />
| <br />
|<br />
| 12<br />
|"Better estimate will be available in fall" <br />
| <br />
|Faisal Reza, faculty organizer, --[[User:Smelissali|Smelissali]] 13:13, 2 August 2006 (EDT)<br />
|-<br />
| [[University of Arizona 2006 | Arizona]]<br />
| 10<br />
|2<br />
| 12<br />
|<br />
| <br />
|Patrick Hollinger (student team leader) --[[User:Smelissali|Smelissali]] 14:07, 4 August 2006 (EDT)<br />
|-<br />
|[[Missouri_Western_State_University_2006| Missouri]]<br />
|7<br />
|2<br />
|9<br />
|Solid<br />
|<br />
|Email from Todd Eckdahl 8/2 --[[User:Ahessel|Ahessel]] 13:53, 2 August 2006 (EDT)<br />
|-<br />
|[[University_of_Oklahoma_2006 | Oklahoma]]<br />
|<br />
|<br />
|4-5<br />
|<br />
|<br />
|Email from Bruce Roe 8/2 --[[User:Ahessel|Ahessel]] 14:20, 2 August 2006 (EDT)<br />
|-<br />
|[[Boston University 2006| Boston]]<br />
|16<br />
|1<br />
|17<br />
|Good<br />
|<br />
|PC Alex Kates, BU<br />
|-<br />
|[[Brown:iGEM portal 2006 |Brown]]<br />
|8-12<br />
|1-3<br />
|9-15<br />
|Students:Good, Staff:Rough<br />
|<br />
|PC- John Cumbers, Brown<br />
|-<br />
|[[University of Calgary 2006|Calgary]]<br />
|7<br />
|3<br />
|10<br />
|Very rough<br />
|<br />
|PC - Christian Jacob, Calgary<br />
|-<br />
|[[Harvard 2006|Harvard]]<br />
|10<br />
|5-8<br />
|15-18<br />
|Students: Accurate Staff: Rough<br />
|<br />
|PC - Nik Stroustrup<br />
|-<br />
|[[University of Michigan 2006|Michigan]]<br />
|8-12<br />
|3-5<br />
|11-17<br />
|Rough<br />
|<br />
|PC - Peter Woolf<br />
|-<br />
|[[MIT 2006|MIT]]<br />
|5<br />
|7<br />
|12<br />
|Good<br />
|<br />
|Obvious<br />
|-<br />
|[[Princeton 2006|Princeton]]<br />
|12<br />
|6<br />
|18<br />
|Solid data<br />
|<br />
|PC - Cil Purnick, updated 10/20/06<br />
|-<br />
|[[University_of_Toronto_2006|Toronto and Waterloo]]<br />
|<br />
|<br />
|20-25<br />
|Good<br />
|<br />
|Emails from Stephen Davies and Nancy Xu --[[User:Ahessel|Ahessel]] 12:26, 8 August 2006 (EDT)<br />
|-<br />
|[[Mississippi_State_University_2006|Mississippi State]]<br />
|8<br />
|1<br />
|9<br />
|Solid<br />
|<br />
|Filip To --[[User:Fto|Filip To]] Oct 20, 2006<br />
|-<br />
|[[McGill_University_2006|McGill]]<br />
|<br />
|<br />
|12<br />
|Good<br />
|<br />
|Email from Jay Nadeau --[[User:Ahessel|Ahessel]] 12:26, 8 August 2006 (EDT)<br />
|-<br />
|[[Prairie_View_A%26M_University_2006|Prairie View]]<br />
|<br />
|<br />
|6<br />
|Good<br />
|<br />
|Email from Raul Cuero --[[User:Ahessel|Ahessel]] 12:26, 8 August 2006 (EDT)<br />
|-<br />
|[[Africa_2006|Africa]]<br />
|<br />
|<br />
|4-5<br />
|Good<br />
|<br />
|Email from Raul Cuero --[[User:Ahessel|Ahessel]] 12:26, 8 August 2006 (EDT)<br />
|-<br />
|[[Latin_America_2006|Latin America]]<br />
|<br />
|<br />
|11-12<br />
|Good<br />
|<br />
|Email from Raul Cuero --[[User:Ahessel|Ahessel]] 12:26, 8 August 2006 (EDT)<br />
|-<br />
|[[Penn State University 2006|Penn State]]<br />
|<br />
|<br />
|8-10<br />
|pretty good<br />
|<br />
|email from Pat Cirino, Tom Richard --[[User:Smelissali|Smelissali]] 20:42, 9 August 2006 (EDT)<br />
|-<br />
|[[University of California Berkeley 2006|UC Berkeley]]<br />
|7<br />
|2<br />
|9<br />
|quite good<br />
|<br />
|Team meeting on 8/07 --[[User:Smelissali|Smelissali]] 20:42, 9 August 2006 (EDT) <br />
|-<br />
|[[University of California San Francisco 2006|UCSF]]<br />
|4<br />
|<br />
|4<br />
|good<br />
|<br />
|team meeting on 8/08 --[[User:Smelissali|Smelissali]] 20:42, 9 August 2006 (EDT)<br />
|-<br />
|[[Rice_University_2006|Rice University]]<br />
|7<br />
|2<br />
|9<br />
|good<br />
|<br />
|Email from Beth Beason -- 9 October 2006 -rr<br />
|-<br />
|[[IPN_UNAM_2006|IPN_UNAM]]<br />
|<br />
|<br />
|8 from IPN, 4 from UNAM = 12 total<br />
|Definite<br />
|Some flights/hotel booked, waiting for definitive yes on visas but shouldn't be a problem<br />
|Email from Genaro ----[[User:Meaganl|Meaganl]] 15:04, 28 September 2006 (EDT)<br />
|-<br />
|[[Davidson_2006|Davidson]]<br />
|4 students<br />
|3 faculty<br />
|7<br />
|certain<br />
|hotel set; travel set<br />
|posted by Malcolm Campbell<br />
<br />
|-<br />
|Chiba<br />
|16<br />
|1-2<br />
|17-18<br />
|fair, depends on money<br />
|money, need accommodation<br />
|Reshma (via emails with team)<br />
|-<br />
|NCBS<br />
|6<br />
|1<br />
|~ 7<br />
|very good<br />
|need accommodation<br />
|Reshma (via emails with team)<br />
|-<br />
|[[Tokyo_Alliance_2006|Tokyo alliance]]<br />
|4<br />
|3<br />
|7<br />
|very good<br />
|hotel set; travel set <br />
|edited by Kiga<br />
|-<br />
|UT Austin<br />
|6<br />
|1-2<br />
|7-8<br />
|very good<br />
|<br />
|Eric Davidson Oct 20<br />
|-<br />
|Chungbuk<br />
|?<br />
|2<br />
|?<br />
|?<br />
|?<br />
|Reshma<br />
|-<br />
|[school]<br />
|[#students]<br />
|[#staff]<br />
|[total attendance]<br />
|[accuracy of guess]<br />
|[issues for travel]<br />
|[source of info]<br />
|}</div>MDolinarhttp://2006.igem.org/wiki/index.php/Background_and_Signalling_PathwayBackground and Signalling Pathway2006-10-21T17:35:17Z<p>MDolinar: </p>
<hr />
<div><center>[[Image:Logo-si.jpg]]<br />
[[Image:line-si.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006/Background|Home]]</th><br />
<th>[[Proposal & Approach]]</th><br />
<th>[[Anticipated Results & Significance]]</th><br />
<th>[[Troubleshooting, References & Sponsors]]</th><br />
<th>[[Team members]]</th></tr></table><br />
[[Image:line-si2.jpg]]<br />
</center><br />
<br />
<br />
<h1>Background</h1><br />
<br />
When bacteria invade the human body, it has to respond quickly by activation of the innate branch of the immune system, which recognizes the broad spectrum of molecules, specifical for pathogenic microorganisms. Cells which represent the first line of defence are macrophages and the way they act is rather complicated. On the surface of these cells (actually inserted into cell membrane) are several types of receptor molecules, commonly abbreviated as TLR (toll-like receptors). These can specifically bind various components of bacteria (e.g. lipopolysaccharides which are part of <teing added constantly. This kind of cell signalling is the basis of our efficient innate immune response (i.e. the fast-acting, not mediated by antibodies) against microbes. In some instances, however, extensive signalling mediated by TLR is not beneficial. Under some not yet fully understood circumstances, bacteria can trigger this same signalling pathway just that it results in excessive inflammatory response. As result, sepsis occurs, which can lead to severe organ failure and in about 20% cases cosequences are fatal.<br />
Response to bacterial stimulus is a double-edged sword - too strong response may lead to sepsis, while the too weak response does not contain the bacterial infection, therefor both extremes have to be in the balance.<br />
<br />
[[Image:scheme2.jpg|center|260 px]]<br />
<br />
<br />
<br />
<h1>Signalling pathway of bacterial recognition of the human innate immune system</h1><br />
<br />
Toll like receptors (TLRs) are key regulators of innate immunity, sensing and responding to invading microorganisms. So far we know eleven different receptors that recognize specific molecular patterns that are present in microbial components, e.g. lipopeptides, double and single stranded RNA of viruses, CpG containing DNA, glycolipids, and variety of others.<br />
We have focused on TLR4 and TLR5 that respond to presence of LPS and flagellin respectively, so both sense the presence of bacteria.<br />
<br />
After binding of bacterial constituents to TLR, its intracellular TIR-domain interacts with either MyD88 (MyD88 dependent way) or TRIF (MyD88 independent way), two signaling molecules in the cytoplasm that transmit signal to other components of the cascade, as shown on picture below. TLR4 can induce response through both ways, TLR5 only through MyD88 depended.<br />
<br />
<center><gallery><br />
Image:pot4.jpg<br />
Image:Scheme1.jpg<br />
</gallery></center><br />
<br />
<h2>MyD88 dependent pathway</h2><br />
<br />
When receptors sense the presence of microbial components, stimulation of TLRs occurs and triggers the association of MyD88 (myeloid differentiation primary-response protein 88), which in turn recruits IRAK4, thereby allowing association with IRAK1. IRAK4 phosphorylates IRAK1 and with phosphorylated IRAK1, TRAF6 (tumour-necrosis-factor receptor- associated factor 6) associates. The complex of phosphorylated IRAK1 and TRAF6 dissociats from the receptor and form a complex with TAK1, TAB1 and TAB2, which induces the phosphorylation of TAB2 and TAK1. This leads to the ubiquitylation of TRAF6, which induces the activation of TAK1. TAK1 phosphorylates IKK complex (inhibitor of nuclear factor &kappa;B (I&kappa;B)-kinase complex), which consists of IKK&alpha;, IKK&beta; and IKK&gamma;. The IKK complex then phosphorylates I&kappa;B, which leads to its ubiquitylation and subsequent degradation. This allows NF&kappa;B to translocate to the nucleus and to induce the expression of its target genes, such as cytokines and chemokines.<br />
Inflammatory cytokines, chemokines and interferons attract cells of immune system (lymphocytes, macrophages) to infected area. This seems to be beneficial to organism, but it can also lead to severe inflammation and sepsis.<br />
Some receptors mediate response through MyD88-independent pathway. A number of genes known to be interferon(IFN)-inducible genes was identified. <br />
<br />
<br />
<hr><br />
komentarji:<br />
# označi za vsako sliko, kaj pomeni in od kod je<br />
#{Marko, 21.10.} V petek na sluzbenem racunalniku so bili naslovi poglavij se vedno stlaceni v 1 vrsti, na prenosniku pa so v redu - preverite, ali je to odvisno od locljivosti ekrana oz. ali je pri vseh (ostalih) razpored poglavij v 2 vrsticah v redu.<br />
<hr><br />
<br />
<center><br />
[[Image:line-si.jpg]]<br />
<table cellpadding="10"><br />
<tr><th>[[Ljubljana, Slovenia 2006/Background|Home]]</th><br />
<th>[[Proposal & Approach]]</th><br />
<th>[[Anticipated Results & Significance]]</th><br />
<th>[[Troubleshooting, References & Sponsors]]</th><br />
<th>[[Team members]]</th></tr></table><br />
[[Image:line-si2.jpg]]<br />
</center></div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/BackgroundLjubljana, Slovenia 2006/Background2006-10-20T07:17:43Z<p>MDolinar: </p>
<hr />
<div><center>[[Image:Logo-si.jpg]]<br />
[[Image:line-si.jpg]]<br />
<table><br />
<tr><td>[[Backround and Signalling Pathway]]</td></tr><br />
<tr><td>[[Proposal & Approach]]</td></tr><br />
<tr><td>[[Anticipated Results & Significance]]</td></tr><br />
<tr><td>[[Troubleshooting, References & Sponsors]]</td></tr><br />
<tr><td>[[Team members]]</td></tr><br />
</table><br />
<br />
[[Image:line-si2.jpg]]<br />
</center><br />
<br />
<h3>Tu še pride pozdravni tekst. Ostale povezave delujejo.</h3></div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/BackgroundLjubljana, Slovenia 2006/Background2006-10-18T15:02:28Z<p>MDolinar: </p>
<hr />
<div>This is an empty page to fit in background information about the project of our team.</div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/NewsLjubljana, Slovenia 2006/News2006-10-18T15:01:21Z<p>MDolinar: </p>
<hr />
<div>(3rd week of October)<br />
BioBricks are ready to be shipped to MIT.<br />
Finishing the content of the wiki (on our internal wiki site). Still needs some polishing...<br />
<br />
(2nd week of October)<br />
Travel arrangements were fixed. We will travel via Frankfurt and hope to be in Boston on 12:15. We will stay in a suburbs hotel and commute to the jamboree by two vans.<br />
<br />
(First week of October)<br />
Last-minute improvements of the layout delayed printing of the brochure for another week.<br />
Jobs for final presentation (wiki, PowerPoint, English brochure, poster) were taken by pairs of team members.<br />
<br />
(September 22)<br />
Deadline for presentation brochure layout - printing due September 25.<br />
<br />
(September 19)<br />
Transfection of human cell line with first 3 vectors.<br />
<br />
(September)<br />
Rok started working with cell lines. Introduction to fluorescence microscopy. Optimizing ELISAs as detection system. Correcting mutations in some of the constructs.<br />
<br />
(late August)<br />
First completed vectors containing all the BioBricks & correctly ligated. Continue working on other constructs.<br />
<br />
(August 2-4)<br />
iGEM ambassador Robin Kuenzler was visiting. Presentations of iGEM concept, ETH 2005 project, work with wikis and introduction to Registry of biological parts were scheduled as was a short presentation of our project and progress in the lab. Click [[Robin's visit|here]] to see some photos.<br />
<br />
(August 1)<br />
Work intensified. First constructs are due August 15 and Jelka, Matej and Jernej moved their samples to the [http://openwetware.org/wiki/FCCT_Biochemistry_Lab FCCT Biochemistry Lab] and continue preparing constructs they are in charge of.<br />
<br />
(July 19) <br />
Supervisors are back from vacation - time for a brief overview of the lab work...<br />
Work actually started on June 7 with basic cloning, vector preparations, PCRs etc. Instructors from Roman's group were great help all the time. First experiments with ELISA detection system were already performed (looks promising). Next team meeting is in one week. iGEM ambassador expected to visit in early August.<br />
<br />
[[Ljubljana, Slovenia 2006|Back home]]</div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006Ljubljana, Slovenia 20062006-10-18T14:26:42Z<p>MDolinar: </p>
<hr />
<div><center>[[Image:LJU2.jpg]]<br />
<div style="padding: 7px; color: #ffffff; background-color: #3674C2; width: 626px"><br />
<br />
[[Ljubljana, Slovenia 2006 | <font face="trebuchet ms" style="color:#ffffff"> '''Home''' </font>]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
[[Ljubljana, Slovenia 2006/News | <font face="trebuchet ms" style="color:#ffffff"> '''News''' </font>]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
[[Ljubljana, Slovenia 2006/Team Members | <font face="trebuchet ms" style="color:#ffffff"> '''Team Members''' </font>]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
[[Ljubljana, Slovenia 2006/Project | <font face="trebuchet ms" style="color:#ffffff"> '''Project''' </font>]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
[[Ljubljana, Slovenia 2006/Progress | <font face="trebuchet ms" style="color:#ffffff"> '''Progress''' </font>]] <br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
[[Ljubljana, Slovenia 2006/Back Door | <font face="trebuchet ms" style="color:#ffffff"> '''Internal''' </font>]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
[[Ljubljana, Slovenia 2006/Slovenia | <font face="trebuchet ms" style="color:#ffffff"> '''Slovenia''' </font>]] &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<br />
</div><br />
</center><br />
<br />
Our team is composed of 9 students of different undergraduate programs within the [http://www.uni-lj.si/English/description.asp University of Ljubljana], 3 instructors from the National Institute of Chemistry and 2 supervisors. Project research will be conducted at the [http://www.ki.si/eng/ki/Introduction.html National Institute of Chemistry] (in the group of Prof. Roman Jerala) and at the [http://www.fkkt.uni-lj.si/en/ Faculty of Chemistry and Chemical Technology], [http://openwetware.org/wiki/FCCT_Biochemistry_Lab Biochemistry Chair] (under supervision of Asst. Prof. Marko Dolinar).<br />
<br />
34 students initially applied for the team. Project proposals were due March 10. We received 13 different project ideas. Both supervisors evaluated the proposed projects and had interviews in the week from April 3 to 7. The 'lab' team members were selected on April 12 and two physics students one week later. In May, we were mainly discussing possible projects and soon focused on a cellular signalisation issue. Laboratory work started on June 7. We were first preparing vector constructs and soon thereafter started testing detection systems (including enzyme-linked immune assays, cell sorter analyses and luminiscence measurements). The model (CellDesigner) of the signalling pathway was developed as well. We work hard to get the anticipated response in genetically modified cells. In parallel, we are preparing the final version of the wiki on another Web server, so expect to have the almost complete version here in the next few days.<br />
----<br />
<small>''In the center of the photograph is the rectorate building of the University of Ljubljana. Right behind, in the white building the first [http://www.fkkt.uni-lj.si/attachments/521/realka.jpg laboratories] of the Chemistry Department were located since 1919.''</small></div>MDolinarhttp://2006.igem.org/wiki/index.php/Jamboree_AttendanceJamboree Attendance2006-10-04T07:14:22Z<p>MDolinar: </p>
<hr />
<div>This page is the place to put information related to the [[Jamboree]] 2006 planning.<br />
<br />
=Attendance planning=<br />
{|| style="width:100%; height:10px" border="1"<br />
!style="background:silver"| school<br />
!style="background:silver"|number of students<br />
!style="background:silver"|number of advisors/staff<br />
!style="background:silver"|total number<br />
!style="background:silver"|accuracy of guess (according to the team)<br />
!style="background:silver"|status of travel planning (visa,...)<br />
!style="background:silver"| source of information<br />
|- <br />
| [[Cambridge_University_2006 | Cambridge]]<br />
| 6<br />
| 2-5<br />
| 8-13<br />
| <br />
| visa applications, as soon as the team is assembled<br />
| mail from Gos Micklem, [[User:Jonas|jonas]] 09:26, 7 July 2006 (EDT)<br />
|- <br />
| [[Imperial_College_2006 | Imperial College]]<br />
| 8<br />
| 2-3<br />
| 10-11<br />
| <br />
| <br />
| mail from Vincent Rouilly, [[User:Jonas|jonas]] 8 July 2006 (EDT)<br />
|- <br />
| [[University_of_Edinburgh_2006 | Edinburgh]]<br />
| 9<br />
| 2-3<br />
| 11-12<br />
| <br />
| <br />
| mail from Chris French, [[User:Tamara|Tamara]] 18 July 2006<br />
|- <br />
| [[Freiburg_University_2006 | Freiburg]]<br />
| 2-3<br />
| 1<br />
| 3-4<br />
| <br />
| <br />
| mail from Andrei Kouznetsov, [[User:Tamara|Tamara]] 18 July 2006<br />
|- <br />
| [[ETH_Zurich_2006 | ETH Zurich]]<br />
| 2-3<br />
| 9<br />
| 12<br />
| 11-12<br />
| <br />
| mail from Sven Panke, [[User:Robin|robin]] 12:16, 12 July 2006 (EDT)<br />
|- <br />
| [[UPV-UV_Valencia%2C_Spain_2006 | Valencia]]<br />
| 10<br />
| 5<br />
| 13-17<br />
| <br />
| <br />
| talked to Alfonso Jaramillo, [[User:Robin|robin]] 12:00, 12 July 2006 (EDT)<br />
|- <br />
| [[Ljubljana, Slovenia 2006 | Ljubljana]]<br />
| 8<br />
| 3<br />
| 11<br />
| high<br />
| <br />
| [[User:MDolinar|Marko]], 4 October 2006 (CET)<br />
|-<br />
| [[Turkey_2006 | EGE Turkey]]<br />
| <br />
|<br />
| 3<br />
| <br />
| <br />
| mail from Bahattin, [[User:Robin|robin]] 13:37, 16 July 2006 (EDT)<br />
|-<br />
| [[Duke_2006 | Duke University]]<br />
| <br />
|<br />
| 12<br />
|"Better estimate will be available in fall" <br />
| <br />
|Faisal Reza, faculty organizer, --[[User:Smelissali|Smelissali]] 13:13, 2 August 2006 (EDT)<br />
|-<br />
| [[University of Arizona 2006 | Arizona]]<br />
| 10<br />
|2<br />
| 12<br />
|<br />
| <br />
|Patrick Hollinger (student team leader) --[[User:Smelissali|Smelissali]] 14:07, 4 August 2006 (EDT)<br />
|-<br />
|[[Missouri_Western_State_University_2006| Missouri]]<br />
|7<br />
|2<br />
|9<br />
|high<br />
|<br />
|Email from Todd Eckdahl 8/2 --[[User:Ahessel|Ahessel]] 13:53, 2 August 2006 (EDT)<br />
|-<br />
|[[University_of_Oklahoma_2006 | Oklahoma]]<br />
|<br />
|<br />
|4-5<br />
|<br />
|<br />
|Email from Bruce Roe 8/2 --[[User:Ahessel|Ahessel]] 14:20, 2 August 2006 (EDT)<br />
|-<br />
|[[Boston University 2006| Boston]]<br />
|16<br />
|1<br />
|17<br />
|Good<br />
|<br />
|PC Alex Kates, BU<br />
|-<br />
|[[Brown:iGEM portal 2006 |Brown]]<br />
|12-14<br />
|0-3<br />
|14-17<br />
|Students:Good, Staff:Rough<br />
|<br />
|PC- John Cumbers, Brown<br />
|-<br />
|[[University of Calgary 2006|Calgary]]<br />
|7<br />
|3<br />
|10<br />
|Very rough<br />
|<br />
|PC - Christian Jacob, Calgary<br />
|-<br />
|[[Harvard 2006|Harvard]]<br />
|10<br />
|5-8<br />
|15-18<br />
|Students: Accurate Staff: Rough<br />
|<br />
|PC - Nik Stroustrup<br />
|-<br />
|[[University of Michigan 2006|Michigan]]<br />
|8-12<br />
|3-5<br />
|11-17<br />
|Rough<br />
|<br />
|PC - Peter Woolf<br />
|-<br />
|[[MIT 2006|MIT]]<br />
|6<br />
|7<br />
|13<br />
|Good<br />
|<br />
|Obvious<br />
|-<br />
|[[Princeton 2006|Princeton]]<br />
|10-14<br />
|3-5<br />
|15-19<br />
|Good<br />
|<br />
|PC - Cil Purnick<br />
|-<br />
|[[University_of_Toronto_2006|Toronto and Waterloo]]<br />
|<br />
|<br />
|20-25<br />
|Good<br />
|<br />
|Emails from Stephen Davies and Nancy Xu --[[User:Ahessel|Ahessel]] 12:26, 8 August 2006 (EDT)<br />
|-<br />
|[[McGill_University_2006|McGill]]<br />
|<br />
|<br />
|12<br />
|Good<br />
|<br />
|Email from Jay Nadeau --[[User:Ahessel|Ahessel]] 12:26, 8 August 2006 (EDT)<br />
|-<br />
|[[Prairie_View_A%26M_University_2006|Prairie View]]<br />
|<br />
|<br />
|6<br />
|Good<br />
|<br />
|Email from Raul Cuero --[[User:Ahessel|Ahessel]] 12:26, 8 August 2006 (EDT)<br />
|-<br />
|[[Africa_2006|Africa]]<br />
|<br />
|<br />
|4-5<br />
|Good<br />
|<br />
|Email from Raul Cuero --[[User:Ahessel|Ahessel]] 12:26, 8 August 2006 (EDT)<br />
|-<br />
|[[Latin_America_2006|Latin America]]<br />
|<br />
|<br />
|11-12<br />
|Good<br />
|<br />
|Email from Raul Cuero --[[User:Ahessel|Ahessel]] 12:26, 8 August 2006 (EDT)<br />
|-<br />
|[[Penn State University 2006|Penn State]]<br />
|<br />
|<br />
|8-10<br />
|pretty good<br />
|<br />
|email from Pat Cirino, Tom Richard --[[User:Smelissali|Smelissali]] 20:42, 9 August 2006 (EDT)<br />
|-<br />
|[[University of California Berkeley 2006|UC Berkeley]]<br />
|7<br />
|2<br />
|9<br />
|quite good<br />
|<br />
|Team meeting on 8/07 --[[User:Smelissali|Smelissali]] 20:42, 9 August 2006 (EDT) <br />
|-<br />
|[[University of California San Francisco 2006|UCSF]]<br />
|4<br />
|<br />
|4<br />
|good<br />
|<br />
|team meeting on 8/08 --[[User:Smelissali|Smelissali]] 20:42, 9 August 2006 (EDT)<br />
|-<br />
|[[Rice_University_2006|Rice University]]<br />
|<br />
|<br />
|8<br />
|good<br />
|<br />
|Email from Beth Beason--[[User:Ahessel|Ahessel]] 17:03, 15 August 2006 (EDT)<br />
|-<br />
|[[IPN_UNAM_2006|IPN_UNAM]]<br />
|<br />
|<br />
|8 from IPN, 4 from UNAM = 12 total<br />
|Definite<br />
|Some flights/hotel booked, waiting for definitive yes on visas but shouldn't be a problem<br />
|Email from Genaro ----[[User:Meaganl|Meaganl]] 15:04, 28 September 2006 (EDT)<br />
|-<br />
|[[Davidson_2006|Davidson]]<br />
|4 students<br />
|3 faculty<br />
|7<br />
|certain<br />
|hotel set; travel set<br />
|posted by Malcolm Campbell<br />
<br />
|-<br />
|Chiba<br />
|16<br />
|1-2<br />
|17-18<br />
|fair, depends on money<br />
|money, need accommodation<br />
|Reshma (via emails with team)<br />
|-<br />
|NCBS<br />
|6<br />
|1<br />
|~ 7<br />
|very good<br />
|need accommodation<br />
|Reshma (via emails with team)<br />
|-<br />
|Tokyo alliance<br />
|4<br />
|2-3<br />
|6-7<br />
|very good<br />
|none<br />
|Reshma (email from Prof Kiga)<br />
|-<br />
|Chungbuk<br />
|?<br />
|2<br />
|?<br />
|?<br />
|?<br />
|Reshma<br />
|-<br />
|[school]<br />
|[#students]<br />
|[#staff]<br />
|[total attendance]<br />
|[accuracy of guess]<br />
|[issues for travel]<br />
|[source of info]<br />
|}</div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/NewsLjubljana, Slovenia 2006/News2006-09-20T14:54:58Z<p>MDolinar: </p>
<hr />
<div>(September 22)<br />
Deadline for presentation brochure layout - printing due September 25.<br />
<br />
(September 19)<br />
Transfection of human cell line with first 3 vectors.<br />
<br />
(September)<br />
Rok started working with cell lines. Introduction to fluorescence microscopy. Optimizing ELISAs as detection system. Correcting mutations in some of the constructs.<br />
<br />
(late August)<br />
First completed vectors containing all the BioBricks & correctly ligated. Continue working on other constructs.<br />
<br />
(August 2-4)<br />
iGEM ambassador Robin Kuenzler was visiting. Presentations of iGEM concept, ETH 2005 project, work with wikis and introduction to Registry of biological parts were scheduled as was a short presentation of our project and progress in the lab. Click [[Robin's visit|here]] to see some photos.<br />
<br />
(August 1)<br />
Work intensified. First constructs are due August 15 and Jelka, Matej and Jernej moved their samples to the [http://openwetware.org/wiki/FCCT_Biochemistry_Lab FCCT Biochemistry Lab] and continue preparing constructs they are in charge of.<br />
<br />
(July 19) <br />
Supervisors are back from vacation - time for a brief overview of the lab work...<br />
Work actually started on June 7 with basic cloning, vector preparations, PCRs etc. Instructors from Roman's group were great help all the time. First experiments with ELISA detection system were already performed (looks promising). Next team meeting is in one week. iGEM ambassador expected to visit in early August.<br />
<br />
[[Ljubljana, Slovenia 2006|Back home]]</div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/ProgressLjubljana, Slovenia 2006/Progress2006-09-20T14:37:04Z<p>MDolinar: </p>
<hr />
<div>We will report here about problems and successes in our lab work. <br />
Don't expect this to be really up to date, but we'll try to give some insight into how we proceed.<br />
<br />
___________<br />
<br />
Lab work started on June 7.<br />
<br />
June - July: primer designs, vector minipreps, transformations, PCRs & control digestions.<br />
<br />
Late July: each team member was assigned a list of constructs and deadlines for their completion. <br />
<br />
August: first constructs were due August 15. They were finished towards the end of September.<br />
<br />
September: several mutations detected in some of the constructs. Repeating joining parts into complex units. Focusing on crucial constructs with confirmed sequence. First transfection of human cell line on September 19.<br />
<br />
<br />
[[Ljubljana, Slovenia 2006|Back home]]</div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Team_MembersLjubljana, Slovenia 2006/Team Members2006-09-20T11:10:02Z<p>MDolinar: </p>
<hr />
<div>[[Image:SLOteam06a.jpg]]<br />
<br />
''From left to right: Alja, Ota, Jelka, Matej, Monika, Jernej, Rok, Simon, Marko, Mojca M., Roman.<br />
''<br />
<br />
----<br />
'''STUDENTS:'''<br />
<br />
*Simon Belak (Physics)<br />
*[[User:Moni|Monika Ciglič (Microbiology)]]<br />
*[[User:Ota|Ota Fekonja]](Microbiology)<br />
*[[User:Jernej|Jernej Kovač]] (Biochemistry)<br />
*[[User:Mojca Miklavec|Mojca Miklavec]] (Physics & Bioinformatics)<br />
*[[User:Alja|Alja Oblak]] (Microbiology)<br />
*[[User:Jelka.pohar|Jelka Pohar]] (Microbiology)<br />
*[[User:Matej|Matej Skočaj]] (Microbiology)<br />
*[[User:rtkavc|Rok Tkavc (Microbiology)]]<br />
<br />
'''SUPERVISORS:'''<br />
<br />
*[[User:MDolinar|Marko Dolinar]] (Faculty of Chemistry & Chemical Technology)<br />
*Roman Jerala (National Institute of Chemistry = NIC)<br />
<br />
'''INSTRUCTORS:'''<br />
*Mojca Benčina (NIC)<br />
*Mateja Manček Keber (NIC)<br />
*Gabriela Panter (NIC)<br />
<br />
----<br />
<br><br />
[[Image:SLOteam06b.jpg]] <br />
<br />
''From left to right: Gabriela, Roman, Jelka, Jernej, Monika, Matej, Alja, Rok, Mojca M., Marko.''<br />
<br />
[[Ljubljana, Slovenia 2006|Back home]]</div>MDolinarhttp://2006.igem.org/wiki/index.php/File:SLOteam06b.jpgFile:SLOteam06b.jpg2006-09-20T10:59:49Z<p>MDolinar: </p>
<hr />
<div>SLO team (September 19, 2006)<br />
<br />
Absent: Ota, Mojca B. & Mateja</div>MDolinarhttp://2006.igem.org/wiki/index.php/File:SLOteam06b.jpgFile:SLOteam06b.jpg2006-09-20T10:55:35Z<p>MDolinar: SLO team (September 20, 2006)</p>
<hr />
<div>SLO team (September 20, 2006)</div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Team_MembersLjubljana, Slovenia 2006/Team Members2006-09-20T10:54:50Z<p>MDolinar: </p>
<hr />
<div>[[Image:SLOteam06a.jpg]]<br />
<br />
''From left to right: Alja, Ota, Jelka, Matej, Monika, Jernej, Rok, Simon, Marko, Mojca M., Roman.<br />
''<br />
<br />
----<br />
'''STUDENTS:'''<br />
<br />
*Simon Belak (Physics)<br />
*[[User:Moni|Monika Ciglič (Microbiology)]]<br />
*[[User:Ota|Ota Fekonja]](Microbiology)<br />
*[[User:Jernej|Jernej Kovač]] (Biochemistry)<br />
*[[User:Mojca Miklavec|Mojca Miklavec]] (Physics & Bioinformatics)<br />
*[[User:Alja|Alja Oblak]] (Microbiology)<br />
*[[User:Jelka.pohar|Jelka Pohar]] (Microbiology)<br />
*[[User:Matej|Matej Skočaj]] (Microbiology)<br />
*[[User:rtkavc|Rok Tkavc (Microbiology)]]<br />
<br />
'''SUPERVISORS:'''<br />
<br />
*[[User:MDolinar|Marko Dolinar]] (Faculty of Chemistry & Chemical Technology)<br />
*Roman Jerala (National Institute of Chemistry = NIC)<br />
<br />
'''INSTRUCTORS:'''<br />
*Mojca Benčina (NIC)<br />
*Mateja Manček Keber (NIC)<br />
*Gabriela Panter (NIC)<br />
<br />
[[Image:SLOteam06b.jpg]] <br />
<br />
[[Ljubljana, Slovenia 2006|Back home]]</div>MDolinarhttp://2006.igem.org/wiki/index.php/Jamboree_AttendanceJamboree Attendance2006-08-23T15:26:57Z<p>MDolinar: </p>
<hr />
<div>This page is the place to put information related to the [[Jamboree]] 2006 planning.<br />
<br />
=Attendance planning=<br />
{|| style="width:100%; height:10px" border="1"<br />
!style="background:silver"| school<br />
!style="background:silver"|number of students<br />
!style="background:silver"|number of advisors/staff<br />
!style="background:silver"|total number<br />
!style="background:silver"|accuracy of guess (according to the team)<br />
!style="background:silver"|status of travel planning (visa,...)<br />
!style="background:silver"| source of information<br />
|- <br />
| [[Cambridge_University_2006 | Cambridge]]<br />
| 6<br />
| 2-5<br />
| 8-13<br />
| <br />
| visa applications, as soon as the team is assembled<br />
| mail from Gos Micklem, [[User:Jonas|jonas]] 09:26, 7 July 2006 (EDT)<br />
|- <br />
| [[Imperial_College_2006 | Imperial College]]<br />
| 8<br />
| 2-3<br />
| 10-11<br />
| <br />
| <br />
| mail from Vincent Rouilly, [[User:Jonas|jonas]] 8 July 2006 (EDT)<br />
|- <br />
| [[University_of_Edinburgh_2006 | Edinburgh]]<br />
| 9<br />
| 2-3<br />
| 11-12<br />
| <br />
| <br />
| mail from Chris French, [[User:Tamara|Tamara]] 18 July 2006<br />
|- <br />
| [[Freiburg_University_2006 | Freiburg]]<br />
| 2-3<br />
| 1<br />
| 3-4<br />
| <br />
| <br />
| mail from Andrei Kouznetsov, [[User:Tamara|Tamara]] 18 July 2006<br />
|- <br />
| [[ETH_Zurich_2006 | ETH Zurich]]<br />
| 2-3<br />
| 9<br />
| 12<br />
| 11-12<br />
| <br />
| mail from Sven Panke, [[User:Robin|robin]] 12:16, 12 July 2006 (EDT)<br />
|- <br />
| [[UPV-UV_Valencia%2C_Spain_2006 | Valencia]]<br />
| 10<br />
| 5<br />
| 13-17<br />
| <br />
| <br />
| talked to Alfonso Jaramillo, [[User:Robin|robin]] 12:00, 12 July 2006 (EDT)<br />
|- <br />
| [[Ljubljana, Slovenia 2006 | Ljubljana]]<br />
| 8<br />
| 5<br />
| 13<br />
| good<br />
| <br />
| [[User:MDolinar|Marko]] 17:23, 23 August 2006 (CET)<br />
|-<br />
| [[Turkey_2006 | EGE Turkey]]<br />
| <br />
|<br />
| 3<br />
| <br />
| <br />
| mail from Bahattin, [[User:Robin|robin]] 13:37, 16 July 2006 (EDT)<br />
|-<br />
| [[Duke_2006 | Duke University]]<br />
| <br />
|<br />
| 12<br />
|"Better estimate will be available in fall" <br />
| <br />
|Faisal Reza, faculty organizer, --[[User:Smelissali|Smelissali]] 13:13, 2 August 2006 (EDT)<br />
|-<br />
| [[University of Arizona 2006 | Arizona]]<br />
| 10<br />
|2<br />
| 12<br />
|<br />
| <br />
|Patrick Hollinger (student team leader) --[[User:Smelissali|Smelissali]] 14:07, 4 August 2006 (EDT)<br />
|-<br />
|[[Missouri_Western_State_University_2006| Missouri]]<br />
|7<br />
|2<br />
|9<br />
|high<br />
|<br />
|Email from Todd Eckdahl 8/2 --[[User:Ahessel|Ahessel]] 13:53, 2 August 2006 (EDT)<br />
|-<br />
|[[University_of_Oklahoma_2006 | Oklahoma]]<br />
|<br />
|<br />
|4-5<br />
|<br />
|<br />
|Email from Bruce Roe 8/2 --[[User:Ahessel|Ahessel]] 14:20, 2 August 2006 (EDT)<br />
|-<br />
|[[Boston University 2006| Boston]]<br />
|16<br />
|1<br />
|17<br />
|Good<br />
|<br />
|PC Alex Kates, BU<br />
|-<br />
|[[Brown:iGEM portal 2006 |Brown]]<br />
|12-14<br />
|0-3<br />
|14-17<br />
|Students:Good, Staff:Rough<br />
|<br />
|PC- John Cumbers, Brown<br />
|-<br />
|[[University of Calgary 2006|Calgary]]<br />
|7<br />
|3<br />
|10<br />
|Very rough<br />
|<br />
|PC - Christian Jacob, Calgary<br />
|-<br />
|[[Harvard 2006|Harvard]]<br />
|10<br />
|5-8<br />
|15-18<br />
|Students: Accurate Staff: Rough<br />
|<br />
|PC - Nik Stroustrup<br />
|-<br />
|[[University of Michigan 2006|Michigan]]<br />
|8-12<br />
|3-5<br />
|11-17<br />
|Rough<br />
|<br />
|PC - Peter Woolf<br />
|-<br />
|[[MIT 2006|MIT]]<br />
|6<br />
|7<br />
|13<br />
|Good<br />
|<br />
|Obvious<br />
|-<br />
|[[Princeton 2006|Princeton]]<br />
|10-14<br />
|3-5<br />
|15-19<br />
|Good<br />
|<br />
|PC - Cil Purnick<br />
|-<br />
|[[University_of_Toronto_2006|Toronto and Waterloo]]<br />
|<br />
|<br />
|20-25<br />
|Good<br />
|<br />
|Emails from Stephen Davies and Nancy Xu --[[User:Ahessel|Ahessel]] 12:26, 8 August 2006 (EDT)<br />
|-<br />
|[[McGill_University_2006|McGill]]<br />
|<br />
|<br />
|12<br />
|Good<br />
|<br />
|Email from Jay Nadeau --[[User:Ahessel|Ahessel]] 12:26, 8 August 2006 (EDT)<br />
|-<br />
|[[Prairie_View_A%26M_University_2006|Prairie View]]<br />
|<br />
|<br />
|6<br />
|Good<br />
|<br />
|Email from Raul Cuero --[[User:Ahessel|Ahessel]] 12:26, 8 August 2006 (EDT)<br />
|-<br />
|[[Africa_2006|Africa]]<br />
|<br />
|<br />
|4-5<br />
|Good<br />
|<br />
|Email from Raul Cuero --[[User:Ahessel|Ahessel]] 12:26, 8 August 2006 (EDT)<br />
|-<br />
|[[Latin_America_2006|Latin America]]<br />
|<br />
|<br />
|11-12<br />
|Good<br />
|<br />
|Email from Raul Cuero --[[User:Ahessel|Ahessel]] 12:26, 8 August 2006 (EDT)<br />
|-<br />
|[[Penn State University 2006|Penn State]]<br />
|<br />
|<br />
|8-10<br />
|pretty good<br />
|<br />
|email from Pat Cirino, Tom Richard --[[User:Smelissali|Smelissali]] 20:42, 9 August 2006 (EDT)<br />
|-<br />
|[[University of California Berkeley 2006|UC Berkeley]]<br />
|7<br />
|2<br />
|9<br />
|quite good<br />
|<br />
|Team meeting on 8/07 --[[User:Smelissali|Smelissali]] 20:42, 9 August 2006 (EDT) <br />
|-<br />
|[[University of California San Francisco 2006|UCSF]]<br />
|4<br />
|<br />
|4<br />
|good<br />
|<br />
|team meeting on 8/08 --[[User:Smelissali|Smelissali]] 20:42, 9 August 2006 (EDT)<br />
|-<br />
|[[Rice_University_2006|Rice University]]<br />
|<br />
|<br />
|8<br />
|good<br />
|<br />
|Email from Beth Beason--[[User:Ahessel|Ahessel]] 17:03, 15 August 2006 (EDT)<br />
|-<br />
|[[IPN_UNAM_2006|IPN_UNAM]]<br />
|7 positive (15 max)<br />
|<br />
|7-15<br />
|Rough, for now<br />
|Working on getting visas<br />
|Email from Genaro --[[User:Meaganl|Meaganl]] 08:59, 17 August 2006 (EDT)<br />
|-<br />
|[school]<br />
|[#students]<br />
|[#staff]<br />
|[total attendance]<br />
|[accuracy of guess]<br />
|[issues for travel]<br />
|[source of info]<br />
|}</div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/ProjectLjubljana, Slovenia 2006/Project2006-08-13T20:25:34Z<p>MDolinar: /* Background */</p>
<hr />
<div>Although in the past years all participating iGEM teams focused on bacteria and yeast, we decided to modify signalling pathways in higher eukaryotes. This is a far more complicated system to understand but we feel that the recently accumulated knowledge enables rational planning of cells with novel properties.<br />
<br />
== Background ==<br />
<br />
When bacteria attack a human body, immune response is the normal reaction of the organism. Cells which represent the first line of defence are macrophages and the way they act is rather complicated. On the surface of these cells (actually inserted into cell membrane) are several types of receptor molecules, commonly abbreviated as TLR (toll-like receptors). These can specifically bind various components of bacteria (e.g. lipopolysaccharides which are part of bacterial outer membranes) and upon binding, they interact with other molecules inside the cell, forming a cell signalling pathway that, after several relays, ends in the cell nucleus and induces production of immune response proteins, such as cytokines and chemokines. The complete pathway is depicted [http://www.komabiotech.co.kr/technical/review/toll_like_receptor.gif here] and is rather complicated. Stages of the signal transfer were elucidated recently. Cell signalling like this is the basis of our efficient innate immune response (i.e. the fast-acting, not mediated by antibodies) against microbes. In some instances, however, signalling mediated by TLR is not beneficial. Under some not yet fully understood circumstances, bacteria can trigger this same signalling pathway just that it results in excessive inflammatory response. As result, sepsis occurs, which can lead to severe organ failure and in about 20% cases cosequences are fatal.<br />
<br />
== Signalling pathway ==<br />
After binding of bacterial constituents to TLR, its intracellular TIR-domain interacts with either MyD88 or TRIF, two signalling molecules in the cytoplasm that relay signal to other components of the pathway as shown [http://www.biken.osaka-u.ac.jp/act/images/akiraB.png here].<br />
<br />
<br />
== Idea ==<br />
<br />
<br />
<br />
== Approach ==<br />
<br />
<br />
== Anticipated results ==<br />
<br />
----<br />
<br />
<br />
''(more to follow)''<br />
<br />
[[University of Ljubljana & National Institute of Chemistry, Ljubljana, Slovenia 2006|Back home]]</div>MDolinarhttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/ProjectLjubljana, Slovenia 2006/Project2006-08-13T20:23:50Z<p>MDolinar: </p>
<hr />
<div>Although in the past years all participating iGEM teams focused on bacteria and yeast, we decided to modify signalling pathways in higher eukaryotes. This is a far more complicated system to understand but we feel that the recently accumulated knowledge enables rational planning of cells with novel properties.<br />
<br />
== Background ==<br />
<br />
When bacteria attack a human body, immune response is the normal reaction of the organism. Cells which represent the first line of defence are macrophages and the way they act is rather complicated. On the surface of these cells (actually inserted into cell membrane) are several types of receptor molecules, commonly abbreviated as TLR (toll-like receptors). These can specifically bind various components of bacteria (e.g. lipopolysaccharides which are part of bacterial outer membranes) and upon binding, they interact with other molecules inside the cell, forming a cell signalling pathway that, after several relays, ends in the cell nucleus and induces production of immune response proteins, such as cytokines and chemokines. The complete pathway is depicted [http://www.komabiotech.co.kr/technical/review/toll_like_receptor.gif here] and is rather complicated. Stages of the signal transfer were elucidated recently. Cell signalling like this is the basis of our efficient innate immune response (i.e. the fast-acting, not mediated by antibodies) against microbes. In some instances, however, signalling mediated by TLR is not beneficial. Under some not yet fully understood circumstances, bacteria can trigger this same signalling pathway which results in excessive inflamatory response. As result, sepsis occurs, which can lead to organ failure and in about 20% cosequences are fatal.<br />
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== Signalling pathway ==<br />
After binding of bacterial constituents to TLR, its intracellular TIR-domain interacts with either MyD88 or TRIF, two signalling molecules in the cytoplasm that relay signal to other components of the pathway as shown [http://www.biken.osaka-u.ac.jp/act/images/akiraB.png here].<br />
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== Idea ==<br />
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== Approach ==<br />
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== Anticipated results ==<br />
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''(more to follow)''<br />
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[[University of Ljubljana & National Institute of Chemistry, Ljubljana, Slovenia 2006|Back home]]</div>MDolinarhttp://2006.igem.org/wiki/index.php/Robin%27s_visitRobin's visit2006-08-09T14:15:01Z<p>MDolinar: 1 photo added</p>
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<div> [[Image:Macek.jpg]]<br />
Jernej, Jelka, Robin, Ota, Matej, Rok (taking the photo)<br />
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[[Image:Ljubljana's castle.jpg]]<br />
Matej, Robin, Jernej, Jelka, Ota, Rok (taking the photo)<br />
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[[Image:LJU_ambassador.jpg]]<br />
Robin, Jernej, Mojca, Rok, Monika, Alja, Ota, Jelka, Matej<br />
(photo: Marko)</div>MDolinar