http://2007.igem.org/wiki/index.php?title=Cambridge/Gram-positive_chassis_project&feed=atom&action=historyCambridge/Gram-positive chassis project - Revision history2024-03-29T10:10:23ZRevision history for this page on the wikiMediaWiki 1.16.5http://2007.igem.org/wiki/index.php?title=Cambridge/Gram-positive_chassis_project&diff=46153&oldid=prevYijin at 00:11, 27 October 20072007-10-27T00:11:33Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Promoters tested:</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Promoters tested:</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#[http://partsregistry.org/Part:BBa_I13604 BBa_I13604]: Tet operator (<del class="diffchange diffchange-inline">[</del>[http://partsregistry.org/Part:BBa_R0040 BBa_R0040]) + YFP(LVA-) reporter, followed by a Lac operator ([http://partsregistry.org/Part:BBa_R0011 BBa_R0011]) + CFP(LVA-) reporter</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#[http://partsregistry.org/Part:BBa_I13604 BBa_I13604]: Tet operator ([http://partsregistry.org/Part:BBa_R0040 BBa_R0040]) + YFP(LVA-) reporter, followed by a Lac operator ([http://partsregistry.org/Part:BBa_R0011 BBa_R0011]) + CFP(LVA-) reporter</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#[http://partsregistry.org/Part:BBa_I13973 BBa_I13973]: cI promoter ([http://partsregistry.org/Part:BBa_R0051 BBa_R0051]) + YFP-aav reporter device</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#[http://partsregistry.org/Part:BBa_I13973 BBa_I13973]: cI promoter ([http://partsregistry.org/Part:BBa_R0051 BBa_R0051]) + YFP-aav reporter device</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#[http://partsregistry.org/Part:BBa_J04450 BBa_J04450]: RFP Coding Device switched on by IPTG, contains LacI promoter ([http://partsregistry.org/Part:BBa_R0010 BBa_R0010])</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#[http://partsregistry.org/Part:BBa_J04450 BBa_J04450]: RFP Coding Device switched on by IPTG, contains LacI promoter ([http://partsregistry.org/Part:BBa_R0010 BBa_R0010])</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#[http://partsregistry.org/Part:BBa_I746105 BBa_I746105]: GFP with agr P2 promoter ([http://partsregistry.org/Part:BBa_I746104 BBa_I746104])</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#[http://partsregistry.org/Part:BBa_I746105 BBa_I746105]: GFP with agr P2 promoter ([http://partsregistry.org/Part:BBa_I746104 BBa_I746104])</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#*Note: the agr P2 promoter is derived from S. aureus, but we were unsure whether it would work in ''B. subtilis'' or ''E. coli'' as it was never previously reported in literature.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#*Note: the agr P2 promoter is derived from S. aureus, but we were unsure whether it would work in ''B. subtilis'' or ''E. coli'' as it was never previously reported in literature.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">#[http://partsregistry.org/Part:BBa_I746666 BBa_I746666]: Pspac-hy ([http://partsregistry.org/Part:BBa_I746665 BBa_I746665]) with GFP ([http://partsregistry.org/Part:BBa_E0840 BBa_E0840])</ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In order to test if promoters in the BioBrick registry functioned in Bacillus, this was the protocol we adopted for the testing:</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In order to test if promoters in the BioBrick registry functioned in Bacillus, this was the protocol we adopted for the testing:</div></td></tr>
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<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># I13604, I13973, J04450 <del class="diffchange diffchange-inline">and </del>I746105 was digested with the appropriate enzymes for at least 2 hours</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div># I13604, I13973, J04450<ins class="diffchange diffchange-inline">, </ins>I746105 <ins class="diffchange diffchange-inline">and I746666 </ins>was digested with the appropriate enzymes for at least 2 hours</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># The products of the digestion were then run on a gel</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># The products of the digestion were then run on a gel</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#* Appropriate bands removed from gel</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#* Appropriate bands removed from gel</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For pNZ8901:</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For pNZ8901:</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>*Fluorescent E. cloni colonies were obtained for BBa_I13604, BBa_J04450 <del class="diffchange diffchange-inline">and </del>BBa_I746105. After miniprepping and transformation into Bacillus, none of the colonies showed fluorescence. In the case of BBa_J04450, after miniprepping the Bacillus colonies and running a double digest to separate insert from vector, some colonies were found to contain 2 bands of the correct size, suggesting that the greater stringency behind use of promoters in Bacillus may be the cause. No DNA was recovered from the miniprep of pNZ + BBa_I13604.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>*Fluorescent E. cloni colonies were obtained for BBa_I13604, BBa_J04450<ins class="diffchange diffchange-inline">, </ins>BBa_I746105 <ins class="diffchange diffchange-inline">and BBa_I746666. For BBa_I746666, the fluorescent E. cloni colonies, which were rather small, failed to grow in liquid culture broth</ins>. After miniprepping and transformation into Bacillus, none of the colonies showed fluorescence. In the case of BBa_J04450, after miniprepping the Bacillus colonies and running a double digest to separate insert from vector, some colonies were found to contain 2 bands of the correct size, suggesting that the greater stringency behind use of promoters in Bacillus may be the cause. No DNA was recovered from the miniprep of pNZ + BBa_I13604.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For pPL82:</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For pPL82:</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Bruckner R. A series of shuttle vectors for Bacillus subtilis and Escherichia coli. Gene. 1992 Dec 1;122(1):187-92. [http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&list_uids=1452028&dopt=Citation]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Bruckner R. A series of shuttle vectors for Bacillus subtilis and Escherichia coli. Gene. 1992 Dec 1;122(1):187-92. [http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&list_uids=1452028&dopt=Citation]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Anagnostopoulos C, Spizizen J. REQUIREMENTS FOR TRANSFORMATION IN BACILLUS SUBTILIS. J Bacteriol. 1961 May;81(5):741-6. [http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&list_uids=16561900&dopt=Citation]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Anagnostopoulos C, Spizizen J. REQUIREMENTS FOR TRANSFORMATION IN BACILLUS SUBTILIS. J Bacteriol. 1961 May;81(5):741-6. [http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&list_uids=16561900&dopt=Citation]</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div># Yansura DG, Henner DJ. Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis. Proc Natl Acad Sci <del class="diffchange diffchange-inline">U S A</del>. 1984 Jan;81(2):439-43. [http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=retrieve&db=pubmed&list_uids=6420789&dopt=Citation]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div># Yansura DG, Henner DJ. Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis. Proc Natl Acad Sci <ins class="diffchange diffchange-inline">USA</ins>. 1984 Jan;81(2):439-43. [http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=retrieve&db=pubmed&list_uids=6420789&dopt=Citation<ins class="diffchange diffchange-inline">]</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 class="diffchange diffchange-inline"># Quisel JD, Burkholder WF, Grossman AD. In vivo effects of sporulation kinases on mutant Spo0A proteins in Bacillus subtilis. J Bacteriol. 2001 Nov;183(22):6573-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=11673427]</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 class="diffchange diffchange-inline"># Peschke U, Beuck V, Bujard H, Gentz R, Le Grice S. Efficient Utilization of E. coli transcriptional signals in B. subtilis. J Mol Biol 1985. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=2419570</ins>]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Bongers RS, Veening JW, Van Wieringen M, Kuipers OP, Kleerebezem M. Development and characterization of a subtilin-regulated expression system in Bacillus subtilis: strict control of gene expression by addition of subtilin. Appl Environ Microbiol. 2005 Dec;71(12):8818-24. [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1317459]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Bongers RS, Veening JW, Van Wieringen M, Kuipers OP, Kleerebezem M. Development and characterization of a subtilin-regulated expression system in Bacillus subtilis: strict control of gene expression by addition of subtilin. Appl Environ Microbiol. 2005 Dec;71(12):8818-24. [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1317459]</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;"># Peschke et. al. Efficient Utilization of Escherichia coli Transcriptional Signals in Bacillus subtilis. J. Mol. Biol. (1985) 186, 547-555. [http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6WK7-4FNGC6X-3B-2&_cdi=6899&_user=1495569&_orig=search&_coverDate=12%2F05%2F1985&_sk=998139996&view=c&wchp=dGLbVzz-zSkzk&md5=f2bb837faf66fe2ce34b84f9f822d688&ie=/sdarticle.pdf]</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;"># Quisel et.al. In Vivo Effects of Sporulation Kinases on Mutant Spo0A Proteins in Bacillus subtilis.J Bacteriol. 2001 November; 183(22): 6573–6578. [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=11673427]</del></div></td><td colspan="2"> </td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Postscript: The great Bacillus competency/transformation assay ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Postscript: The great Bacillus competency/transformation assay ==</div></td></tr>
</table>Yijinhttp://2007.igem.org/wiki/index.php?title=Cambridge/Gram-positive_chassis_project&diff=45998&oldid=prevSheilasoh: /* References */2007-10-26T23:41:41Z<p><span class="autocomment">References</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Yansura DG, Henner DJ. Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis. Proc Natl Acad Sci U S A. 1984 Jan;81(2):439-43. [http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=retrieve&db=pubmed&list_uids=6420789&dopt=Citation]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Yansura DG, Henner DJ. Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis. Proc Natl Acad Sci U S A. 1984 Jan;81(2):439-43. [http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=retrieve&db=pubmed&list_uids=6420789&dopt=Citation]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Bongers RS, Veening JW, Van Wieringen M, Kuipers OP, Kleerebezem M. Development and characterization of a subtilin-regulated expression system in Bacillus subtilis: strict control of gene expression by addition of subtilin. Appl Environ Microbiol. 2005 Dec;71(12):8818-24. [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1317459]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Bongers RS, Veening JW, Van Wieringen M, Kuipers OP, Kleerebezem M. Development and characterization of a subtilin-regulated expression system in Bacillus subtilis: strict control of gene expression by addition of subtilin. Appl Environ Microbiol. 2005 Dec;71(12):8818-24. [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1317459]</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;"># Peschke et. al. Efficient Utilization of Escherichia coli Transcriptional Signals in Bacillus subtilis. J. Mol. Biol. (1985) 186, 547-555. [http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6WK7-4FNGC6X-3B-2&_cdi=6899&_user=1495569&_orig=search&_coverDate=12%2F05%2F1985&_sk=998139996&view=c&wchp=dGLbVzz-zSkzk&md5=f2bb837faf66fe2ce34b84f9f822d688&ie=/sdarticle.pdf]</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;"># Quisel et.al. In Vivo Effects of Sporulation Kinases on Mutant Spo0A Proteins in Bacillus subtilis.J Bacteriol. 2001 November; 183(22): 6573–6578. [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=11673427]</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>== Postscript: The great Bacillus competency/transformation assay ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Postscript: The great Bacillus competency/transformation assay ==</div></td></tr>
</table>Sheilasohhttp://2007.igem.org/wiki/index.php?title=Cambridge/Gram-positive_chassis_project&diff=44112&oldid=prevYijin: /* Details */2007-10-26T19:43:19Z<p><span class="autocomment">Details</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In addition, for the [[Cambridge/Signalling project|signalling project]] we intend to transfer our ''agr'' BioBricks into ''B. subtilis'' and later, using the FepA permeability device, into ''E. coli'' - if successful, this will allow cross-talk between two very different bacterial species.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In addition, for the [[Cambridge/Signalling project|signalling project]] we intend to transfer our ''agr'' BioBricks into ''B. subtilis'' and later, using the FepA permeability device, into ''E. coli'' - if successful, this will allow cross-talk between two very different bacterial species.</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>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Acquiring our Bacillus strains: the ''B. subtilis'' shopping list ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Acquiring our Bacillus strains: the ''B. subtilis'' shopping list ==</div></td></tr>
</table>Yijinhttp://2007.igem.org/wiki/index.php?title=Cambridge/Gram-positive_chassis_project&diff=44110&oldid=prevYijin: /* Research on ''B. subtilis'' */2007-10-26T19:43:04Z<p><span class="autocomment">Research on ''B. subtilis''</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In addition, for the [[Cambridge/Signalling project|signalling project]] we intend to transfer our ''agr'' BioBricks into ''B. subtilis'' and later, using the FepA permeability device, into ''E. coli'' - if successful, this will allow cross-talk between two very different bacterial species.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In addition, for the [[Cambridge/Signalling project|signalling project]] we intend to transfer our ''agr'' BioBricks into ''B. subtilis'' and later, using the FepA permeability device, into ''E. coli'' - if successful, this will allow cross-talk between two very different bacterial species.</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;">== Research on ''B. subtilis'' ==</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: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">Check out the papers we've trawled through [http://www.ccbi.cam.ac.uk/iGEM2007/index.php/Detailed_background_information_-_B._subtilis here].</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>== Acquiring our Bacillus strains: the ''B. subtilis'' shopping list ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Acquiring our Bacillus strains: the ''B. subtilis'' shopping list ==</div></td></tr>
</table>Yijinhttp://2007.igem.org/wiki/index.php?title=Cambridge/Gram-positive_chassis_project&diff=44102&oldid=prevYijin: /* References */2007-10-26T19:41:47Z<p><span class="autocomment">References</span></p>
<table style="background-color: white; color:black;">
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Yansura DG, Henner DJ. Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis. Proc Natl Acad Sci U S A. 1984 Jan;81(2):439-43. [http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=retrieve&db=pubmed&list_uids=6420789&dopt=Citation]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Yansura DG, Henner DJ. Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis. Proc Natl Acad Sci U S A. 1984 Jan;81(2):439-43. [http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=retrieve&db=pubmed&list_uids=6420789&dopt=Citation]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Bongers RS, Veening JW, Van Wieringen M, Kuipers OP, Kleerebezem M. Development and characterization of a subtilin-regulated expression system in Bacillus subtilis: strict control of gene expression by addition of subtilin. Appl Environ Microbiol. 2005 Dec;71(12):8818-24. [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1317459]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Bongers RS, Veening JW, Van Wieringen M, Kuipers OP, Kleerebezem M. Development and characterization of a subtilin-regulated expression system in Bacillus subtilis: strict control of gene expression by addition of subtilin. Appl Environ Microbiol. 2005 Dec;71(12):8818-24. [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1317459]</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;">For a more detailed view of our research, please visit [http://www.ccbi.cam.ac.uk/iGEM2007/index.php/Detailed_background_information_-_B._subtilis Detailed background information - B. subtilis].</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>== Postscript: The great Bacillus competency/transformation assay ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Postscript: The great Bacillus competency/transformation assay ==</div></td></tr>
</table>Yijinhttp://2007.igem.org/wiki/index.php?title=Cambridge/Gram-positive_chassis_project&diff=38635&oldid=prevYijin: /* References */2007-10-25T22:46:59Z<p><span class="autocomment">References</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Bongers RS, Veening JW, Van Wieringen M, Kuipers OP, Kleerebezem M. Development and characterization of a subtilin-regulated expression system in Bacillus subtilis: strict control of gene expression by addition of subtilin. Appl Environ Microbiol. 2005 Dec;71(12):8818-24. [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1317459]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Bongers RS, Veening JW, Van Wieringen M, Kuipers OP, Kleerebezem M. Development and characterization of a subtilin-regulated expression system in Bacillus subtilis: strict control of gene expression by addition of subtilin. Appl Environ Microbiol. 2005 Dec;71(12):8818-24. [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1317459]</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>For a more detailed view of our research, please visit [<del class="diffchange diffchange-inline">[</del>Detailed background information - B. subtilis]<del class="diffchange diffchange-inline">]</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>For a more detailed view of our research, please visit [<ins class="diffchange diffchange-inline">http://www.ccbi.cam.ac.uk/iGEM2007/index.php/Detailed_background_information_-_B._subtilis </ins>Detailed background information - B. subtilis]<ins class="diffchange diffchange-inline">.</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>== Postscript: The great Bacillus competency/transformation assay ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Postscript: The great Bacillus competency/transformation assay ==</div></td></tr>
</table>Yijinhttp://2007.igem.org/wiki/index.php?title=Cambridge/Gram-positive_chassis_project&diff=38633&oldid=prevYijin: /* Introduction */2007-10-25T22:46:33Z<p><span class="autocomment">Introduction</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We have received a lot of help from a Bacillus lab in Newcastle - they sent us the protocol they use in making ''B. subtilis'' competent, as well as their transformation protocol. Both protocols were largely similar, hence the resulting protocol used in the assay is a merged version of both protocols.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We have received a lot of help from a Bacillus lab in Newcastle - they sent us the protocol they use in making ''B. subtilis'' competent, as well as their transformation protocol. Both protocols were largely similar, hence the resulting protocol used in the assay is a merged version of both protocols.</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>For experimental details, please refer to the article on [<del class="diffchange diffchange-inline">[</del>the great Bacillus competency/transformation assay<del class="diffchange diffchange-inline">]</del>].</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>For experimental details, please refer to the article on [<ins class="diffchange diffchange-inline">http://www.ccbi.cam.ac.uk/iGEM2007/index.php/The_great_Bacillus_competency/transformation_assay </ins>the great Bacillus competency/transformation assay].</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== Conclusions ===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== Conclusions ===</div></td></tr>
</table>Yijinhttp://2007.igem.org/wiki/index.php?title=Cambridge/Gram-positive_chassis_project&diff=38631&oldid=prevYijin: /* Achievements */2007-10-25T22:45:47Z<p><span class="autocomment">Achievements</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== Achievements ===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== Achievements ===</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 can say for sure that we have successfully introduced J04450, containing R0010 (the lacI promoter), into ''B. subtilis'', and it did not work. The problem lies more with the promoter than the fluorescent proteins, as other fluorescent proteins (GFP, CFP, YFP) have been shown to work in ''B. subtilis'' [http://aem.asm.org/cgi/content/full/68/5/2624?view=long&pmid=11976148].</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We can say for sure that we have successfully introduced J04450, containing R0010 (the lacI promoter), into ''B. subtilis'', and it did not work. The problem lies more with the promoter than the fluorescent proteins, as other fluorescent proteins (GFP, CFP, YFP) have been shown to work in ''B. subtilis'' <ins class="diffchange diffchange-inline">(</ins>[http://aem.asm.org/cgi/content/full/68/5/2624?view=long&pmid=11976148]<ins class="diffchange diffchange-inline">)</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>For the other promoters, we have attempted to introduce it into ''B. subtilis'' but had never observed expression of fluorescent proteins, and have yet to be able to confirm that the introduced plasmid (which conferred antibiotic resistance) could be stably inherited.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For the other promoters, we have attempted to introduce it into ''B. subtilis'' but had never observed expression of fluorescent proteins, and have yet to be able to confirm that the introduced plasmid (which conferred antibiotic resistance) could be stably inherited.</div></td></tr>
</table>Yijinhttp://2007.igem.org/wiki/index.php?title=Cambridge/Gram-positive_chassis_project&diff=38627&oldid=prevYijin: /* Summary table of vectors tested */2007-10-25T22:45:03Z<p><span class="autocomment">Summary table of vectors tested</span></p>
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<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>| pBS42</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| pBS42</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>| Unsure, discussed in next section ([<del class="diffchange diffchange-inline">[Media</del>: <del class="diffchange diffchange-inline">PBS42 shuttle vector</del>.txt<del class="diffchange diffchange-inline">|</del>GenBank format<del class="diffchange diffchange-inline">]</del>], [<del class="diffchange diffchange-inline">[Media</del>: <del class="diffchange diffchange-inline">PBS42 shuttle vector</del>.pdf<del class="diffchange diffchange-inline">|</del>pdf<del class="diffchange diffchange-inline">]</del>])</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>| Unsure, discussed in next section ([<ins class="diffchange diffchange-inline">http</ins>:<ins class="diffchange diffchange-inline">//www.ccbi.cam.ac.uk/iGEM2007/images/3/35/PBS42_shuttle_vector</ins>.txt GenBank format], [<ins class="diffchange diffchange-inline">http</ins>:<ins class="diffchange diffchange-inline">//www.ccbi.cam.ac.uk/iGEM2007/images/0/08/PBS42_shuttle_vector</ins>.pdf pdf])</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| Yes</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| Yes</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| Yes (but not when it's modified)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| Yes (but not when it's modified)</div></td></tr>
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<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>| pNZ8901</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| pNZ8901</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>| Yes ([<del class="diffchange diffchange-inline">[Media</del>:<del class="diffchange diffchange-inline">PNZ8901 shuttle vector</del>.txt<del class="diffchange diffchange-inline">|</del>GenBank format<del class="diffchange diffchange-inline">]</del>], [<del class="diffchange diffchange-inline">[Media</del>:<del class="diffchange diffchange-inline">PNZ8901 shuttle vector</del>.pdf<del class="diffchange diffchange-inline">|</del>pdf<del class="diffchange diffchange-inline">]</del>])</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>| Yes ([<ins class="diffchange diffchange-inline">http</ins>:<ins class="diffchange diffchange-inline">//www.ccbi.cam.ac.uk/iGEM2007/images/d/da/PNZ8901_shuttle_vector</ins>.txt GenBank format], [<ins class="diffchange diffchange-inline">http</ins>:<ins class="diffchange diffchange-inline">//www.ccbi.cam.ac.uk/iGEM2007/images/4/4c/PNZ8901_shuttle_vector</ins>.pdf pdf])</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| Yes (2 days)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| Yes (2 days)</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| Yes (Overnight)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| Yes (Overnight)</div></td></tr>
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<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>| pPL82</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| pPL82</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>| Yes ([<del class="diffchange diffchange-inline">[Media</del>:<del class="diffchange diffchange-inline">PDR67 vector</del>.txt<del class="diffchange diffchange-inline">|</del>Similar to pDR67<del class="diffchange diffchange-inline">]</del>], [<del class="diffchange diffchange-inline">[Media</del>: <del class="diffchange diffchange-inline">PDR67 vector</del>.pdf<del class="diffchange diffchange-inline">|</del>pdf<del class="diffchange diffchange-inline">]</del>])</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>| Yes ([<ins class="diffchange diffchange-inline">http</ins>:<ins class="diffchange diffchange-inline">//www.ccbi.cam.ac.uk/iGEM2007/images/c/c3/PDR67_vector</ins>.txt Similar to pDR67], [<ins class="diffchange diffchange-inline">http</ins>:<ins class="diffchange diffchange-inline">//www.ccbi.cam.ac.uk/iGEM2007/images/3/39/PDR67_vector</ins>.pdf pdf])</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| Yes (2 days)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| Yes (2 days)</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| Yes (Overnight)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| Yes (Overnight)</div></td></tr>
</table>Yijinhttp://2007.igem.org/wiki/index.php?title=Cambridge/Gram-positive_chassis_project&diff=38616&oldid=prevYijin: /* Research on ''B. subtilis'' */2007-10-25T22:42:15Z<p><span class="autocomment">Research on ''B. subtilis''</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Research on ''B. subtilis'' ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Research on ''B. subtilis'' ==</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>Check out the papers we've trawled through [<del class="diffchange diffchange-inline">[Detailed background information </del>- <del class="diffchange diffchange-inline">B</del>. <del class="diffchange diffchange-inline">subtilis|</del>here<del class="diffchange diffchange-inline">]</del>].</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Check out the papers we've trawled through [<ins class="diffchange diffchange-inline">http://www.ccbi.cam.ac.uk/iGEM2007/index.php/Detailed_background_information_</ins>-<ins class="diffchange diffchange-inline">_B</ins>.<ins class="diffchange diffchange-inline">_subtilis </ins>here].</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>== Acquiring our Bacillus strains: the ''B. subtilis'' shopping list ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Acquiring our Bacillus strains: the ''B. subtilis'' shopping list ==</div></td></tr>
</table>Yijin