http://2006.igem.org/wiki/index.php?title=Special:Contributions/Alja&feed=atom&limit=50&target=Alja&year=&month=2006.igem.org - User contributions [en]2024-03-29T02:06:12ZFrom 2006.igem.orgMediaWiki 1.16.5http://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Results_%26_ConclusionsLjubljana, Slovenia 2006/Results & Conclusions2006-11-01T19:16:45Z<p>Alja: </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 by 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|center|thumb|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 />
<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>Aljahttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Results_%26_ConclusionsLjubljana, Slovenia 2006/Results & Conclusions2006-11-01T19:15:40Z<p>Alja: </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 by 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|center|thumb|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 />
<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>Aljahttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/MethodsLjubljana, Slovenia 2006/Methods2006-10-30T19:17:23Z<p>Alja: </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/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 to 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 Signalling 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>Aljahttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Results_%26_ConclusionsLjubljana, Slovenia 2006/Results & Conclusions2006-10-30T18:24:15Z<p>Alja: </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>Phosphorylation of ERK kinases reflects the activation of the TLR signaling </h2><br />
<br />
<br style="clear:both;"/><br />
<br />
[[Image:cito1.gif|cito1.gif|left|thumb|400px|Picture 22: Levels of phosphorylated ERK kinases in dnMyD88 transfected cells]][[Image:cito3.gif|right|thumb|355px|Picture 23: Levels of Phosphorylated ERK kinases in wild type cells]]<br />
<br />
<br style="clear:both;"/><br />
<br />
Our Flow Cytometry experiments showed that 20 minute stimulation of cells transfected with dnMyD88 has little efect, but after 80 minute stimulation the increase in the level of phosphorylated ERK kinases was evident(Picture 22).<br />
Untransfected cells gave similar results, as shown on Picture 23.<br />
Further optimization of the method would be necessary in order to see a difference between dnMyD88 transfected cells and wild type.<br />
<br />
<h2>Inducible transcription by 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. This promoter is inducible and we detected the R-luciferase activity only in stimulated cells with significant activity appearing a few hours after the cell stimulation.</p><br />
<p>We also measured F-luciferase activity. We expected a decrease in activity after a certain amount of time. We did not detect it, which could be due to our experiment not lasting long enough or perhaps the fusion of dnMyD88 with the renilla luciferase stericali hindered the activity of dnMyD88. That is why our future experiments were conducted only with dnMyD88.<br />
<br />
<br style="clear:both;"/><br />
<br />
<center><br />
[[Image:graf_gabi.gif|left|thumb|400px|Figure 19: <b>Transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with Rluc reporter is induced by bacterial flagellin </b> Activity of Rluciferase 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.]]</center><br />
[[Image:graf17.gif|right|thumb|600px|Figure 20:Transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with Rluc reporter is induced by bacterial flagellin. Activity of Fluciferase stimulated by the addition of two pulses of [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]]. ]]<br />
<br />
<br style="clear:both;"/><br />
<br />
<h2>Inhibition of cell signaling by a feedback device</h2><br />
<br />
<p>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 signalling 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 will 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.jpg|center|thumb|450px|Figure 20:<b> [[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 />
<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 />
<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|Picture 21: <p><b>Testing the effect of PEST fusions</b></p>]]<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 />
<h2>Detection of free NF-kB in whole cell extracts dependent on time after stimulation with LPS with ELISA method</h2><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>Aljahttp://2006.igem.org/wiki/index.php/File:Graf17.gifFile:Graf17.gif2006-10-30T18:19:51Z<p>Alja: </p>
<hr />
<div></div>Aljahttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Results_%26_ConclusionsLjubljana, Slovenia 2006/Results & Conclusions2006-10-30T18:18:38Z<p>Alja: </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>Phosphorylation of ERK kinases reflects the activation of the TLR signaling </h2><br />
<br />
[[Image:cito1.gif|cito1.gif|left|thumb|400px|Picture 22: Levels of phosphorylated ERK kinases in dnMyD88 transfected cells]][[Image:cito3.gif|right|thumb|355px|Picture 23: Levels of Phosphorylated ERK kinases in wild type cells]]<br />
Our Flow Cytometry experiments showed that 20 minute stimulation of cells transfected with dnMyD88 has little efect, but after 80 minute stimulation the increase in the level of phosphorylated ERK kinases was evident(Picture 22).<br />
Untransfected cells gave similar results, as shown on Picture 23.<br />
Further optimization of the method would be necessary in order to see a difference between dnMyD88 transfected cells and wild type.<br />
<br />
<h2>Inducible transcription by 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. This promoter is inducible and we detected the R-luciferase activity only in stimulated cells with significant activity appearing a few hours after the cell stimulation.</p><br />
<p>We also measured F-luciferase activity. We expected a decrease in activity after a certain amount of time. We did not detect it, which could be due to our experiment not lasting long enough or perhaps the fusion of dnMyD88 with the renilla luciferase stericali hindered the activity of dnMyD88. That is why our future experiments were conducted only with dnMyD88.<br />
<br />
<br />
<center><br />
[[Image:graf_gabi.gif|left|thumb|450px|Figure 19: <b>Transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with Rluc reporter is induced by bacterial flagellin </b> Activity of Rluciferase stimulated by the addition of two pulses of [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]] (at 0 and 4 hours). [[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.]]</center><br />
[[Image:graf17.gif|right|thumb|450px|Figure 20:]]<br />
<br />
<h2>Inhibition of cell signaling by a feedback device</h2><br />
<br />
<p>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 signalling 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 will 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.jpg|center|thumb|450px|Figure 20:<b> [[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 />
<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 />
<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|Picture 21: <p><b>Testing the effect of PEST fusions</b></p>]]<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 />
<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>Aljahttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Results_%26_ConclusionsLjubljana, Slovenia 2006/Results & Conclusions2006-10-30T18:10:48Z<p>Alja: </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>Phosphorylation of ERK kinases reflects the activation of the TLR signaling </h2><br />
<br />
[[Image:cito1.gif|cito1.gif|left|thumb|400px|Picture 22: Levels of phosphorylated ERK kinases in dnMyD88 transfected cells]][[Image:cito3.gif|right|thumb|355px|Picture 23: Levels of Phosphorylated ERK kinases in wild type cells]]<br />
Our Flow Cytometry experiments showed that 20 minute stimulation of cells transfected with dnMyD88 has little efect, but after 80 minute stimulation the increase in the level of phosphorylated ERK kinases was evident(Picture 22).<br />
Untransfected cells gave similar results, as shown on Picture 23.<br />
Further optimization of the method would be necessary in order to see a difference between dnMyD88 transfected cells and wild type.<br />
<br />
<h2>Inducible transcription by 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. This promoter is inducible and we detected the R-luciferase activity only in stimulated cells with significant activity appearing a few hours after the cell stimulation.</p><br />
<p>We also measured F-luciferase activity. We expected a decrease in activity after a certain amount of time. We did not detect it, which could be due to our experiment not lasting long enough or perhaps the fusion of dnMyD88 with the renilla luciferase stericali hindered the activity of dnMyD88. That is why our future experiments were conducted only with dnMyD88.<br />
<br />
<br />
<center><br />
[[Image:graf_gabi.gif|left|thumb|450px|Figure 19: <b>Transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with Rluc reporter is induced by bacterial flagellin </b> Activity of Rluciferase stimulated by the addition of two pulses of [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]] (at 0 and 4 hours). [[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.]]</center><br />
<br />
<h2>Inhibition of cell signaling by a feedback device</h2><br />
<br />
<p>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 signalling 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 will 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.jpg|center|thumb|450px|Figure 20:<b> [[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 />
<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 />
<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|Picture 21: <p><b>Testing the effect of PEST fusions</b></p>]]<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 />
<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>Aljahttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Results_%26_ConclusionsLjubljana, Slovenia 2006/Results & Conclusions2006-10-30T18:10:12Z<p>Alja: </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>Phosphorylation of ERK kinases reflects the activation of the TLR signaling </h2><br />
<br />
[[Image:cito1.gif|cito1.gif|left|thumb|400px|Picture 22: Levels of phosphorylated ERK kinases in dnMyD88 transfected cells]][[Image:cito3.gif|right|thumb|355px|Picture 23: Levels of Phosphorylated ERK kinases in wild type cells]]<br />
Our Flow Cytometry experiments showed that 20 minute stimulation of cells transfected with dnMyD88 has little efect, but after 80 minute stimulation the increase in the level of phosphorylated ERK kinases was evident(Picture 22).<br />
Untransfected cells gave similar results, as shown on Picture 23.<br />
Further optimization of the method would be necessary in order to see a difference between dnMyD88 transfected cells and wild type.<br />
<br />
<h2>Inducible transcription by 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. This promoter is inducible and we detected the R-luciferase activity only in stimulated cells with significant activity appearing a few hours after the cell stimulation.</p><br />
<p>We also measured F-luciferase activity. We expected a decrease in activity after a certain amount of time. We did not detect it, which could be due to our experiment not lasting long enough or perhaps the fusion of dnMyD88 with the renilla luciferase stericali hindered the activity of dnMyD88. That is why our future experiments were conducted only with dnMyD88.<br />
<br />
<br />
<center><br />
[[Image:graf_gabi.gif|center|thumb|450px|Figure 19: <b>Transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with Rluc reporter is induced by bacterial flagellin </b> Activity of Rluciferase stimulated by the addition of two pulses of [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]] (at 0 and 4 hours). [[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.]]</center><br />
<br />
<h2>Inhibition of cell signaling by a feedback device</h2><br />
<br />
<p>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 signalling 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 will 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.jpg|center|thumb|450px|Figure 20:<b> [[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 />
<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 />
<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|Picture 21: <p><b>Testing the effect of PEST fusions</b></p>]]<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 />
<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>Aljahttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Results_%26_ConclusionsLjubljana, Slovenia 2006/Results & Conclusions2006-10-30T17:58:03Z<p>Alja: </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>Phosphorylation of ERK kinases reflects the activation of the TLR signaling </h2><br />
<br />
[[Image:cito1.gif|cito1.gif|left|thumb|400px|Picture 22: Levels of phosphorylated ERK kinases in dnMyD88 transfected cells]][[Image:cito3.gif|right|thumb|355px|Picture 23: Levels of Phosphorylated ERK kinases in wild type cells]]<br />
Our Flow Cytometry experiments showed that 20 minute stimulation of cells transfected with dnMyD88 has little efect, but after 80 minute stimulation the increase in the level of phosphorylated ERK kinases was evident(Picture 22).<br />
Untransfected cells gave similar results, as shown on Picture 23.<br />
Further optimization of the method would be necessary in order to see a difference between dnMyD88 transfected cells and wild type.<br />
<br />
<h2>Inducible transcription by 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 gene activation and 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. This promoter is inducible as we have detected the R-luciferase activity only in stimulated cells with significant activity appearing four hours after the cell stimulation.</p><br />
<br />
<br />
<center><br />
[[Image:graf_gabi.gif|center|thumb|450px|Figure 19: <b>Transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with Rluc reporter is induced by bacterial flagellin </b> Activity of Rluciferase stimulated by the addition of two pulses of [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]] (at 0 and 4 hours). [[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.]]</center><br />
<br />
<h2>Inhibition of cell signaling by a feedback device</h2><br />
<br />
<p>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 signalling 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 will 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.jpg|center|thumb|450px|Figure 20:<b> [[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 />
<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 />
<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|Picture 21: <p><b>Testing the effect of PEST fusions</b></p>]]<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 />
<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>Aljahttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Results_%26_ConclusionsLjubljana, Slovenia 2006/Results & Conclusions2006-10-30T17:57:15Z<p>Alja: </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>Phosphorylation of ERK kinases reflects the activation of the TLR signaling </h2><br />
<br />
[[Image:cito1.gif|cito1.gif|left|thumb|400px|Picture 22: Levels of phosphorylated ERK kinases in dnMyD88 transfected cells]][[Image:cito3.gif|right|thumb|355px|Picture 23: Levels of Phosphorylated ERK kinases in wild type cells]]<br />
Our Flow Cytometry experiments showed that 20 minute stimulation of cells transfected with dnMyD88 has little efect, but after 80 minute stimulation the increase in the level of phosphorylated ERK kinases was evident(Picture 22).<br />
Untransfected cells gave similar results, as shown on Picture 23.<br />
Further optimization of the method would be necessary in order to see a difference between dnMyD88 transfected cells and wild type.<br />
<br />
<h2>Inducible transcription by 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 gene activation and 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. This promoter is inducible as we have detected the R-luciferase activity only in stimulated cells with significant activity appearing four hours after the cell stimulation.</p><br />
<br />
<br />
<center><br />
[[Image:graf_gabi.gif|center|thumb|450px|Figure 19: <b>Transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with Rluc reporter is induced by bacterial flagellin </b> Activity of Rluciferase stimulated by the addition of two pulses of [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]] (at 0 and 4 hours). [[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.]]</center><br />
<br />
<h2>Inhibition of cell signaling by a feedback device</h2><br />
<br />
<p>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 signalling 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 will 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.jpg|center|thumb|450px|Figure 20:<b> [[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 />
<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 />
<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|Picture 21: <p><b>Testing the effect of PEST fusions</b></p>]]<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 />
<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>Aljahttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Results_%26_ConclusionsLjubljana, Slovenia 2006/Results & Conclusions2006-10-30T17:56:49Z<p>Alja: </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>Phosphorylation of ERK kinases reflects the activation of the TLR signaling </h2><br />
<br />
[[Image:cito1.gif|cito1.gif|left|thumb|400px|Picture 22: Levels of phosphorylated ERK kinases in dnMyD88 transfected cells]][[Image:cito3.gif|right|thumb|355px|Picture 23: Levels of Phosphorylated ERK kinases in wild type cells]]<br />
Our Flow Cytometry experiments showed that 20 minute stimulation of cells transfected with dnMyD88 has little efect, but after 80 minute stimulation the increase in the level of phosphorylated ERK kinases was evident(Picture 22).<br />
Untransfected cells gave similar results, as shown on Picture 23.<br />
Further optimization of the method would be necessary in order to see a difference between dnMyD88 transfected cells and wild type.<br />
<br />
<h2>Inducible transcription by 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 gene activation and 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. This promoter is inducible as we have detected the R-luciferase activity only in stimulated cells with significant activity appearing four hours after the cell stimulation.</p><br />
<br />
<br />
<center><br />
[[Image:graf_gabi.gif|center|thumb|450px|Figure 19: <b>Transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with Rluc reporter is induced by bacterial flagellin </b> Activity of Rluciferase stimulated by the addition of two pulses of [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]] (at 0 and 4 hours). [[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.]]</center><br />
<br />
<h2>Inhibition of cell signaling by a feedback device</h2><br />
<br />
<p>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 signalling 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 will 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.jpg|center|thumb|450px|Figure 20:<b> [[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 />
<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 />
<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|Picture 21: <p><b>Testing the effect of PEST fusions</b></p>]]<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 />
<h2>Phosphorylation of ERK kinases reflects the activation of the TLR signaling </h2><br />
<br />
[[Image:cito1.gif|cito1.gif|left|thumb|400px|Picture 22: Levels of phosphorylated ERK kinases in dnMyD88 transfected cells]][[Image:cito3.gif|right|thumb|355px|Picture 23: Levels of Phosphorylated ERK kinases in wild type cells]]<br />
Our Flow Cytometry experiments showed that 20 minute stimulation of cells transfected with dnMyD88 has little efect, but after 80 minute stimulation the increase in the level of phosphorylated ERK kinases was evident(Picture 22).<br />
Untransfected cells gave similar results, as shown on Picture 23.<br />
Further optimization of the method would be necessary in order to see a difference between dnMyD88 transfected cells and wild type.<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>Aljahttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Results_%26_ConclusionsLjubljana, Slovenia 2006/Results & Conclusions2006-10-30T17:56:28Z<p>Alja: </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>Phosphorylation of ERK kinases reflects the activation of the TLR signaling </h2><br />
<br />
[[Image:cito1.gif|cito1.gif|left|thumb|400px|Picture 22: Levels of phosphorylated ERK kinases in dnMyD88 transfected cells]][[Image:cito3.gif|right|thumb|355px|Picture 23: Levels of Phosphorylated ERK kinases in wild type cells]]<br />
Our Flow Cytometry experiments showed that 20 minute stimulation of cells transfected with dnMyD88 has little efect, but after 80 minute stimulation the increase in the level of phosphorylated ERK kinases was evident(Picture 22).<br />
Untransfected cells gave similar results, as shown on Picture 23.<br />
Further optimization of the method would be necessary in order to see a difference between dnMyD88 transfected cells and wild type.<br />
<br />
<h2>Inducible transcription by 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 gene activation and 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. This promoter is inducible as we have detected the R-luciferase activity only in stimulated cells with significant activity appearing four hours after the cell stimulation.</p><br />
<br />
<br />
<center><br />
[[Image:graf_gabi.gif|center|thumb|450px|Figure 19: <b>Transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with Rluc reporter is induced by bacterial flagellin </b> Activity of Rluciferase stimulated by the addition of two pulses of [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]] (at 0 and 4 hours). [[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.]]</center><br />
<br />
<div style="float:left; background-color:#cedff2; padding: .2em .6em; border: 1px solid black">'''<br />
<br />
<h2>Inhibition of cell signaling by a feedback device</h2><br />
<br />
<p>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 signalling 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 will 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.jpg|center|thumb|450px|Figure 20:<b> [[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 />
<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 />
<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|Picture 21: <p><b>Testing the effect of PEST fusions</b></p>]]<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 />
<h2>Phosphorylation of ERK kinases reflects the activation of the TLR signaling </h2><br />
<br />
[[Image:cito1.gif|cito1.gif|left|thumb|400px|Picture 22: Levels of phosphorylated ERK kinases in dnMyD88 transfected cells]][[Image:cito3.gif|right|thumb|355px|Picture 23: Levels of Phosphorylated ERK kinases in wild type cells]]<br />
Our Flow Cytometry experiments showed that 20 minute stimulation of cells transfected with dnMyD88 has little efect, but after 80 minute stimulation the increase in the level of phosphorylated ERK kinases was evident(Picture 22).<br />
Untransfected cells gave similar results, as shown on Picture 23.<br />
Further optimization of the method would be necessary in order to see a difference between dnMyD88 transfected cells and wild type.<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>Aljahttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Results_%26_ConclusionsLjubljana, Slovenia 2006/Results & Conclusions2006-10-30T17:52:42Z<p>Alja: </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>Phosphorylation of ERK kinases reflects the activation of the TLR signaling </h2><br />
<br />
[[Image:cito1.gif|cito1.gif|left|thumb|400px|Picture 22: Levels of phosphorylated ERK kinases in dnMyD88 transfected cells]][[Image:cito3.gif|right|thumb|355px|Picture 23: Levels of Phosphorylated ERK kinases in wild type cells]]<br />
Our Flow Cytometry experiments showed that 20 minute stimulation of cells transfected with dnMyD88 has little efect, but after 80 minute stimulation the increase in the level of phosphorylated ERK kinases was evident(Picture 22).<br />
Untransfected cells gave similar results, as shown on Picture 23.<br />
Further optimization of the method would be necessary in order to see a difference between dnMyD88 transfected cells and wild type.<br />
<br />
<br />
<div style="float:left; background-color:#cedff2; padding: .2em .6em; border: 1px solid black">'''<br />
<br />
<br />
<h2>Inducible transcription by 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 gene activation and 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. This promoter is inducible as we have detected the R-luciferase activity only in stimulated cells with significant activity appearing four hours after the cell stimulation.</p><br />
<br />
<br />
<center><br />
[[Image:graf_gabi.gif|center|thumb|450px|Figure 19: <b>Transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with Rluc reporter is induced by bacterial flagellin </b> Activity of Rluciferase stimulated by the addition of two pulses of [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]] (at 0 and 4 hours). [[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.]]</center><br />
<br />
<div style="float:left; background-color:#cedff2; padding: .2em .6em; border: 1px solid black">'''<br />
<br />
<h2>Inhibition of cell signaling by a feedback device</h2><br />
<br />
<p>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 signalling 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 will 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.jpg|center|thumb|450px|Figure 20:<b> [[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 />
<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 />
<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|Picture 21: <p><b>Testing the effect of PEST fusions</b></p>]]<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 />
<h2>Phosphorylation of ERK kinases reflects the activation of the TLR signaling </h2><br />
<br />
[[Image:cito1.gif|cito1.gif|left|thumb|400px|Picture 22: Levels of phosphorylated ERK kinases in dnMyD88 transfected cells]][[Image:cito3.gif|right|thumb|355px|Picture 23: Levels of Phosphorylated ERK kinases in wild type cells]]<br />
Our Flow Cytometry experiments showed that 20 minute stimulation of cells transfected with dnMyD88 has little efect, but after 80 minute stimulation the increase in the level of phosphorylated ERK kinases was evident(Picture 22).<br />
Untransfected cells gave similar results, as shown on Picture 23.<br />
Further optimization of the method would be necessary in order to see a difference between dnMyD88 transfected cells and wild type.<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>Aljahttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Results_%26_ConclusionsLjubljana, Slovenia 2006/Results & Conclusions2006-10-30T17:51:11Z<p>Alja: </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>Phosphorylation of ERK kinases reflects the activation of the TLR signaling </h2><br />
<br />
[[Image:cito1.gif|cito1.gif|left|thumb|400px|Picture 22: Levels of phosphorylated ERK kinases in dnMyD88 transfected cells]][[Image:cito3.gif|right|thumb|355px|Picture 23: Levels of Phosphorylated ERK kinases in wild type cells]]<br />
Our Flow Cytometry experiments showed that 20 minute stimulation of cells transfected with dnMyD88 has little efect, but after 80 minute stimulation the increase in the level of phosphorylated ERK kinases was evident(Picture 22).<br />
Untransfected cells gave similar results, as shown on Picture 23.<br />
Further optimization of the method would be necessary in order to see a difference between dnMyD88 transfected cells and wild type.<br />
<br />
<br />
<div style="float:left; background-color:#cedff2; padding: .2em .6em; border: 1px solid black">'''<br />
<br />
<br />
<h2>Inducible transcription by 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 gene activation and 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. This promoter is inducible as we have detected the R-luciferase activity only in stimulated cells with significant activity appearing four hours after the cell stimulation.</p><br />
<br />
<br />
<center><br />
[[Image:graf_gabi.gif|center|thumb|450px|Figure 19: <b>Transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with Rluc reporter is induced by bacterial flagellin </b> Activity of Rluciferase stimulated by the addition of two pulses of [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]] (at 0 and 4 hours). [[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.]]</center><br />
<br />
<h2>Inhibition of cell signaling by a feedback device</h2><br />
<br />
<p>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 signalling 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 will 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.jpg|center|thumb|450px|Figure 20:<b> [[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 />
<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 />
<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|Picture 21: <p><b>Testing the effect of PEST fusions</b></p>]]<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 />
<h2>Phosphorylation of ERK kinases reflects the activation of the TLR signaling </h2><br />
<br />
[[Image:cito1.gif|cito1.gif|left|thumb|400px|Picture 22: Levels of phosphorylated ERK kinases in dnMyD88 transfected cells]][[Image:cito3.gif|right|thumb|355px|Picture 23: Levels of Phosphorylated ERK kinases in wild type cells]]<br />
Our Flow Cytometry experiments showed that 20 minute stimulation of cells transfected with dnMyD88 has little efect, but after 80 minute stimulation the increase in the level of phosphorylated ERK kinases was evident(Picture 22).<br />
Untransfected cells gave similar results, as shown on Picture 23.<br />
Further optimization of the method would be necessary in order to see a difference between dnMyD88 transfected cells and wild type.<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>Aljahttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Results_%26_ConclusionsLjubljana, Slovenia 2006/Results & Conclusions2006-10-30T17:31:06Z<p>Alja: </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>Inducible transcription by 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 gene activation and 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. This promoter is inducible as we have detected the R-luciferase activity only in stimulated cells with significant activity appearing four hours after the cell stimulation.</p><br />
<br />
<br />
<center><br />
[[Image:rezultati2a.jpg|center|thumb|450px|Figure 19: <b>Transcription of [[Ljubljana, Slovenia 2006/Terms & References#Terms|dnMyD88]] with Rluc reporter is induced by bacterial flagellin </b> Activity of Rluciferase stimulated by the addition of two pulses of [[Ljubljana, Slovenia 2006/Terms & References#Terms|flagellin]] (at 0 and 4 hours). [[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.]]</center><br />
<br />
<h2>Inhibition of cell signaling by a feedback device</h2><br />
<br />
<p>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 signalling 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 will 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.jpg|center|thumb|450px|Figure 20:<b> [[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 />
<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 />
<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 />
[[Image:graf1.gif|center|800px|]]<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 />
<h2>Phosphorylation of ERK kinases reflects the activation of the TLR signaling </h2><br />
<br />
[[Image:cito1.gif|cito1.gif]][[Image:cito3.gif]]<br />
Our Flow Cytometry experiments showed that 20 minute stimulation of cells transfected with dnMyD88 has little efect, but after 80 minute stimulation the increase in the level of phosphorylated ERK kinases was evident(left chart).<br />
Untransfected cells gave similar results, as shown on the right chart.<br />
Further optimization of the method would be necessary in order to see a difference between dnMyD88 transfected cells and wild type.<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>Aljahttp://2006.igem.org/wiki/index.php/User:AljaUser:Alja2006-10-29T16:45:24Z<p>Alja: </p>
<hr />
<div>== Alja Oblak ==<br />
[[Image:Alja.jpg]]<br />
<br />
<br />
Member of the [http://2006.igem.org/wiki/index.php/University_of_Ljubljana_%26_National_Institute_of_Chemistry%2C_Ljubljana%2C_Slovenia_2006 iGEM 2006 team from Slovenia]<br />
<br />
<br />
[http://www.uni-lj.si University of Ljubljana]<br />
<br />
[http://www.bf.uni-lj.si Biotechnical faculty]<br />
<br />
Microbiology, 4th year<br />
<br />
e-mail: alja.oblak@campus.bf.uni-lj.si<br />
<br />
<br />
<br />
<br />
Interests: [http://dgt-si.org/index.html music]</div>Aljahttp://2006.igem.org/wiki/index.php/User:AljaUser:Alja2006-08-10T10:03:20Z<p>Alja: </p>
<hr />
<div>== Alja Oblak ==<br />
[[Image:Alja.jpg]]<br />
<br />
<br />
Member of the [http://2006.igem.org/wiki/index.php/University_of_Ljubljana_%26_National_Institute_of_Chemistry%2C_Ljubljana%2C_Slovenia_2006 iGEM 2006 team from Slovenia]<br />
<br />
<br />
[http://www.uni-lj.si University of Ljubljana]<br />
<br />
[http://www.bf.uni-lj.si Biotechnical faculty]<br />
<br />
Microbiology, 3rd year<br />
<br />
e-mail: alja.oblak@campus.bf.uni-lj.si<br />
<br />
<br />
<br />
<br />
Interests: [http://dgt-si.org/index.html music]</div>Aljahttp://2006.igem.org/wiki/index.php/User:AljaUser:Alja2006-08-10T10:01:27Z<p>Alja: </p>
<hr />
<div>== Alja Oblak ==<br />
[[Image:Alja.jpg]]<br />
<br />
<br />
Member of the [http://2006.igem.org/wiki/index.php/University_of_Ljubljana_%26_National_Institute_of_Chemistry%2C_Ljubljana%2C_Slovenia_2006 iGEM 2006 team from Slovenia]<br />
<br />
<br />
[http://www.uni-lj.si University of Ljubljana]<br />
<br />
[http://www.bf.uni-lj.si Biotechnical faculty]<br />
<br />
Microbiology, 3rd year<br />
<br />
<br />
<br />
<br />
Interests: [http://dgt-si.org/index.html music]</div>Aljahttp://2006.igem.org/wiki/index.php/User:AljaUser:Alja2006-08-09T10:51:44Z<p>Alja: </p>
<hr />
<div><br />
== Alja Oblak ==<br />
[[Image:Alja.jpg]]<br />
<br />
<br />
Member of the [http://2006.igem.org/wiki/index.php/University_of_Ljubljana_%26_National_Institute_of_Chemistry%2C_Ljubljana%2C_Slovenia_2006 iGEM 2006 team from Slovenia]<br />
<br />
<br />
[http://www.uni-lj.si University of Ljubljana]<br />
<br />
[http://www.bf.uni-lj.si Biotechnical faculty]<br />
<br />
Microbiology, 3rd year</div>Aljahttp://2006.igem.org/wiki/index.php/File:Alja.jpgFile:Alja.jpg2006-08-05T15:14:19Z<p>Alja: </p>
<hr />
<div></div>Aljahttp://2006.igem.org/wiki/index.php/User:AljaUser:Alja2006-08-05T15:10:11Z<p>Alja: </p>
<hr />
<div>== Alja Oblak ==<br />
<br />
<br />
Member of the [http://2006.igem.org/wiki/index.php/University_of_Ljubljana_%26_National_Institute_of_Chemistry%2C_Ljubljana%2C_Slovenia_2006 iGEM 2006 team from Slovenia]<br />
<br />
<br />
[http://www.uni-lj.si University of Ljubljana]<br />
<br />
[http://www.bf.uni-lj.si Biotechnical faculty]<br />
<br />
Microbiology, 3rd year</div>Aljahttp://2006.igem.org/wiki/index.php/User:AljaUser:Alja2006-08-05T15:07:14Z<p>Alja: </p>
<hr />
<div>== Alja Oblak ==<br />
<br />
<br />
Member of the [http://2006.igem.org/wiki/index.php/University_of_Ljubljana_%26_National_Institute_of_Chemistry%2C_Ljubljana%2C_Slovenia_2006 iGEM 2006 team from Slovenia]<br />
<br />
<br />
[http://www.uni-lj.si University of Ljubljana]<br />
<br />
<br />
[http://www.bf.uni-lj.si Biotechnical faculty]<br />
<br />
Microbiology, 3rd year</div>Aljahttp://2006.igem.org/wiki/index.php/User:AljaUser:Alja2006-08-05T15:04:10Z<p>Alja: </p>
<hr />
<div>== Alja Oblak ==<br />
<br />
<br />
Member of the iGEM 2006 team from Slovenia<br />
[http://2006.igem.org/wiki/index.php/University_of_Ljubljana_%26_National_Institute_of_Chemistry%2C_Ljubljana%2C_Slovenia_2006]<br />
<br />
<br />
University of Ljubljana[http://www.uni-lj.si]<br />
<br />
<br />
Biotechnical faculty[http://www.bf.uni-lj.si]<br />
<br />
Microbiology, 3rd year</div>Aljahttp://2006.igem.org/wiki/index.php/User:AljaUser:Alja2006-08-04T07:36:50Z<p>Alja: </p>
<hr />
<div>== Headline text ==<br />
Alja Oblak<br />
<br />
Member of the iGEM 2006 team from Slovenia<br />
[http://2006.igem.org/wiki/index.php/University_of_Ljubljana_%26_National_Institute_of_Chemistry%2C_Ljubljana%2C_Slovenia_2006]<br />
<br />
<br />
University of Ljubljana[http://www.uni-lj.si]<br />
<br />
<br />
Biotechnical faculty[http://www.bf.uni-lj.si]<br />
<br />
Microbiology, 3rd year</div>Aljahttp://2006.igem.org/wiki/index.php/User:AljaUser:Alja2006-08-04T07:36:16Z<p>Alja: </p>
<hr />
<div>== Headline text ==<br />
Alja Oblak<br />
<br />
Member of the iGEM 2006 team from Slovenia<br />
[http://2006.igem.org/wiki/index.php/University_of_Ljubljana_%26_National_Institute_of_Chemistry%2C_Ljubljana%2C_Slovenia_2006]<br />
<br />
<br />
University of Ljubljana[[http://www.uni-lj.si]]<br />
<br />
<br />
Biotechnical faculty[[http://www.bf.uni-lj.si]]<br />
<br />
Microbiology, 3rd year</div>Aljahttp://2006.igem.org/wiki/index.php/User:AljaUser:Alja2006-08-04T07:35:15Z<p>Alja: </p>
<hr />
<div>== Headline text ==<br />
Alja Oblak<br />
<br />
Member of the iGEM 2006 team from Slovenia<br />
[http://2006.igem.org/wiki/index.php/University_of_Ljubljana_%26_National_Institute_of_Chemistry%2C_Ljubljana%2C_Slovenia_2006]<br />
<br />
<br />
University of Ljubljana<br />
<br />
Biotechnical faculty[[http://www.bf.uni-lj.si]]<br />
<br />
Microbiology, 3rd year</div>Aljahttp://2006.igem.org/wiki/index.php/Ljubljana,_Slovenia_2006/Team_MembersLjubljana, Slovenia 2006/Team Members2006-08-04T07:33:19Z<p>Alja: </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 />
*Monika Ciglič (Microbiology)<br />
*Ota Fekonja (Microbiology)<br />
*Jernej Kovač (Biochemistry)<br />
*Mojca Miklavec (Physics & Bioinformatics)<br />
*[[User:Alja|Alja Oblak]] (Microbiology)<br />
*Jelka Pohar (Microbiology)<br />
*Matej Skočaj (Microbiology)<br />
*[[User:rtkavc|Rok Tkavc (Microbiology)]]<br />
<br />
'''SUPERVISORS:'''<br />
<br />
*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 />
[http://2006.igem.org/University_of_Ljubljana_%26_National_Institute_of_Chemistry%2C_Ljubljana%2C_Slovenia_2006 Back home]</div>Aljahttp://2006.igem.org/wiki/index.php/User:AljaUser:Alja2006-08-04T07:28:38Z<p>Alja: </p>
<hr />
<div>== Headline text ==<br />
Alja Oblak<br />
<br />
Member of the iGEM 2006 team from Slovenia<br />
[http://2006.igem.org/wiki/index.php/University_of_Ljubljana_%26_National_Institute_of_Chemistry%2C_Ljubljana%2C_Slovenia_2006]<br />
<br />
University of Ljubljana<br />
<br />
Biotechnical faculty<br />
<br />
Microbiology, 3rd year</div>Alja