http://2008.igem.org/wiki/index.php?title=Team:Guelph/Project&feed=atom&action=historyTeam:Guelph/Project - Revision history2024-03-28T16:17:16ZRevision history for this page on the wikiMediaWiki 1.16.5http://2008.igem.org/wiki/index.php?title=Team:Guelph/Project&diff=110826&oldid=prev38.122.127.2: Undo revision 106639 by iGEM HQ2018-05-30T17:48:34Z<p>Undo revision 106639 by iGEM HQ</p>
<a href="http://2008.igem.org/wiki/index.php?title=Team:Guelph/Project&diff=110826&oldid=106639">Show changes</a>38.122.127.2http://2008.igem.org/wiki/index.php?title=Team:Guelph/Project&diff=106639&oldid=prev185.106.92.123: PRmkJToSNCUwNuPl2016-03-29T00:10:26Z<p>PRmkJToSNCUwNuPl</p>
<a href="http://2008.igem.org/wiki/index.php?title=Team:Guelph/Project&diff=106639&oldid=105632">Show changes</a>185.106.92.123http://2008.igem.org/wiki/index.php?title=Team:Guelph/Project&diff=105632&oldid=prevDivDevDav at 07:34, 30 October 20082008-10-30T07:34:30Z<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 07:34, 30 October 2008</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 148:</td>
<td colspan="2" class="diff-lineno">Line 148:</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;"></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 are planning to order a 200 bp ''Zea mays'' <del class="diffchange diffchange-inline">Actin2 </del>intron construct from our sponsoring DNA synthesis company, IDT, which will consist of shuffled Biobrick ends (XbaI and PstI will switch spots) which will allow RNAi compatibility with any pre-existing Biobrick in the registry. We will program the ribosome binding site sequence AGGAGG into the middle of the intron, as well as the 3' and 5' ends of the construct to improve RNA stability by ribosome binding. We will also program NdeI and AvrII (compatible end to XbaI and SpeI) restriction sites into the intron where we will clone a GFP gene for monitoring of RNAi construct transcription in vivo. The strong synthetic transcription termination signal from Biobrick BBa_B1006 will also be programmed into the construct. Cloning will take place with the antisense biobrick going in first using NotI and SpeI sites, followed by the sense construct and XbaI and EcoRI restriction sites. WIth both orientations in place the constructs will be ligated to the strong constitutive PbsA promotor in the broad host range proprietary vector pDSK-GFPuv and then electroporated into the corn endophyte, ''Klebsiella pneumonii'' 342. GFP silencing will be tested by innoculation of ''Arabidopsis'' plants constitutively expressing GFP, while corn gene silencing activity will be monitored by testing endophyte silencing efficiency of the TB1 gene whose expression inhibits branch formation in corn plants. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We are planning to order a 200 bp ''Zea mays'' <ins class="diffchange diffchange-inline">Actin1 </ins>intron construct from our sponsoring DNA synthesis company, IDT, which will consist of shuffled Biobrick ends (XbaI and PstI will switch spots) which will allow RNAi compatibility with any pre-existing Biobrick in the registry. We will program the ribosome binding site sequence AGGAGG into the middle of the intron, as well as the 3' and 5' ends of the construct to improve RNA stability by ribosome binding. We will also program NdeI and AvrII (compatible end to XbaI and SpeI) restriction sites into the intron where we will clone a GFP gene for monitoring of RNAi construct transcription in vivo. The strong synthetic transcription termination signal from Biobrick BBa_B1006 will also be programmed into the construct. Cloning will take place with the antisense biobrick going in first using NotI and SpeI sites, followed by the sense construct and XbaI and EcoRI restriction sites. WIth both orientations in place the constructs will be ligated to the strong constitutive PbsA promotor in the broad host range proprietary vector pDSK-GFPuv and then electroporated into the corn endophyte, ''Klebsiella pneumonii'' 342. GFP silencing will be tested by innoculation of ''Arabidopsis'' plants constitutively expressing GFP, while corn gene silencing activity will be monitored by testing endophyte silencing efficiency of the TB1 gene whose expression inhibits branch formation in corn plants. </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>Our strategy: [[Image:RNAi_strategy.jpg|700px]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Our strategy: [[Image:RNAi_strategy.jpg|700px]]</div></td></tr>
</table>DivDevDavhttp://2008.igem.org/wiki/index.php?title=Team:Guelph/Project&diff=105471&oldid=prevDivDevDav: /* The Experiments */2008-10-30T06:39:45Z<p><span class="autocomment">The Experiments</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 06:39, 30 October 2008</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 89:</td>
<td colspan="2" class="diff-lineno">Line 89:</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 promoter that first interested us was the strong constitutive promoter from </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The promoter that first interested us was the strong constitutive promoter from </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>herbicide tolerant Amaranthus weeds which is unregulated (and therefore constitutive) in prokaryotes. To get this we should PCR up the promoter from the pDSK-GFPuv plasmid (introducing an SpeI site after the native NdeI), and put it into <del class="diffchange diffchange-inline">pSB1A2-E0240 </del>to test promoter activity by visually checking GFP levels. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>herbicide tolerant Amaranthus weeds which is unregulated (and therefore constitutive) in prokaryotes. To get this we should PCR up the promoter from the pDSK-GFPuv plasmid (introducing an SpeI site after the native NdeI), and put it into <ins class="diffchange diffchange-inline">BBa_E0240 </ins>to test promoter activity by visually checking GFP levels. </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;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 122:</td>
<td colspan="2" class="diff-lineno">Line 122:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>http://www.isb.vt.edu/articles/feb0802.htm</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>http://www.isb.vt.edu/articles/feb0802.htm</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>These two complete operons (both ending in the transcriptional terminator containing GFP biobrick <del class="diffchange diffchange-inline">E0240</del>) will then be put under control of the PSBA promoter or the arabinose inducible promoter, and the whole transcriptional cassette cut out to be inserted into broad host range plasmids like pDKS-GFPuv or pTG262 which will be electroporated into biologically relevant microbes such as probiotic lactobacilli, corn endophytic ''Klebsiella pneumonii'' 342, or ''E. coli'' Nissle 1917 which is sold in some countries as a probiotic for intestinal health. We will then test these microbes for beta carotene production (which ever we end up being able to get to work) in conditions simulating those we are interested in, such as human intestines. Take a look at our strategy :</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>These two complete operons (both ending in the transcriptional terminator containing GFP biobrick <ins class="diffchange diffchange-inline">BBa_E0240</ins>) will then be put under control of the PSBA promoter or the arabinose inducible promoter, and the whole transcriptional cassette cut out to be inserted into broad host range plasmids like pDKS-GFPuv or pTG262 which will be electroporated into biologically relevant microbes such as probiotic lactobacilli, corn endophytic ''Klebsiella pneumonii'' 342, or ''E. coli'' Nissle 1917 which is sold in some countries as a probiotic for intestinal health. We will then test these microbes for beta carotene production (which ever we end up being able to get to work) in conditions simulating those we are interested in, such as human intestines. Take a look at our strategy :</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>Our strategy : </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Our strategy : </div></td></tr>
</table>DivDevDavhttp://2008.igem.org/wiki/index.php?title=Team:Guelph/Project&diff=105308&oldid=prevDivDevDav: /* The Experiments */2008-10-30T05:33:17Z<p><span class="autocomment">The Experiments</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 05:33, 30 October 2008</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 111:</td>
<td colspan="2" class="diff-lineno">Line 111:</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;"></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>At this point we will attempt to add other genes to the sequence, such as the fumarate reductase operon since its overexpression results in increased cell membrane, which is where lipid soluble carotenoids are stored<del class="diffchange diffchange-inline">. GFP which could work as a reporter to let us know the transcript is OK as well as having transcriptional terminators attached to it to ensure mRNA stability</del>. Here's the link the paper on fumarate reductase overexpression: </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>At this point we will attempt to add other genes to the sequence, such as the fumarate reductase operon since its overexpression results in increased cell membrane, which is where lipid soluble carotenoids are stored. Here's the link the paper on fumarate reductase overexpression: </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;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 122:</td>
<td colspan="2" class="diff-lineno">Line 122:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>http://www.isb.vt.edu/articles/feb0802.htm</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>http://www.isb.vt.edu/articles/feb0802.htm</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>These two complete operons (both ending in GFP) will then be put under control of the PSBA promoter or the arabinose inducible promoter, and the whole transcriptional cassette cut out to be inserted into broad host range plasmids like pDKS-GFPuv or pTG262 which will be electroporated into biologically relevant microbes such as probiotic lactobacilli, corn endophytic ''Klebsiella pneumonii'' 342, or ''E. coli'' Nissle 1917 which is sold in some countries as a probiotic for intestinal health. We will then test these microbes for beta carotene production (which ever we end up being able to get to work) in conditions simulating those we are interested in, such as human intestines. Take a look at our strategy :</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>These two complete operons (both ending in <ins class="diffchange diffchange-inline">the transcriptional terminator containing </ins>GFP <ins class="diffchange diffchange-inline">biobrick E0240</ins>) will then be put under control of the PSBA promoter or the arabinose inducible promoter, and the whole transcriptional cassette cut out to be inserted into broad host range plasmids like pDKS-GFPuv or pTG262 which will be electroporated into biologically relevant microbes such as probiotic lactobacilli, corn endophytic ''Klebsiella pneumonii'' 342, or ''E. coli'' Nissle 1917 which is sold in some countries as a probiotic for intestinal health. We will then test these microbes for beta carotene production (which ever we end up being able to get to work) in conditions simulating those we are interested in, such as human intestines. Take a look at our strategy :</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>Our strategy : </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Our strategy : </div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 137:</td>
<td colspan="2" class="diff-lineno">Line 137:</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;"></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 propose a happy medium which would be relatively easy and cheap, stable during the whole lifecycle of the plant, Biobrick compatible (allowing RNAi constructs to be made from any biobrick compatible fragment) and importantly, quick and effective. Bacterial <del class="diffchange diffchange-inline">induced gene silencing </del>(BIGS) will employ an endophytic microbe (lives mutualistically inside a plant) to deliver an RNAi signal against a plant gene in order to study that gene's function. We are hoping that as these microbes die, they will release these molecules into the medium, and since plants are very sensitive to these double stranded RNA molecules, they will sense AND systemically amplify the signal across the entire plant body. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We propose a happy medium which would be relatively easy and cheap, stable during the whole lifecycle of the plant, Biobrick compatible (allowing RNAi constructs to be made from any biobrick compatible fragment) and importantly, quick and effective. Bacterial <ins class="diffchange diffchange-inline">Induced Gene Silencing </ins>(BIGS) will employ an endophytic microbe (lives mutualistically inside a plant) to deliver an RNAi signal against a plant gene in order to study that gene's function. We are hoping that as these microbes die, they will release these molecules into the medium, and since plants are very sensitive to these double stranded RNA molecules, they will sense AND systemically amplify the signal across the entire plant body. </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;"></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>There are relevant precedants for this area of research, but it has never before been done using endophytes in plants which <del class="diffchange diffchange-inline">can </del>produce this signal during the entire life cycle of the host. Here are some relevant publications supporting the potential of our work which we will not further discuss: </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>There are relevant precedants for this area of research, but it has never before been done using endophytes in plants which <ins class="diffchange diffchange-inline">could theorhetically </ins>produce this signal during the entire life cycle of the host. Here are some relevant publications supporting the potential of our work which we will not further discuss: </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;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 148:</td>
<td colspan="2" class="diff-lineno">Line 148:</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;"></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 are planning to order a 200 bp Zea mays <del class="diffchange diffchange-inline">actin2 </del>intron construct from our sponsoring DNA synthesis company, IDT, which will consist of shuffled Biobrick ends (XbaI and PstI will switch spots) which will allow RNAi compatibility with any pre-existing Biobrick in the registry. We will program the ribosome binding site sequence AGGAGG into the middle of the intron, as well as the 3' and 5' ends of the construct to improve RNA stability by ribosome binding. We will also program NdeI and AvrII (compatible end to XbaI and SpeI) restriction sites into the intron where we will clone a GFP gene for monitoring of RNAi construct transcription in vivo. The strong synthetic transcription termination signal from Biobrick BBa_B1006 will also be programmed into the construct. Cloning will take place with the antisense biobrick going in first using NotI and SpeI sites, followed by the sense construct and XbaI and EcoRI restriction sites. WIth both orientations in place the constructs will be ligated to the strong constitutive PbsA promotor in the broad host range proprietary vector pDSK-GFPuv and then electroporated into the corn endophyte, ''Klebsiella pneumonii'' 342. GFP silencing will be tested by innoculation of ''Arabidopsis'' plants constitutively expressing GFP, while corn gene silencing activity will be monitored by testing endophyte silencing efficiency of the TB1 gene whose expression inhibits branch formation in corn plants. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We are planning to order a 200 bp <ins class="diffchange diffchange-inline">''</ins>Zea mays<ins class="diffchange diffchange-inline">'' Actin2 </ins>intron construct from our sponsoring DNA synthesis company, IDT, which will consist of shuffled Biobrick ends (XbaI and PstI will switch spots) which will allow RNAi compatibility with any pre-existing Biobrick in the registry. We will program the ribosome binding site sequence AGGAGG into the middle of the intron, as well as the 3' and 5' ends of the construct to improve RNA stability by ribosome binding. We will also program NdeI and AvrII (compatible end to XbaI and SpeI) restriction sites into the intron where we will clone a GFP gene for monitoring of RNAi construct transcription in vivo. The strong synthetic transcription termination signal from Biobrick BBa_B1006 will also be programmed into the construct. Cloning will take place with the antisense biobrick going in first using NotI and SpeI sites, followed by the sense construct and XbaI and EcoRI restriction sites. WIth both orientations in place the constructs will be ligated to the strong constitutive PbsA promotor in the broad host range proprietary vector pDSK-GFPuv and then electroporated into the corn endophyte, ''Klebsiella pneumonii'' 342. GFP silencing will be tested by innoculation of ''Arabidopsis'' plants constitutively expressing GFP, while corn gene silencing activity will be monitored by testing endophyte silencing efficiency of the TB1 gene whose expression inhibits branch formation in corn plants. </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>Our strategy: [[Image:RNAi_strategy.jpg|700px]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Our strategy: [[Image:RNAi_strategy.jpg|700px]]</div></td></tr>
</table>DivDevDavhttp://2008.igem.org/wiki/index.php?title=Team:Guelph/Project&diff=105283&oldid=prevDivDevDav: /* Background and Justification */2008-10-30T05:25:24Z<p><span class="autocomment">Background and Justification</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 05:25, 30 October 2008</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 65:</td>
<td colspan="2" class="diff-lineno">Line 65:</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;"></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>2) BIGS for functional genomics: Functional genomics is an area of genetics that uses genomic information to help understand gene function. Currently, the most straightforward way to do this for any given gene is to analyze a single gene mutant to discover that organism's deficiency, and thus the gene's normal function. If you're lucky, you can find a natural mutant, or make one using random mutagenisis. However, in complex eukaryotes with tens of thousands of genes, it is impossible to expect that you can get knock outs for any particular gene you want to study. As of a decade ago, it began to be possible to use RNAi to selectively knock out your particular gene by making a transgenic organism expressing double stranded RNA with the same sequence of the target gene. For a eukaryote like corn, this makes it possible knock out one particular gene you want to study, but it does require about a year for that transgenic corn to be made. An alternative in some eukaryotes is Viral Induced Gene Silencing, or VIGS, whereby the double stranded RNA is transiently delivered into the organism by a virus. VIGS has only been developed for certain eukaryotes however (not including corn), and even so is only a transient way to silence a gene, giving a scientist only a portion of the eukaryote's life cycle where gene function might be studied. We ask ourselves, can a system be made which could effectively silence eukaryotic genes using RNAi, but more quickly and cheaply than making a transgenic eukaryote and more stably and long term than by using VIGS? Our idea is that a normal endosymbiont of an organism could do this if it can be coaxed to produce this RNAi signal. Read on to find out how we'll test this idea...</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>2) BIGS for functional genomics: Functional genomics is an area of genetics that uses genomic information to help understand gene function. Currently, the most straightforward way to do this for any given gene is to analyze a single gene mutant to discover that organism's deficiency, and thus the gene's normal function. If you're lucky, you can find a natural mutant, or make one using random mutagenisis. However, in complex eukaryotes with tens of thousands of genes, it is impossible to expect that you can get knock outs for any particular gene you want to study. As of a decade ago, it began to be possible to use RNAi to selectively knock out your particular gene by making a transgenic organism expressing double stranded RNA with the same sequence of the target gene. For a eukaryote like corn, this makes it possible <ins class="diffchange diffchange-inline">to </ins>knock out one particular gene you want to study, but it does require about a year for that transgenic corn to be made. An alternative in some eukaryotes is Viral Induced Gene Silencing, or VIGS, whereby the double stranded RNA is transiently delivered into the organism by a virus. VIGS has only been developed for certain eukaryotes however (not including corn), and even so is only a transient way to silence a gene, giving a scientist only a portion of the eukaryote's life cycle where gene function might be studied. We ask ourselves, can a system be made which could effectively silence eukaryotic genes using RNAi, but more quickly and cheaply than making a transgenic eukaryote and more stably and long term than by using VIGS? Our idea is that a normal endosymbiont of an organism could do this if it can be coaxed to produce this RNAi signal. Read on to find out how we'll test this idea...</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>=== The Experiments ===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== The Experiments ===</div></td></tr>
</table>DivDevDavhttp://2008.igem.org/wiki/index.php?title=Team:Guelph/Project&diff=105277&oldid=prevDivDevDav: /* Overall project */2008-10-30T05:23:17Z<p><span class="autocomment">Overall project</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 05:23, 30 October 2008</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 42:</td>
<td colspan="2" class="diff-lineno">Line 42:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== '''Overall project''' ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== '''Overall project''' ==</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>Microbes are found in every nook and cranny of the planet, and multicellular organisms are no exception. Plants and animals are found to contain huge numbers of bacteria and fungi that help with nutrient absorption, producing beneficial compounds, fighting off pathogens, or often are pathogens themselves. We are interested in taking advantage of some of these microbes to deliver transgenic payloads for the benefit or modification of the host organism. These might be called GM endosymbionts. On the human side, we would like to introduce the carotendoid metabolic genes from a well studied soil microbe called Erwinia <del class="diffchange diffchange-inline">urodevora </del>into human probiotic microbes which will survive and colonize the intestine for stable production of the essential human nutrient, pro-vitamin A. Time permitting, we will attempt to enhance carotenoid accumulation by increasing plasma membrane sink by overexpression of the fumarate reductase operon. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Microbes are found in every nook and cranny of the planet, and multicellular organisms are no exception. Plants and animals are found to contain huge numbers of bacteria and fungi that help with nutrient absorption, producing beneficial compounds, fighting off pathogens, or often are pathogens themselves. We are interested in taking advantage of some of these microbes to deliver transgenic payloads for the benefit or modification of the host organism. These might be called GM endosymbionts. On the human side, we would like to introduce the carotendoid metabolic genes from a well studied soil microbe called <ins class="diffchange diffchange-inline">''</ins>Erwinia <ins class="diffchange diffchange-inline">urodovora'' </ins>into human probiotic microbes which will survive and colonize the intestine for stable production of the essential human nutrient, pro-vitamin A. Time permitting, we will attempt to enhance carotenoid accumulation by increasing plasma membrane sink by overexpression of the fumarate reductase operon. </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>Millions of humans suffer from vitamin A deficiencies across the world, resulting in blindness and death which could be mitigated by symbitic production of this important vitamin. We will also attempt to get corn endosymbionts to produce pro-vitamin A, a function usually performed by the plant itself. In both cases, the ethical and practical implications of this technology will affect how it might ever be implemented, so we are very excited to be collaborating with the Calgary Ethics team to ruminate on the possibilities and pitfalls of this bit of synthetic biology. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Millions of humans suffer from vitamin A deficiencies across the world, resulting in blindness and death which could be mitigated by symbitic production of this important vitamin. We will also attempt to get corn endosymbionts to produce pro-vitamin A, a function usually performed by the plant itself. In both cases, the ethical and practical implications of this technology will affect how it might ever be implemented, so we are very excited to be collaborating with the Calgary Ethics team to ruminate on the possibilities and pitfalls of this bit of synthetic biology. </div></td></tr>
</table>DivDevDavhttp://2008.igem.org/wiki/index.php?title=Team:Guelph/Project&diff=105268&oldid=prevDivDevDav: /* Project Abstract */2008-10-30T05:21:09Z<p><span class="autocomment">Project Abstract</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 05:21, 30 October 2008</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 28:</td>
<td colspan="2" class="diff-lineno">Line 28:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== '''Project Abstract''' ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== '''Project Abstract''' ==</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 class="diffchange diffchange-inline">In humans </del>microbes help digest our food and produce vitamins to suppliment </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">Humans contain billions of </ins>microbes <ins class="diffchange diffchange-inline">which </ins>help digest our food and produce vitamins to suppliment </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>our diet, while plants such as corn harbour microbes within their tissues </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>our diet, while plants such as corn harbour microbes within their tissues </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>which can extend the metabolic capacity of their host. In <del class="diffchange diffchange-inline">order </del>to </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>which can extend the metabolic capacity of their host. In <ins class="diffchange diffchange-inline">an attempt </ins>to </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>exploit these patterns of microbial habitation, we <del class="diffchange diffchange-inline">attempted to modify </del>the broad host range </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>exploit these patterns of microbial habitation, we <ins class="diffchange diffchange-inline">built constructs which were inserted into </ins>the broad host range plasmid pDSK-GFPuv to contain either a synthetic operon of metabolic genes </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>plasmid pDSK-GFPuv to contain either a synthetic operon of metabolic genes </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>belonging to the soil microbe <ins class="diffchange diffchange-inline">''</ins>Erwinia uredovora<ins class="diffchange diffchange-inline">''</ins>, or Biobrick compatible </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>belonging to the soil microbe Erwinia uredovora, or Biobrick compatible </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>RNAi constructs targetting <ins class="diffchange diffchange-inline">the plant </ins>expression of either GFP or corn TB1 genes. These plasmids were</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>RNAi constructs targetting expression of either GFP or corn TB1 genes. These plasmids were</div></td><td class='diff-marker'>+</td><td style="background: #cfc; 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>to be electroporated into either probiotic Escherichia coli Nissle 1917 or endophytic </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>to be electroporated into either probiotic Escherichia coli Nissle 1917 or endophytic </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Klebsiella pneumonii 342. Assays will then show whether a genetically modified </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Klebsiella pneumonii 342. Assays will then show whether a genetically modified </div></td></tr>
</table>DivDevDavhttp://2008.igem.org/wiki/index.php?title=Team:Guelph/Project&diff=105187&oldid=prevDivDevDav at 05:05, 30 October 20082008-10-30T05:05:57Z<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 05:05, 30 October 2008</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 87:</td>
<td colspan="2" class="diff-lineno">Line 87:</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;"></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>However, this was not a perfectly Biobrick compliant strategy, and there were unecessary extra steps we'd have to follow so we reworked our strategy. <del class="diffchange diffchange-inline">Now </del>we <del class="diffchange diffchange-inline">will put into </del>a <del class="diffchange diffchange-inline">Biobrick vector a strong constitutive promoter from </del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>However, this was not a perfectly Biobrick compliant strategy, and there were unecessary extra steps we'd have to follow so we reworked our strategy. <ins class="diffchange diffchange-inline">Instead the strategy </ins>we <ins class="diffchange diffchange-inline">followed depended on sequentially adding genes to an operon by cutting the </ins>a <ins class="diffchange diffchange-inline">biobrick plasmid containing CrtE with SpeI </ins>and <ins class="diffchange diffchange-inline">PstI, and adding each consecutive XbaI / PstI cut gene via ligation</ins>. <ins class="diffchange diffchange-inline">The last gene will have transcription terminators on </ins>the <ins class="diffchange diffchange-inline">end. Next we would add promoters</ins>, and <ins class="diffchange diffchange-inline">uing EcoRI and XbaI, then cut the transcriptional unit out and transfer </ins>to <ins class="diffchange diffchange-inline">other plasmids using EcoRI and PstI</ins>. <ins class="diffchange diffchange-inline"> </ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">herbicide tolerant Amaranthus weeds which is unregulated (</del>and <del class="diffchange diffchange-inline">therefore constitutive) in prokaryotes</del>. <del class="diffchange diffchange-inline"> To do this we should PCR up the promoter from </del>the <del class="diffchange diffchange-inline">pDSK-GFPuv plasmid (introducing an SpeI site after the native NdeI)</del>, and <del class="diffchange diffchange-inline">put it into pSB1A2-E0240 </del>to <del class="diffchange diffchange-inline">test promoter activity by visually checking GFP levels</del>. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></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 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;">The promoter that first interested us was the strong constitutive promoter from </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;">herbicide tolerant Amaranthus weeds which is unregulated (and therefore constitutive) in prokaryotes. To get this we should PCR up the promoter from the pDSK-GFPuv plasmid (introducing an SpeI site after the native NdeI), and put it into pSB1A2-E0240 to test promoter activity by visually checking GFP levels. </ins></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>With a strong, constitutive, biobricked promoter having an NdeI on the start codon, we're ready to <del class="diffchange diffchange-inline">clone </del>the <del class="diffchange diffchange-inline">carotenoid genes into the NdeI space in between promoter and GFP</del>. The gene order for the operon has already been optimized. This paper explains we should have E-B-I-Y. Check it out:</div></td><td class='diff-marker'>+</td><td style="background: #cfc; 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>With a strong, constitutive, biobricked promoter having an NdeI on the start codon <ins class="diffchange diffchange-inline">followed by an SpeI site</ins>, we're ready to <ins class="diffchange diffchange-inline">build </ins>the <ins class="diffchange diffchange-inline">operon</ins>. The gene order for the operon has already been optimized. This paper explains we should have E-B-I-Y. Check it out:</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;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 110:</td>
<td colspan="2" class="diff-lineno">Line 112:</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;"></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>At this point we will attempt to add other genes to the sequence, such as <del class="diffchange diffchange-inline">GFP which could work as a reporter to let us know the transcript is OK or </del>the fumarate reductase operon since its overexpression results in increased cell membrane, which is where lipid soluble carotenoids are stored. Here's the link the paper: </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>At this point we will attempt to add other genes to the sequence, such as the fumarate reductase operon since its overexpression results in increased cell membrane, which is where lipid soluble carotenoids are stored<ins class="diffchange diffchange-inline">. GFP which could work as a reporter to let us know the transcript is OK as well as having transcriptional terminators attached to it to ensure mRNA stability</ins>. Here's the link the paper <ins class="diffchange diffchange-inline">on fumarate reductase overexpression</ins>: </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;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 121:</td>
<td colspan="2" class="diff-lineno">Line 123:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>http://www.isb.vt.edu/articles/feb0802.htm</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>http://www.isb.vt.edu/articles/feb0802.htm</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 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;">These two complete operons (both ending in GFP) will then be put under control of the PSBA promoter or the arabinose inducible promoter, and the whole transcriptional cassette cut out to be inserted into broad host range plasmids like pDKS-GFPuv or pTG262 which will be electroporated into biologically relevant microbes such as probiotic lactobacilli, corn endophytic ''Klebsiella pneumonii'' 342, or ''E. coli'' Nissle 1917 which is sold in some countries as a probiotic for intestinal health. We will then test these microbes for beta carotene production (which ever we end up being able to get to work) in conditions simulating those we are interested in, such as human intestines. Take a look at our strategy :</ins></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>Our strategy : </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Our strategy : </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:BetaCaroteneStrategy.jpg|700px]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:BetaCaroteneStrategy.jpg|700px]]</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;">Assay for beta-carotene production.</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: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">Transfer to gram positive and gram negative plasmids.</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: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">Electroporate into different probiotic strains. </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: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">Monitor beta carotene production and GFP in biological system/model.</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: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">Celebrate a succesful new technology for improved human health!</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;"></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;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
</table>DivDevDavhttp://2008.igem.org/wiki/index.php?title=Team:Guelph/Project&diff=59797&oldid=prevDivDevDav at 19:21, 21 October 20082008-10-21T19:21:39Z<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 19:21, 21 October 2008</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 77:</td>
<td colspan="2" class="diff-lineno">Line 77:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>So what is the stategy we should follow? Initially this </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>So what is the stategy we should follow? Initially this </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Hydrogen synthetic operon paper was inspiring us: </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Hydrogen synthetic operon paper was inspiring us: </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;"></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;"></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>http://www.ncbi.nlm.nih.gov/pubmed/17996187</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>http://www.ncbi.nlm.nih.gov/pubmed/17996187</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;"></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>Its specific for a bacterial synthetic </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Its specific for a bacterial synthetic </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>operon and if we followed it we wouldn't have to rework things too hard in order to follow it.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>operon and if we followed it we wouldn't have to rework things too hard in order to follow it.</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;"></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>However, this was not a perfectly Biobrick compliant strategy, and there were unecessary extra steps we'd have to follow so we reworked our strategy. Now we will put into a Biobrick vector a strong constitutive promoter from </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>However, this was not a perfectly Biobrick compliant strategy, and there were unecessary extra steps we'd have to follow so we reworked our strategy. Now we will put into a Biobrick vector a strong constitutive promoter from </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>herbicide tolerant Amaranthus weeds which is unregulated (and therefore </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>herbicide tolerant Amaranthus weeds which is unregulated (and therefore constitutive) in prokaryotes. To do this we should PCR up the promoter from the pDSK-GFPuv plasmid (introducing an SpeI site after the native NdeI), and put it into pSB1A2-E0240 to test promoter activity by visually checking GFP levels. </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>constitutive) in prokaryotes. To do this we should PCR up the promoter </div></td><td class='diff-marker'>+</td><td style="background: #cfc; 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>from the pDSK-GFPuv plasmid (introducing an SpeI site after the native NdeI), and put it into pSB1A2-E0240 to test promoter activity by visually checking GFP levels. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; 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 class="diffchange diffchange-inline">With a strong, constitutive, biobricked promoter having an NdeI on the start codon, we're ready to clone the carotenoid genes into the NdeI space in between promoter and GFP. The gene order for the operon has already been optimized. This paper explains we should have E-B-I-Y. Check it out:</ins></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;">With a strong, constitutive, biobricked promoter having an NdeI on the start codon, we're ready to clone the carotenoid genes into the NdeI space in between promoter and GFP. The gene order for the operon has already been optimized. This paper </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;">explains we should have E-B-I-Y. Check it out:</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>http://aem.asm.org/cgi/content/abstract/AEM.02268-06v1</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>http://aem.asm.org/cgi/content/abstract/AEM.02268-06v1</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;"></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 initial gene, crt-E, on plasmid p3-10-10 will be amplified and appropriate flanking , XbaI and NdeI (on or before the start codon) and SpeI (after the stop codon) restriction sites introduced. There is also a ribosome binding site present in front of each of the carotenoid genes, and thus we will conserve them in our cloning strategy. We will cut the plasmid with XbaI and SpeI then dephosphorylate it with antartic alkaline phosphatase, kindly provided by our sponsor New England Biolabs. Likewise, the PCR product will be cut with XbaI and SpeI, and ligated to the dephosphorylated plasmid. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The initial gene, crt-E, on plasmid p3-10-10 will be amplified and appropriate flanking , XbaI and NdeI (on or before the start codon) and SpeI (after the stop codon) restriction sites introduced. There is also a ribosome binding site present in front of each of the carotenoid genes, and thus we will conserve them in our cloning strategy. We will cut the plasmid with XbaI and SpeI then dephosphorylate it with antartic alkaline phosphatase, kindly provided by our sponsor New England Biolabs. Likewise, the PCR product will be cut with XbaI and SpeI, and ligated to the dephosphorylated plasmid. </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;"></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 Biobricked crt-B PCR product will also be flanked with XbaI and SpeI, and ligated into its own pSB1A2 plasmid where it will attain the EcoRI and PstI sites as well. To insert this crt-B after crt-E, we will cut it with XbaI and Pst, while cutting the plasmid with SpeI and PstI. This will destroy the SpeI site on the plasmid and the XbaI site on the PCR product, but these complementary sticky ends have the benefit of decreasing the rate of background ligations; the complementary sticky ends act as 'heat seeking missiles', seeking out their complementary mates and ignoring non-complementary overhangs or blunt end. The PstI site will be conserved for future use. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The Biobricked crt-B PCR product will also be flanked with XbaI and SpeI, and ligated into its own pSB1A2 plasmid where it will attain the EcoRI and PstI sites as well. To insert this crt-B after crt-E, we will cut it with XbaI and Pst, while cutting the plasmid with SpeI and PstI. This will destroy the SpeI site on the plasmid and the XbaI site on the PCR product, but these complementary sticky ends have the benefit of decreasing the rate of background ligations; the complementary sticky ends act as 'heat seeking missiles', seeking out their complementary mates and ignoring non-complementary overhangs or blunt end. The PstI site will be conserved for future use. </div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 107:</td>
<td colspan="2" class="diff-lineno">Line 111:</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>At this point we will attempt to add other genes to the sequence, such as GFP which could work as a reporter to let us know the transcript is OK or the fumarate reductase operon since its overexpression results in increased cell membrane, which is where lipid soluble carotenoids are stored. Here's the link the paper: </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>At this point we will attempt to add other genes to the sequence, such as GFP which could work as a reporter to let us know the transcript is OK or the fumarate reductase operon since its overexpression results in increased cell membrane, which is where lipid soluble carotenoids are stored. Here's the link the paper: </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;"></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>http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=215469</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=215469</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;"></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>In plants it has been shown that sink strength is a limiting factor to carotenoid accumulation, so giving bacteria more carotenoid storage space may result in much more carotenoid accumulation. Here's a discussion about engineering plant sinks for increased carotenoid accumulation: </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In plants it has been shown that sink strength is a limiting factor to carotenoid accumulation, so giving bacteria more carotenoid storage space may result in much more carotenoid accumulation. Here's a discussion about engineering plant sinks for increased carotenoid accumulation: </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;"></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>http://www.isb.vt.edu/articles/feb0802.htm</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>http://www.isb.vt.edu/articles/feb0802.htm</div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 134:</td>
<td colspan="2" class="diff-lineno">Line 141:</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>Functional genomics is the study of gene function in an organism through (genomics) informed study of transcription, translation, interactions, and phenotype. A common way to do this is by making mutants and assaying for different phenotypes; a very convenient way to accomplish this is by using RNA interferance (RNAi). Usually this is done by making transgenic plants expressing RNAi constructs, and is stable but limited by the difficulty and length of time it takes to produce the transgenic plant. In the case of corn, this means almost a full year before you can test your gene function. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Functional genomics is the study of gene function in an organism through (genomics) informed study of transcription, translation, interactions, and phenotype. A common way to do this is by making mutants and assaying for different phenotypes; a very convenient way to accomplish this is by using RNA interferance (RNAi). Usually this is done by making transgenic plants expressing RNAi constructs, and is stable but limited by the difficulty and length of time it takes to produce the transgenic plant. In the case of corn, this means almost a full year before you can test your gene function. </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;"></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>An alternative is called viral induced gene silencing (VIGS) which can uses a plant specific virus to deliver and transiently silence the gene. An obvious limitation here is that it is a transient process even if it has been developed for a particular plant, which it has not been for corn. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>An alternative is called viral induced gene silencing (VIGS) which can uses a plant specific virus to deliver and transiently silence the gene. An obvious limitation here is that it is a transient process even if it has been developed for a particular plant, which it has not been for corn. </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;"></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 propose a happy medium which would be relatively easy and cheap, stable during the whole lifecycle of the plant, Biobrick compatible (allowing RNAi constructs to be made from any biobrick compatible fragment) and importantly, quick and effective. Bacterial induced gene silencing (BIGS) will employ an endophytic microbe (lives mutualistically inside a plant) to deliver an RNAi signal against a plant gene in order to study that gene's function. We are hoping that as these microbes die, they will release these molecules into the medium, and since plants are very sensitive to these double stranded RNA molecules, they will sense AND systemically amplify the signal across the entire plant body. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We propose a happy medium which would be relatively easy and cheap, stable during the whole lifecycle of the plant, Biobrick compatible (allowing RNAi constructs to be made from any biobrick compatible fragment) and importantly, quick and effective. Bacterial induced gene silencing (BIGS) will employ an endophytic microbe (lives mutualistically inside a plant) to deliver an RNAi signal against a plant gene in order to study that gene's function. We are hoping that as these microbes die, they will release these molecules into the medium, and since plants are very sensitive to these double stranded RNA molecules, they will sense AND systemically amplify the signal across the entire plant body. </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;"></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>There are relevant precedants for this area of research, but it has never before been done using endophytes in plants which can produce this signal during the entire life cycle of the host. Here are some relevant publications supporting the potential of our work which we will not further discuss: </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>There are relevant precedants for this area of research, but it has never before been done using endophytes in plants which can produce this signal during the entire life cycle of the host. Here are some relevant publications supporting the potential of our work which we will not further discuss: </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;"></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>Spraying RNAi E. Coli onto plants to protect against viruses - http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=153545</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Spraying RNAi E. Coli onto plants to protect against viruses - http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=153545</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>Using E. coli to deliver RNAi signals to tumors in mouse colons - http://www.ncbi.nlm.nih.gov/books/bv.fcgi?indexed=google&rid=eurekah.section.75458 </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Using E. coli to deliver RNAi signals to tumors in mouse colons - http://www.ncbi.nlm.nih.gov/books/bv.fcgi?indexed=google&rid=eurekah.section.75458 </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;"></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 are planning to order a 200 bp Zea mays actin2 intron construct from our sponsoring DNA synthesis company, IDT, which will consist of shuffled Biobrick ends (XbaI and PstI will switch spots) which will allow RNAi compatibility with any pre-existing Biobrick in the registry. We will program the ribosome binding site sequence AGGAGG into the middle of the intron, as well as the 3' and 5' ends of the construct to improve RNA stability by ribosome binding. We will also program NdeI and AvrII (compatible end to XbaI and SpeI) restriction sites into the intron where we will clone a GFP gene for monitoring of RNAi construct transcription in vivo. The strong synthetic transcription termination signal from Biobrick BBa_B1006 will also be programmed into the construct. Cloning will take place with the antisense biobrick going in first using NotI and SpeI sites, followed by the sense construct and XbaI and EcoRI restriction sites. WIth both orientations in place the constructs will be ligated to the strong constitutive PbsA promotor in the broad host range proprietary vector pDSK-GFPuv and then electroporated into the corn endophyte, ''Klebsiella pneumonii'' 342. GFP silencing will be tested by innoculation of ''Arabidopsis'' plants constitutively expressing GFP, while corn gene silencing activity will be monitored by testing endophyte silencing efficiency of the TB1 gene whose expression inhibits branch formation in corn plants. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We are planning to order a 200 bp Zea mays actin2 intron construct from our sponsoring DNA synthesis company, IDT, which will consist of shuffled Biobrick ends (XbaI and PstI will switch spots) which will allow RNAi compatibility with any pre-existing Biobrick in the registry. We will program the ribosome binding site sequence AGGAGG into the middle of the intron, as well as the 3' and 5' ends of the construct to improve RNA stability by ribosome binding. We will also program NdeI and AvrII (compatible end to XbaI and SpeI) restriction sites into the intron where we will clone a GFP gene for monitoring of RNAi construct transcription in vivo. The strong synthetic transcription termination signal from Biobrick BBa_B1006 will also be programmed into the construct. Cloning will take place with the antisense biobrick going in first using NotI and SpeI sites, followed by the sense construct and XbaI and EcoRI restriction sites. WIth both orientations in place the constructs will be ligated to the strong constitutive PbsA promotor in the broad host range proprietary vector pDSK-GFPuv and then electroporated into the corn endophyte, ''Klebsiella pneumonii'' 342. GFP silencing will be tested by innoculation of ''Arabidopsis'' plants constitutively expressing GFP, while corn gene silencing activity will be monitored by testing endophyte silencing efficiency of the TB1 gene whose expression inhibits branch formation in corn plants. </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>Our strategy: [[Image:RNAi_strategy.jpg|700px]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Our strategy: [[Image:RNAi_strategy.jpg|700px]]</div></td></tr>
</table>DivDevDav