http://2006.igem.org/wiki/index.php?title=Berkeley2006-PromoterMain&feed=atom&action=historyBerkeley2006-PromoterMain - Revision history2024-03-29T11:38:00ZRevision history for this page on the wikiMediaWiki 1.16.5http://2006.igem.org/wiki/index.php?title=Berkeley2006-PromoterMain&diff=20077&oldid=prevJCAnderson: /* */2006-10-30T17:17:59Z<p><span class="autocomment"></span></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 17:17, 30 October 2006</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 13:</td>
<td colspan="2" class="diff-lineno">Line 13:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Berkeley2006_Promoter.GIF]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Berkeley2006_Promoter.GIF]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We therefore developed a set of constitutive promoter parts that vary the transcription rate. The matching of rates can then be achieved by identifying a promoter that satisfies the system's requirements. To make these parts, we adopted a combinatorial approach. A synthetic promoter sequence was targetted with saturation mutagenesis employing a binary swap of the -10 and -35 sequences. Here, 8 positions were simultaneously varied to be either the consensus sequence for that position, or not. We constructed the library fused to an RFP reporter gene containing a strong ribosome binding site, the mRFP1 gene, and a terminator. We inserted the 256 member library into the RFP reporter plasmid and picked 96 visibly white colonies and 96 visibly red colonies. The cells were grown to saturation and then assayed for fluorescence. From this screen, we identified unique promoter variants <del class="diffchange diffchange-inline">that </del>each <del class="diffchange diffchange-inline">display </del>a different level of <del class="diffchange diffchange-inline">fluorescence </del>activity spanning the physiological range between the level observed with cells containing no mRFP gene and values higher than those observed with a Ptet promoter (r0040). Additionally, we constructed the "consensus" promoter basic part, the strongest constitutive promoter. These parts will find general use in tuning the expression level of Biobrick devices. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We therefore developed a set of constitutive promoter parts that vary the transcription rate. The matching of rates can then be achieved by identifying a promoter that satisfies the system's requirements. To make these parts, we adopted a combinatorial approach. A synthetic promoter sequence was targetted with saturation mutagenesis employing a binary swap of the -10 and -35 sequences. Here, 8 positions were simultaneously varied to be either the consensus sequence for that position, or not. We constructed the library fused to an RFP reporter gene containing a strong ribosome binding site, the mRFP1 gene, and a terminator. We inserted the 256 member library into the RFP reporter plasmid and picked 96 visibly white colonies and 96 visibly red colonies. The cells were grown to saturation and then assayed for fluorescence. From this screen, we identified unique promoter variants <ins class="diffchange diffchange-inline">(parts J23100-J23119) </ins>each <ins class="diffchange diffchange-inline">displaying </ins>a different level of activity spanning the physiological range between the level observed with cells containing no mRFP gene and values higher than those observed with a Ptet promoter (r0040). Additionally, we constructed the "consensus" promoter basic part, the strongest constitutive promoter. These parts will find general use in tuning the expression level of Biobrick devices. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>----</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>----</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Next Section: [[Berkeley2006-PlasmidPartsMain | New Biobrick-Compatible Plasmids]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Next Section: [[Berkeley2006-PlasmidPartsMain | New Biobrick-Compatible Plasmids]]</div></td></tr>
</table>JCAndersonhttp://2006.igem.org/wiki/index.php?title=Berkeley2006-PromoterMain&diff=19315&oldid=prevJCAnderson: /* */2006-10-29T18:14:19Z<p><span class="autocomment"></span></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 18:14, 29 October 2006</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 9:</td>
<td colspan="2" class="diff-lineno">Line 9:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== ===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== ===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Isolatation of a family of constitutive promoter parts from a combinatorial library to vary transcription rates'''<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Isolatation of a family of constitutive promoter parts from a combinatorial library to vary transcription rates'''<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>When placing genes under new regulation, the rates of transcription and translation must be tuned to match the levels necessary for activation of the device. In other words, too much protein in the "off" state can lead to a situation where the observed phenotype occurs <del class="diffchange diffchange-inline">independent </del>of the key. Similarly, too little protein in the "on" state can result in a scenario where the bacteria do not conjugate even when the key is present. When controlling a gene transcriptionally, this matching of rates can be achieved by manipulating the ribosome binding site of the gene. In translation control, however, the sequence of the ribosome binding site is an essential feature of the mode of regulation and cannot be changed independently. In this scenario, the transcription rate of the device must instead be changed.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>When placing genes under new regulation, the rates of transcription and translation must be tuned to match the levels necessary for activation of the device. In other words, too much protein in the "off" state can lead to a situation where the observed phenotype occurs <ins class="diffchange diffchange-inline">independently </ins>of the key. Similarly, too little protein in the "on" state can result in a scenario where the bacteria do not conjugate even when the key is present. When controlling a gene transcriptionally, this matching of rates can be achieved by manipulating the ribosome binding site of the gene. In translation control, however, the sequence of the ribosome binding site is an essential feature of the mode of regulation and cannot be changed independently. In this scenario, the transcription rate of the device must instead be changed.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Berkeley2006_Promoter.GIF]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Berkeley2006_Promoter.GIF]]</div></td></tr>
</table>JCAndersonhttp://2006.igem.org/wiki/index.php?title=Berkeley2006-PromoterMain&diff=19035&oldid=prevJCAnderson at 06:21, 29 October 20062006-10-29T06:21:35Z<p></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 06:21, 29 October 2006</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 4:</td>
<td colspan="2" class="diff-lineno">Line 4:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The ability to send messages is linked to the presence of the key by placing critical components of the transfer machinery under lock control. We have identified the ''traG'' gene of the R system and the ''trbC'' gene of the F system as genes critical for transfer. We deleted these genes and observed no transfer of oriT-containing plasmids. We have contructed Biobrick parts for these genes and are currently constructing locked variants to complement their function.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The ability to send messages is linked to the presence of the key by placing critical components of the transfer machinery under lock control. We have identified the ''traG'' gene of the R system and the ''trbC'' gene of the F system as genes critical for transfer. We deleted these genes and observed no transfer of oriT-containing plasmids. We have contructed Biobrick parts for these genes and are currently constructing locked variants to complement their function.<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The ability to maintain a message must similarly be under lock control. Within a network, cells will be constantly receiving messages, and these plasmids will accumulate if allowed to replicate. Our future solution to this problem will employ the R6K/''pir'' system. Here, the protein product of the ''pir'' gene is necessary to replicate plasmids with an R6K origin of replication. We will place ''pir'' under lock control so that only plasmids containing a cognate <del class="diffchange diffchange-inline">lock </del>can activate ''pir'' and replicate.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The ability to maintain a message must similarly be under lock control. Within a network, cells will be constantly receiving messages, and these plasmids will accumulate if allowed to replicate. Our future solution to this problem will employ the R6K/''pir'' system. Here, the protein product of the ''pir'' gene is necessary to replicate plasmids with an R6K origin of replication. We will place ''pir'' under lock control so that only plasmids containing a cognate <ins class="diffchange diffchange-inline">key </ins>can activate ''pir'' and replicate.<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Finally, the ability to receive additional messages <del class="diffchange diffchange-inline">can </del>be placed under lock control. Various genes, including ''dnaB'' are necessary to receive a plasmid by conjugation. We will place these genes similarly under lock control.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Finally, the ability to receive additional messages <ins class="diffchange diffchange-inline">will </ins>be placed under lock control. Various genes, including ''dnaB'' are necessary to receive a plasmid by conjugation. We will place these genes similarly under lock control.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== ===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== ===</div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 13:</td>
<td colspan="2" class="diff-lineno">Line 13:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Berkeley2006_Promoter.GIF]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Berkeley2006_Promoter.GIF]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We <del class="diffchange diffchange-inline">thereore </del>developed a set of <del class="diffchange diffchange-inline">transcriptional </del>promoter parts that vary the transcription rate. The matching of rates can then be achieved by identifying a promoter <del class="diffchange diffchange-inline">providing the proper transcription rate to match </del>the <del class="diffchange diffchange-inline">systems </del>requirements. To make these parts, we adopted a combinatorial approach. A synthetic promoter sequence was targetted with saturation mutagenesis employing a binary swap <del class="diffchange diffchange-inline">at 8 specific positions within </del>the -10 and -35 sequences. <del class="diffchange diffchange-inline">In this way</del>, 8 positions were simultaneously varied to be either the consensus sequence for that position, or not. We constructed the library fused to an RFP reporter gene containing a strong ribosome binding site, the mRFP1 gene, and a terminator. We inserted the 256 member library into the RFP reporter plasmid and picked 96 visibly white colonies and 96 visibly red colonies. The cells were grown to saturation and then assayed for fluorescence. From this screen, we identified unique promoter variants that each display a different level of <del class="diffchange diffchange-inline">fluorescense </del>activity spanning the physiological range between the level observed with cells containing no mRFP gene and values higher than those observed with a Ptet promoter (r0040). Additionally, we constructed the "consensus" promoter basic part, the strongest constitutive promoter. These parts will find general use in tuning the expression level of Biobrick devices. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We <ins class="diffchange diffchange-inline">therefore </ins>developed a set of <ins class="diffchange diffchange-inline">constitutive </ins>promoter parts that vary the transcription rate. The matching of rates can then be achieved by identifying a promoter <ins class="diffchange diffchange-inline">that satisfies </ins>the <ins class="diffchange diffchange-inline">system's </ins>requirements. To make these parts, we adopted a combinatorial approach. A synthetic promoter sequence was targetted with saturation mutagenesis employing a binary swap <ins class="diffchange diffchange-inline">of </ins>the -10 and -35 sequences. <ins class="diffchange diffchange-inline">Here</ins>, 8 positions were simultaneously varied to be either the consensus sequence for that position, or not. We constructed the library fused to an RFP reporter gene containing a strong ribosome binding site, the mRFP1 gene, and a terminator. We inserted the 256 member library into the RFP reporter plasmid and picked 96 visibly white colonies and 96 visibly red colonies. The cells were grown to saturation and then assayed for fluorescence. From this screen, we identified unique promoter variants that each display a different level of <ins class="diffchange diffchange-inline">fluorescence </ins>activity spanning the physiological range between the level observed with cells containing no mRFP gene and values higher than those observed with a Ptet promoter (r0040). Additionally, we constructed the "consensus" promoter basic part, the strongest constitutive promoter. These parts will find general use in tuning the expression level of Biobrick devices. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>----</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>----</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Next Section: [[Berkeley2006-PlasmidPartsMain | New Biobrick-Compatible Plasmids]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Next Section: [[Berkeley2006-PlasmidPartsMain | New Biobrick-Compatible Plasmids]]</div></td></tr>
</table>JCAndersonhttp://2006.igem.org/wiki/index.php?title=Berkeley2006-PromoterMain&diff=19034&oldid=prevJCAnderson: /* */2006-10-29T06:16:13Z<p><span class="autocomment"></span></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 06:16, 29 October 2006</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 8:</td>
<td colspan="2" class="diff-lineno">Line 8:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== ===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== ===</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">'''Isolatation of a family of constitutive promoter parts from a combinatorial library to vary transcription rates'''<br></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>When placing genes under new regulation, the rates of transcription and translation must be tuned to match the levels necessary for activation of the device. In other words, too much protein in the "off" state can lead to a situation where the observed phenotype occurs independent of the key. Similarly, too little protein in the "on" state can result in a scenario where the bacteria do not conjugate even when the key is present. When controlling a gene transcriptionally, this matching of rates can be achieved by manipulating the ribosome binding site of the gene. In translation control, however, the sequence of the ribosome binding site is an essential feature of the mode of regulation and cannot be changed independently. In this scenario, the transcription rate of the device must instead be changed.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>When placing genes under new regulation, the rates of transcription and translation must be tuned to match the levels necessary for activation of the device. In other words, too much protein in the "off" state can lead to a situation where the observed phenotype occurs independent of the key. Similarly, too little protein in the "on" state can result in a scenario where the bacteria do not conjugate even when the key is present. When controlling a gene transcriptionally, this matching of rates can be achieved by manipulating the ribosome binding site of the gene. In translation control, however, the sequence of the ribosome binding site is an essential feature of the mode of regulation and cannot be changed independently. In this scenario, the transcription rate of the device must instead be changed.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">'''Isolatation of a family of constitutive promoter parts from a combinatorial library to vary transcription rates'''</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Berkeley2006_Promoter.GIF]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Berkeley2006_Promoter.GIF]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
</table>JCAndersonhttp://2006.igem.org/wiki/index.php?title=Berkeley2006-PromoterMain&diff=19031&oldid=prevJCAnderson at 06:15, 29 October 20062006-10-29T06:15:26Z<p></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 06:15, 29 October 2006</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 3:</td>
<td colspan="2" class="diff-lineno">Line 3:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Berkeley2006_Conjugation7.GIF]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Berkeley2006_Conjugation7.GIF]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The ability to send messages is linked to the presence of the key by placing critical components of the transfer machinery under lock control. We have identified the traG gene of the R system and the trbC gene of the F system as genes critical for transfer. We deleted these genes and observed no transfer of oriT-containing plasmids. We have contructed Biobrick parts for these genes and are currently constructing locked variants to complement their function.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The ability to send messages is linked to the presence of the key by placing critical components of the transfer machinery under lock control. We have identified the <ins class="diffchange diffchange-inline">''</ins>traG<ins class="diffchange diffchange-inline">'' </ins>gene of the R system and the <ins class="diffchange diffchange-inline">''</ins>trbC<ins class="diffchange diffchange-inline">'' </ins>gene of the F system as genes critical for transfer. We deleted these genes and observed no transfer of oriT-containing plasmids. We have contructed Biobrick parts for these genes and are currently constructing locked variants to complement their function.<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The ability to maintain a message must similarly be under lock control. Within a network, cells will be constantly receiving messages, and these plasmids will accumulate if allowed to replicate. Our future solution to this problem will employ the R6K/pir system. Here, the protein product of the pir gene is necessary to replicate plasmids with an R6K origin of replication. We will place pir under lock control so that only plasmids containing a <del class="diffchange diffchange-inline">cogate </del>lock can activate pir and replicate.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The ability to maintain a message must similarly be under lock control. Within a network, cells will be constantly receiving messages, and these plasmids will accumulate if allowed to replicate. Our future solution to this problem will employ the R6K/<ins class="diffchange diffchange-inline">''</ins>pir<ins class="diffchange diffchange-inline">'' </ins>system. Here, the protein product of the <ins class="diffchange diffchange-inline">''</ins>pir<ins class="diffchange diffchange-inline">'' </ins>gene is necessary to replicate plasmids with an R6K origin of replication. We will place <ins class="diffchange diffchange-inline">''</ins>pir<ins class="diffchange diffchange-inline">'' </ins>under lock control so that only plasmids containing a <ins class="diffchange diffchange-inline">cognate </ins>lock can activate <ins class="diffchange diffchange-inline">''</ins>pir<ins class="diffchange diffchange-inline">'' </ins>and replicate.<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Finally, the ability to receive additional messages can be placed under lock control. Various genes, including dnaB are necessary to receive a plasmid by conjugation. We will place these genes similarly under lock control.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Finally, the ability to receive additional messages can be placed under lock control. Various genes, including <ins class="diffchange diffchange-inline">''</ins>dnaB<ins class="diffchange diffchange-inline">'' </ins>are necessary to receive a plasmid by conjugation. We will place these genes similarly under lock control.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== ===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== ===</div></td></tr>
</table>JCAndersonhttp://2006.igem.org/wiki/index.php?title=Berkeley2006-PromoterMain&diff=19030&oldid=prevJCAnderson at 06:13, 29 October 20062006-10-29T06:13:53Z<p></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 06:13, 29 October 2006</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 1:</td>
<td colspan="2" class="diff-lineno">Line 1:</td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>'''To extend addressable conjugation to a bacterial network, the ability to maintain, send, and receive plasmids must be linked to the transfer of a cognate key sequence in a recipient cell. In this way, whenever a cell recieves a matching key, it becomes activated and responds by sendng <del class="diffchange diffchange-inline">its own </del>message.'''<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>'''To extend addressable conjugation to a bacterial network, the ability to maintain, send, and receive plasmids must be linked to the transfer of a cognate key sequence in a recipient cell. In this way, whenever a cell recieves a matching key, it becomes activated and responds by sendng <ins class="diffchange diffchange-inline">a </ins>message.'''<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Berkeley2006_Conjugation7.GIF]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Berkeley2006_Conjugation7.GIF]]</div></td></tr>
</table>JCAndersonhttp://2006.igem.org/wiki/index.php?title=Berkeley2006-PromoterMain&diff=18982&oldid=prevJCAnderson at 03:53, 29 October 20062006-10-29T03:53:26Z<p></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 03:53, 29 October 2006</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 14:</td>
<td colspan="2" class="diff-lineno">Line 14:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We thereore developed a set of transcriptional promoter parts that vary the transcription rate. The matching of rates can then be achieved by identifying a promoter providing the proper transcription rate to match the systems requirements. To make these parts, we adopted a combinatorial approach. A synthetic promoter sequence was targetted with saturation mutagenesis employing a binary swap at 8 specific positions within the -10 and -35 sequences. In this way, 8 positions were simultaneously varied to be either the consensus sequence for that position, or not. We constructed the library fused to an RFP reporter gene containing a strong ribosome binding site, the mRFP1 gene, and a terminator. We inserted the 256 member library into the RFP reporter plasmid and picked 96 visibly white colonies and 96 visibly red colonies. The cells were grown to saturation and then assayed for fluorescence. From this screen, we identified unique promoter variants that each display a different level of fluorescense activity spanning the physiological range between the level observed with cells containing no mRFP gene and values higher than those observed with a Ptet promoter (r0040). Additionally, we constructed the "consensus" promoter basic part, the strongest constitutive promoter. These parts will find general use in tuning the expression level of Biobrick devices. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We thereore developed a set of transcriptional promoter parts that vary the transcription rate. The matching of rates can then be achieved by identifying a promoter providing the proper transcription rate to match the systems requirements. To make these parts, we adopted a combinatorial approach. A synthetic promoter sequence was targetted with saturation mutagenesis employing a binary swap at 8 specific positions within the -10 and -35 sequences. In this way, 8 positions were simultaneously varied to be either the consensus sequence for that position, or not. We constructed the library fused to an RFP reporter gene containing a strong ribosome binding site, the mRFP1 gene, and a terminator. We inserted the 256 member library into the RFP reporter plasmid and picked 96 visibly white colonies and 96 visibly red colonies. The cells were grown to saturation and then assayed for fluorescence. From this screen, we identified unique promoter variants that each display a different level of fluorescense activity spanning the physiological range between the level observed with cells containing no mRFP gene and values higher than those observed with a Ptet promoter (r0040). Additionally, we constructed the "consensus" promoter basic part, the strongest constitutive promoter. These parts will find general use in tuning the expression level of Biobrick devices. </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>----</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>----</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Next Section: [[Berkeley2006-<del class="diffchange diffchange-inline">NandMain </del>| <del class="diffchange diffchange-inline">Logic Computation in Cellular Networks</del>]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Next Section: [[Berkeley2006-<ins class="diffchange diffchange-inline">PlasmidPartsMain </ins>| <ins class="diffchange diffchange-inline">New Biobrick-Compatible Plasmids</ins>]]</div></td></tr>
</table>JCAndersonhttp://2006.igem.org/wiki/index.php?title=Berkeley2006-PromoterMain&diff=18978&oldid=prevJCAnderson at 03:39, 29 October 20062006-10-29T03:39:00Z<p></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 03:39, 29 October 2006</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 2:</td>
<td colspan="2" class="diff-lineno">Line 2:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Berkeley2006_Conjugation7.GIF]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Berkeley2006_Conjugation7.GIF]]</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The ability to send messages is linked to the presence of the key by placing critical components of the transfer machinery under lock control. We have identified the traG gene of the R system and the trbC gene of the F system as genes critical for transfer. We deleted these genes and observed no transfer of oriT-containing plasmids. We have contructed Biobrick parts for these genes and are currently constructing locked variants to complement their function.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The ability to send messages is linked to the presence of the key by placing critical components of the transfer machinery under lock control. We have identified the traG gene of the R system and the trbC gene of the F system as genes critical for transfer. We deleted these genes and observed no transfer of oriT-containing plasmids. We have contructed Biobrick parts for these genes and are currently constructing locked variants to complement their function.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The ability to maintain a message must similarly be under lock control. Within a network, cells will be constantly receiving messages, and these plasmids will accumulate if allowed to replicate. Our future solution to this problem will employ the R6K/pir system. Here, the protein product of the pir gene is necessary to replicate plasmids with an R6K origin of replication. We will place pir under lock control so that only plasmids containing a cogate lock can activate pir and replicate.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The ability to maintain a message must similarly be under lock control. Within a network, cells will be constantly receiving messages, and these plasmids will accumulate if allowed to replicate. Our future solution to this problem will employ the R6K/pir system. Here, the protein product of the pir gene is necessary to replicate plasmids with an R6K origin of replication. We will place pir under lock control so that only plasmids containing a cogate lock can activate pir and replicate.<br></div></td></tr>
</table>JCAndersonhttp://2006.igem.org/wiki/index.php?title=Berkeley2006-PromoterMain&diff=18977&oldid=prevJCAnderson at 03:38, 29 October 20062006-10-29T03:38:41Z<p></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 03:38, 29 October 2006</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 1:</td>
<td colspan="2" class="diff-lineno">Line 1:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''To extend addressable conjugation to a bacterial network, the ability to maintain, send, and receive plasmids must be linked to the transfer of a cognate key sequence in a recipient cell. In this way, whenever a cell recieves a matching key, it becomes activated and responds by sendng its own message.'''<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''To extend addressable conjugation to a bacterial network, the ability to maintain, send, and receive plasmids must be linked to the transfer of a cognate key sequence in a recipient cell. In this way, whenever a cell recieves a matching key, it becomes activated and responds by sendng its own message.'''<br></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">[[Image:Berkeley2006_Conjugation7.GIF]]</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The ability to send messages is linked to the presence of the key by placing critical components of the transfer machinery under lock control. We have identified the traG gene of the R system and the trbC gene of the F system as genes critical for transfer. We deleted these genes and observed no transfer of oriT-containing plasmids. We have contructed Biobrick parts for these genes and are currently constructing locked variants to complement their function.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The ability to send messages is linked to the presence of the key by placing critical components of the transfer machinery under lock control. We have identified the traG gene of the R system and the trbC gene of the F system as genes critical for transfer. We deleted these genes and observed no transfer of oriT-containing plasmids. We have contructed Biobrick parts for these genes and are currently constructing locked variants to complement their function.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The ability to maintain a message must similarly be under lock control. Within a network, cells will be constantly receiving messages, and these plasmids will accumulate if allowed to replicate. Our future solution to this problem will employ the R6K/pir system. Here, the protein product of the pir gene is necessary to replicate plasmids with an R6K origin of replication. We will place pir under lock control so that only plasmids containing a cogate lock can activate pir and replicate.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The ability to maintain a message must similarly be under lock control. Within a network, cells will be constantly receiving messages, and these plasmids will accumulate if allowed to replicate. Our future solution to this problem will employ the R6K/pir system. Here, the protein product of the pir gene is necessary to replicate plasmids with an R6K origin of replication. We will place pir under lock control so that only plasmids containing a cogate lock can activate pir and replicate.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Finally, the ability to receive additional messages can be placed under lock control. Various genes, including dnaB are necessary to receive a plasmid by conjugation. We will place these genes similarly under lock control.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Finally, the ability to receive additional messages can be placed under lock control. Various genes, including dnaB are necessary to receive a plasmid by conjugation. We will place these genes similarly under lock control.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">[[Image:Berkeley2006_Conjugation7.GIF]]</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== ===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== ===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>When placing genes under new regulation, the rates of transcription and translation must be tuned to match the levels necessary for activation of the device. In other words, too much protein in the "off" state can lead to a situation where the observed phenotype occurs independent of the key. Similarly, too little protein in the "on" state can result in a scenario where the bacteria do not conjugate even when the key is present. When controlling a gene transcriptionally, this matching of rates can be achieved by manipulating the ribosome binding site of the gene. In translation control, however, the sequence of the ribosome binding site is an essential feature of the mode of regulation and cannot be changed independently. In this scenario, the transcription rate of the device must instead be changed.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>When placing genes under new regulation, the rates of transcription and translation must be tuned to match the levels necessary for activation of the device. In other words, too much protein in the "off" state can lead to a situation where the observed phenotype occurs independent of the key. Similarly, too little protein in the "on" state can result in a scenario where the bacteria do not conjugate even when the key is present. When controlling a gene transcriptionally, this matching of rates can be achieved by manipulating the ribosome binding site of the gene. In translation control, however, the sequence of the ribosome binding site is an essential feature of the mode of regulation and cannot be changed independently. In this scenario, the transcription rate of the device must instead be changed.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">We thereore developed a set of transcriptional promoter parts that vary the transcription rate. The matching of rates can then be achieved by identifying a promoter providing the proper transcription rate to match the systems requirements. To make these parts, we adopted a combinatorial approach. A synthetic promoter sequence was targetted with saturation mutagenesis employing a binary swap at 8 specific positions within the -10 and -35 sequences. In this way, 8 positions were simultaneously varied to be either the consensus sequence for that position, or not. We constructed the library fused to an RFP reporter gene containing a strong ribosome binding site, the mRFP1 gene, and a terminator. We inserted the 256 member library into the RFP reporter plasmid and picked 96 visibly white colonies and 96 visibly red colonies. The cells were grown to saturation and then assayed for fluorescence. From this screen, we identified unique promoter variants that each display a different level of fluorescense activity spanning the physiological range between the level observed with cells containing no mRFP gene and values higher than those observed with a Ptet promoter (r0040). Additionally, we constructed the "consensus" promoter basic part, the strongest constitutive promoter. These parts will find general use in tuning the expression level of Biobrick devices. </del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Isolatation of a family of constitutive promoter parts from a combinatorial library to vary transcription rates'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Isolatation of a family of constitutive promoter parts from a combinatorial library to vary transcription rates'''</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Berkeley2006_Promoter.GIF]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Berkeley2006_Promoter.GIF]]</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">We thereore developed a set of transcriptional promoter parts that vary the transcription rate. The matching of rates can then be achieved by identifying a promoter providing the proper transcription rate to match the systems requirements. To make these parts, we adopted a combinatorial approach. A synthetic promoter sequence was targetted with saturation mutagenesis employing a binary swap at 8 specific positions within the -10 and -35 sequences. In this way, 8 positions were simultaneously varied to be either the consensus sequence for that position, or not. We constructed the library fused to an RFP reporter gene containing a strong ribosome binding site, the mRFP1 gene, and a terminator. We inserted the 256 member library into the RFP reporter plasmid and picked 96 visibly white colonies and 96 visibly red colonies. The cells were grown to saturation and then assayed for fluorescence. From this screen, we identified unique promoter variants that each display a different level of fluorescense activity spanning the physiological range between the level observed with cells containing no mRFP gene and values higher than those observed with a Ptet promoter (r0040). Additionally, we constructed the "consensus" promoter basic part, the strongest constitutive promoter. These parts will find general use in tuning the expression level of Biobrick devices. </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>----</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>----</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Next Section: [[Berkeley2006-NandMain | Logic Computation in Cellular Networks]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Next Section: [[Berkeley2006-NandMain | Logic Computation in Cellular Networks]]</div></td></tr>
</table>JCAndersonhttp://2006.igem.org/wiki/index.php?title=Berkeley2006-PromoterMain&diff=18976&oldid=prevJCAnderson at 03:28, 29 October 20062006-10-29T03:28:41Z<p></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 03:28, 29 October 2006</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 8:</td>
<td colspan="2" class="diff-lineno">Line 8:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>When placing genes under new regulation, the rates of transcription and translation must be tuned to match the levels necessary for activation of the device. In other words, too much protein in the "off" state can lead to a situation where the observed phenotype occurs independent of the key. Similarly, too little protein in the "on" state can result in a scenario where the bacteria do not conjugate even when the key is present. When controlling a gene transcriptionally, this matching of rates can be achieved by manipulating the ribosome binding site of the gene. In translation control, however, the sequence of the ribosome binding site is an essential feature of the mode of regulation and cannot be changed independently. In this scenario, the transcription rate of the device must instead be changed.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>When placing genes under new regulation, the rates of transcription and translation must be tuned to match the levels necessary for activation of the device. In other words, too much protein in the "off" state can lead to a situation where the observed phenotype occurs independent of the key. Similarly, too little protein in the "on" state can result in a scenario where the bacteria do not conjugate even when the key is present. When controlling a gene transcriptionally, this matching of rates can be achieved by manipulating the ribosome binding site of the gene. In translation control, however, the sequence of the ribosome binding site is an essential feature of the mode of regulation and cannot be changed independently. In this scenario, the transcription rate of the device must instead be changed.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We thereore developed a set of transcriptional promoter parts that vary the transcription rate. The matching of rates can then be achieved by identifying a promoter providing the proper transcription rate to match the systems requirements. To make these parts, we adopted a combinatorial approach. A synthetic promoter sequence was targetted with saturation mutagenesis employing a binary swap at 8 specific positions within the -10 and -35 sequences. In this way, 8 positions were simultaneously varied to be either the consensus sequence for that position, or not. We constructed the library fused to an RFP reporter gene containing a strong ribosome binding site, the mRFP1 gene, and a terminator. We inserted the 256 member library into the RFP reporter plasmid and picked 96 visibly white colonies and 96 visibly red colonies. The cells were grown to saturation and then assayed for fluorescence. From this screen, we identified <del class="diffchange diffchange-inline">18 </del>unique promoter variants that each display a different level of fluorescense activity spanning the physiological range between the level observed with cells containing no mRFP gene and values higher than those observed with a Ptet promoter (r0040). <del class="diffchange diffchange-inline">These </del>promoter parts will find general use in tuning the expression level of Biobrick devices. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We thereore developed a set of transcriptional promoter parts that vary the transcription rate. The matching of rates can then be achieved by identifying a promoter providing the proper transcription rate to match the systems requirements. To make these parts, we adopted a combinatorial approach. A synthetic promoter sequence was targetted with saturation mutagenesis employing a binary swap at 8 specific positions within the -10 and -35 sequences. In this way, 8 positions were simultaneously varied to be either the consensus sequence for that position, or not. We constructed the library fused to an RFP reporter gene containing a strong ribosome binding site, the mRFP1 gene, and a terminator. We inserted the 256 member library into the RFP reporter plasmid and picked 96 visibly white colonies and 96 visibly red colonies. The cells were grown to saturation and then assayed for fluorescence. From this screen, we identified unique promoter variants that each display a different level of fluorescense activity spanning the physiological range between the level observed with cells containing no mRFP gene and values higher than those observed with a Ptet promoter (r0040). <ins class="diffchange diffchange-inline">Additionally, we constructed the "consensus" </ins>promoter <ins class="diffchange diffchange-inline">basic part, the strongest constitutive promoter. These </ins>parts will find general use in tuning the expression level of Biobrick devices. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Isolatation of a family of constitutive promoter parts from a combinatorial library to vary transcription rates'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Isolatation of a family of constitutive promoter parts from a combinatorial library to vary transcription rates'''</div></td></tr>
</table>JCAnderson