http://2008.igem.org/wiki/index.php?title=Team:Heidelberg/Project/Killing_I&feed=atom&action=historyTeam:Heidelberg/Project/Killing I - Revision history2024-03-29T09:03:52ZRevision history for this page on the wikiMediaWiki 1.16.5http://2008.igem.org/wiki/index.php?title=Team:Heidelberg/Project/Killing_I&diff=91679&oldid=prevYincai: /* Outlook */2008-10-29T16:49:59Z<p><span class="autocomment">Outlook</span></p>
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<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;">== '''Acknowledgements ''' ==</ins></div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">We would heartily like to extend our thanks to Prof. M. Bagdasarian 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;">Michigan State University, to Prof. K. M. Derbyshire from Wadsworth </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;">Center, New York State Department of Health and Prof. E. Lanka 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;">Bundesinstitute für Risikobewertung (BfR), Berlin for their precious and </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;">unconditional help by giving us a lot of information about conjugation </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;">and sending us the plasmids timely. They were all extremely helpful and </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;">even if in the end we couldn’t use all of their provided materials we </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;">profited from our correspondence to all of them.</ins></div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">We would also like to thank Dr. A. Trendl-Kern, Dr. P. Prior and Dr. W. </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;">Reiser at the Practical Laboratory, Zentrum für Molekularbiologie </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;">Heidelberg (ZMBH) for their aspiring support throughout our work, who </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;">shared their knowledge with us and provided us with different λ phages </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;">and materials.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">We would also like to thank Sabine Aschenbrenner for helping us with </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;">technical assistance.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">We can’t imagine realizing our project without their helps. Thank you </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;">very much!</ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== '''References''' ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== '''References''' ==</div></td></tr>
</table>Yincaihttp://2008.igem.org/wiki/index.php?title=Team:Heidelberg/Project/Killing_I&diff=91403&oldid=prevMaximilian.hoerner: /* Phages - Project Description */2008-10-29T16:32:50Z<p><span class="autocomment">Phages - Project Description</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>3. An accumulation of Q makes the RNA polymerase resistant to the tR’ terminator and thereby allows expression of the late genes, which are essential for the lytic cycle. They encode for the viral coat proteins and for lysis proteins. These late genes are expressed with a delay after the delayed early genes, which results from the threshold concentration of Q that has to be accumulated to overcome the tR’ terminator.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>3. An accumulation of Q makes the RNA polymerase resistant to the tR’ terminator and thereby allows expression of the late genes, which are essential for the lytic cycle. They encode for the viral coat proteins and for lysis proteins. These late genes are expressed with a delay after the delayed early genes, which results from the threshold concentration of Q that has to be accumulated to overcome the tR’ terminator.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:Schema_regulatorgene.png|thumb|600px|'''Figure 1.2:''' Regulatory genes of the lambda phage]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:Schema_regulatorgene.png|thumb|600px|'''Figure 1.2:''' Regulatory genes of the lambda phage<ins class="diffchange diffchange-inline">.</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 contrast, the lysogenic state is also initiated during the transcription of the early delayed genes:</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In contrast, the lysogenic state is also initiated during the transcription of the early delayed genes:</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Image:Lambda_vector_pre.png|'''Figure 1.3:''' The original lambda genome</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Image:Lambda_vector_pre.png|'''Figure 1.3:''' The original lambda genome<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>Image:Lambda_vector_end.png|'''Figure 1.4:''' The engineered lambda genome</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Image:Lambda_vector_end.png|'''Figure 1.4:''' The engineered lambda genome <ins class="diffchange diffchange-inline">including the oriT, chloramphenicol resistance and GFP.</ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To avoid that our probiotic bacteria, which would contain and transfer the phage to the target pathogenic bacteria, would also be killed by the phage, we used the natural way to make them immune. Therefore we took the lambda cI gene and put it behind a constitutive promoter. The cI protein will then prevent the expression of the lambda genome in our bacteria and prevent them from being lysed.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To avoid that our probiotic bacteria, which would contain and transfer the phage to the target pathogenic bacteria, would also be killed by the phage, we used the natural way to make them immune. Therefore we took the lambda cI gene and put it behind a constitutive promoter. The cI protein will then prevent the expression of the lambda genome in our bacteria and prevent them from being lysed.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[https://2008.igem.org/Team:Heidelberg/Project/Killing_I back]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[https://2008.igem.org/Team:Heidelberg/Project/Killing_I back]]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Cloning strategy===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Cloning strategy===</div></td></tr>
</table>Maximilian.hoernerhttp://2008.igem.org/wiki/index.php?title=Team:Heidelberg/Project/Killing_I&diff=91365&oldid=prevMaximilian.hoerner: /* Cloning strategy */2008-10-29T16:30:18Z<p><span class="autocomment">Cloning strategy</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Cloning strategy===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Cloning strategy===</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Our goal to clone a modified bacteriophage λ which can be transported by conjugation, allows the selection of infected cells through a chloamphenicol resistance and harbours GFP for visualization was designed in two strategies whereas strategy two builds on strategy one. A detailed description of the two strategies can be seen below (see 1.<del class="diffchange diffchange-inline">1 </del>and 1.<del class="diffchange diffchange-inline">2</del>). The used λ cI protein generator was synthesised by geneart and just transferred in a standard plasmid as it </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Our goal to clone a modified bacteriophage λ which can be transported by conjugation, allows the selection of infected cells through a chloamphenicol resistance and harbours GFP for visualization was designed in two strategies whereas strategy two builds on strategy one. A detailed description of the two strategies can be seen below (see <ins class="diffchange diffchange-inline">figure </ins>1.<ins class="diffchange diffchange-inline">6 </ins>and 1.<ins class="diffchange diffchange-inline">7</ins>). The used λ cI protein generator was synthesised by geneart and just transferred in a standard plasmid as it </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>can be seen in 1.<del class="diffchange diffchange-inline">3</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>can be seen in <ins class="diffchange diffchange-inline">figure </ins>1.<ins class="diffchange diffchange-inline">8</ins>.</div></td></tr>
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</table>Maximilian.hoernerhttp://2008.igem.org/wiki/index.php?title=Team:Heidelberg/Project/Killing_I&diff=91288&oldid=prevMaximilian.hoerner at 16:24, 29 October 20082008-10-29T16:24:21Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For this test ''E. coli'' Top10 cells harboring our helper plasmid pUB307 which were not able to grow on ampicillin or chloramphenicol were used to conjugate this plasmid in ''E. coli'' Top10 infected with our modified phage which therefore were able to grow on chloramphenicol. The culture of cells infected with the phage did only grow in chloramphenicol but not in ampicillin or kanamycin what proofs that our insert was introduced in the λ phage and did not still exist in its cloning vector pBluescript SK II since this one should also provide the cells with a resistance to ampicillin. The conjugation leads to cells able to grow on kanamycin and chloramphenicol but not on ampicillin. In another conjugation step it could be shown that the λ phage can be transported by conjugation in E. coli Top10 harboring the part BBa_T9002 on pSB1A3 since the resulting bacteria were able to grow on chloramphenicol and ampicillin. Summarizing we could proof that our modified λ phage can be transported by conjugation in presence of the helper plasmid pUB307.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For this test ''E. coli'' Top10 cells harboring our helper plasmid pUB307 which were not able to grow on ampicillin or chloramphenicol were used to conjugate this plasmid in ''E. coli'' Top10 infected with our modified phage which therefore were able to grow on chloramphenicol. The culture of cells infected with the phage did only grow in chloramphenicol but not in ampicillin or kanamycin what proofs that our insert was introduced in the λ phage and did not still exist in its cloning vector pBluescript SK II since this one should also provide the cells with a resistance to ampicillin. The conjugation leads to cells able to grow on kanamycin and chloramphenicol but not on ampicillin. In another conjugation step it could be shown that the λ phage can be transported by conjugation in E. coli Top10 harboring the part BBa_T9002 on pSB1A3 since the resulting bacteria were able to grow on chloramphenicol and ampicillin. Summarizing we could proof that our modified λ phage can be transported by conjugation in presence of the helper plasmid pUB307.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:HD_conjugationtest.png|center|thumb|600px|'''Figure 2.1.7:''' <del class="diffchange diffchange-inline">schematic </del>visualization of the proof that our modified λ phage is transported by conjugation. The resulting cells harboring the modified λ phage as well as the part BBa_T9002 on pSB1A3 were able to grow on chloramphenicol and ampicillin.]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:HD_conjugationtest.png|center|thumb|600px|'''Figure 2.1.7:''' <ins class="diffchange diffchange-inline">Schematic </ins>visualization of the proof that our modified λ phage is transported by conjugation. The resulting cells harboring the modified λ phage as well as the part BBa_T9002 on pSB1A3 were able to grow on chloramphenicol and ampicillin.]]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Image:Hd-phages-oriT-pcr.jpg|'''Figure 2.2.1:''' PCR products of RP4 oriT with pMMB863 as template.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Image:Hd-phages-oriT-pcr.jpg|'''Figure 2.2.1:''' PCR products of RP4 oriT with pMMB863 as template.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Image:Hd-phages-oriT-digestion.jpg|'''Figure 2.2.2:''' <del class="diffchange diffchange-inline">control </del>digestion of the oriT cloned in pSB1A2 standard plasmid with EcoRI/PstI. The sizes of the insert (~400kb) as well as the pSB1A2 backbone (~2kb) are correct. Lanes were merged on the computer to remove lanes that were not part of this project. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Image:Hd-phages-oriT-digestion.jpg|'''Figure 2.2.2:''' <ins class="diffchange diffchange-inline">Control </ins>digestion of the oriT cloned in pSB1A2 standard plasmid with EcoRI/PstI. The sizes of the insert (~400kb) as well as the pSB1A2 backbone (~2kb) are correct. Lanes were merged on the computer to remove lanes that were not part of this project. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Image:Hd-phages-terminator-pcr.jpg|'''Figure 2.3.2:''' PCR product of the terminator BBa_B0015 with the correct size of 150 bp amplified out its standard plasmid.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Image:Hd-phages-terminator-pcr.jpg|'''Figure 2.3.2:''' PCR product of the terminator BBa_B0015 with the correct size of 150 bp amplified out its standard plasmid.</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>Image:Hd-phages-CmR-pBlue-digestion.jpg|'''Figure 2.3.3:''' <del class="diffchange diffchange-inline">control </del>digestion to proof that the ligation of the coding chloramphenicol resistance sequence inclduding its promotor and ribosome binding site (see figure 2.3.1) with the terminator BBa_B0015 (see figure2.3.2) was successful.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Image:Hd-phages-CmR-pBlue-digestion.jpg|'''Figure 2.3.3:''' <ins class="diffchange diffchange-inline">Control </ins>digestion to proof that the ligation of the coding chloramphenicol resistance sequence inclduding its promotor and ribosome binding site (see figure 2.3.1) with the terminator BBa_B0015 (see figure2.3.2) was successful.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Image:Hd-phages-CmR-with-terminator-pcr.jpg|'''Figure 2.3.4:''' PCR product of the complete chloramphenicol resistance cassette with standard prefix and suffix.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Image:Hd-phages-CmR-with-terminator-pcr.jpg|'''Figure 2.3.4:''' PCR product of the complete chloramphenicol resistance cassette with standard prefix and suffix.</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>Image:Hd-phages-CmR-pSB1A2-digestion.jpg|'''Figure 2.3.5:''' <del class="diffchange diffchange-inline">control </del>digestion of the complete unmutated chloramphenicol resistance cassette in the standard plasmid pSB1A2 with EcoRI and PstI. An EcoRI restriction site within the cassette, which isn't allowed by BioBrick standard, yields to three instead of the two expected bands. The 2 kb band represents the pSB1A2 backbone.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Image:Hd-phages-CmR-pSB1A2-digestion.jpg|'''Figure 2.3.5:''' <ins class="diffchange diffchange-inline">Control </ins>digestion of the complete unmutated chloramphenicol resistance cassette in the standard plasmid pSB1A2 with EcoRI and PstI. An EcoRI restriction site within the cassette, which isn't allowed by BioBrick standard, yields to three instead of the two expected bands. The 2 kb band represents the pSB1A2 backbone.</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>Image:Hd-phages-CmR-pSB1A2-mut-digestion.jpg|'''Figure 2.3.6:''' <del class="diffchange diffchange-inline">control </del>digestion of the complete mutated chloramphenicol resistance cassette in the standard plasmid pSB1A2 with EcoRI and PstI. The EcoRI restricition site, led to the unwanted third band in figure 2.3.5 was removed by mutagenesis PCR. The sizes of the insert (~1kb) as well as the pSB1A2 backbone (~2kb) are correct.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Image:Hd-phages-CmR-pSB1A2-mut-digestion.jpg|'''Figure 2.3.6:''' <ins class="diffchange diffchange-inline">Control </ins>digestion of the complete mutated chloramphenicol resistance cassette in the standard plasmid pSB1A2 with EcoRI and PstI. The EcoRI restricition site, led to the unwanted third band in figure 2.3.5 was removed by mutagenesis PCR. The sizes of the insert (~1kb) as well as the pSB1A2 backbone (~2kb) are correct.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><gallery widths ="408" heights = "350" perrow="2"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><gallery widths ="408" heights = "350" perrow="2"></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>Image:Hd-phage-pBlue mutkpn.jpg|'''Figure 2.4.1:''' <del class="diffchange diffchange-inline">success </del>of the mutagenesis PCR to remove an unwanted KpnI restriction site.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Image:Hd-phage-pBlue mutkpn.jpg|'''Figure 2.4.1:''' <ins class="diffchange diffchange-inline">Success </ins>of the mutagenesis PCR to remove an unwanted KpnI restriction site.</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>Image:Hd-phage-pBlue insert2-controldigestion.jpg|'''Figure 2.4.2:''' <del class="diffchange diffchange-inline">control </del>digestion of the new insert in pBluescript SK II with different restriction enzymes lead to the correct band pattern. So one can conclude that this strategy also succeeded and the insert is ready for cloning in the λ phage.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Image:Hd-phage-pBlue insert2-controldigestion.jpg|'''Figure 2.4.2:''' <ins class="diffchange diffchange-inline">Control </ins>digestion of the new insert in pBluescript SK II with different restriction enzymes lead to the correct band pattern. So one can conclude that this strategy also succeeded and the insert is ready for cloning in the λ phage.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Image:Hd-phages-cI-J01003.jpg|'''Figure 2.5.1:''' <del class="diffchange diffchange-inline">restriction </del>digestion of bacteriophage λ cI in pMK as synthesised by Geneart and BBa_J01003 in pSB1A2 for gel extraction and ligation. From lane 1 and 2 the 1 kb fragment (cI cassette) and from lane 3 and 4 the 2 kb fragment (pSB1A2 backbone) was extracted from the gel.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Image:Hd-phages-cI-J01003.jpg|'''Figure 2.5.1:''' <ins class="diffchange diffchange-inline">Restriction </ins>digestion of bacteriophage λ cI in pMK as synthesised by Geneart and BBa_J01003 in pSB1A2 for gel extraction and ligation. From lane 1 and 2 the 1 kb fragment (cI cassette) and from lane 3 and 4 the 2 kb fragment (pSB1A2 backbone) was extracted from the gel.</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>Image:Hd-phages-cI-pSB1A2.jpg|'''Figure 2.5.2:''' <del class="diffchange diffchange-inline">control </del>digestion of bacteriophage λ cI in pSB1A2 with EcoRI/PstI after mutagenesis PCR to eliminate an unwanted EcoRI site.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Image:Hd-phages-cI-pSB1A2.jpg|'''Figure 2.5.2:''' <ins class="diffchange diffchange-inline">Control </ins>digestion of bacteriophage λ cI in pSB1A2 with EcoRI/PstI after mutagenesis PCR to eliminate an unwanted EcoRI site.</div></td></tr>
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</table>Maximilian.hoernerhttp://2008.igem.org/wiki/index.php?title=Team:Heidelberg/Project/Killing_I&diff=91214&oldid=prevMaximilian.hoerner at 16:20, 29 October 20082008-10-29T16:20:02Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Chloramphenicol resistance cassette===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Chloramphenicol resistance cassette===</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The conjugation system may allow a broader application of our system in terms of species, since the RP-plasmids are broad host range plasmids and can therefore transfer genetic information to a variety of gram negative and gram positive bacteria. There is also evidence for conjugative transfer between bacteria and yeast cells [[Team:Heidelberg/Project/Killing_I#References|[17]]]. If one could use this system to transfer genetic information into eukaryotic cells, this could be an application to cure plant diseases or even human diseases by injecting for example antisense RNA to silence viral replication or oncogenes.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The conjugation system may allow a broader application of our system in terms of species, since the RP-plasmids are broad host range plasmids and can therefore transfer genetic information to a variety of gram negative and gram positive bacteria. There is also evidence for conjugative transfer between bacteria and yeast cells [[Team:Heidelberg/Project/Killing_I#References|[17]]]. If one could use this system to transfer genetic information into eukaryotic cells, this could be an application to cure plant diseases or even human diseases by injecting for example antisense RNA to silence viral replication or oncogenes.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[https://2008.igem.org/Team:Heidelberg/Project/Killing_I back]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[https://2008.igem.org/Team:Heidelberg/Project/Killing_I back]]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== '''References''' ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== '''References''' ==</div></td></tr>
</table>Maximilian.hoernerhttp://2008.igem.org/wiki/index.php?title=Team:Heidelberg/Project/Killing_I&diff=91193&oldid=prevMaximilian.hoerner at 16:18, 29 October 20082008-10-29T16:18:44Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Our goal to clone a modified bacteriophage λ which can be transported by conjugation, allows the selection of infected cells through a chloamphenicol resistance and harbours GFP for visualization was designed in two strategies whereas strategy two builds on strategy one. A detailed description of the two strategies can be seen below (see 1.1 and 1.2). The used λ cI protein generator was synthesised by geneart and just transferred in a standard plasmid as it </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Our goal to clone a modified bacteriophage λ which can be transported by conjugation, allows the selection of infected cells through a chloamphenicol resistance and harbours GFP for visualization was designed in two strategies whereas strategy two builds on strategy one. A detailed description of the two strategies can be seen below (see 1.1 and 1.2). The used λ cI protein generator was synthesised by geneart and just transferred in a standard plasmid as it </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>can be seen in 1.3.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>can be seen in 1.3.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:HD_phage2_klein.png|center|thumb|500px|'''Figure 1.7:''' Overview of the phage cloning strategy two.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:HD_phage2_klein.png|center|thumb|500px|'''Figure 1.7:''' Overview of the phage cloning strategy two.]]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== '''Results''' ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== '''Results''' ==</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>====proof of conjugation====</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>====proof of conjugation====</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:HD_conjugationtest.png|center|thumb|600px|'''Figure 2.1.7:''' schematic visualization of the proof that our modified λ phage is transported by conjugation. The resulting cells harboring the modified λ phage as well as the part BBa_T9002 on pSB1A3 were able to grow on chloramphenicol and ampicillin.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:HD_conjugationtest.png|center|thumb|600px|'''Figure 2.1.7:''' schematic visualization of the proof that our modified λ phage is transported by conjugation. The resulting cells harboring the modified λ phage as well as the part BBa_T9002 on pSB1A3 were able to grow on chloramphenicol and ampicillin.]]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== oriT ===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== oriT ===</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[https://2008.igem.org/Team:Heidelberg/Project/Killing_I back]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[https://2008.igem.org/Team:Heidelberg/Project/Killing_I back]]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== phage cloning strategy two===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== phage cloning strategy two===</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></gallery></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></gallery></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== cI ===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== cI ===</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== '''Outlook''' ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== '''Outlook''' ==</div></td></tr>
</table>Maximilian.hoernerhttp://2008.igem.org/wiki/index.php?title=Team:Heidelberg/Project/Killing_I&diff=91155&oldid=prevMaximilian.hoerner: /* Introduction */2008-10-29T16:16:46Z<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>To avoid that our probiotic bacteria, which would contain and transfer the phage to the target pathogenic bacteria, would also be killed by the phage, we used the natural way to make them immune. Therefore we took the lambda cI gene and put it behind a constitutive promoter. The cI protein will then prevent the expression of the lambda genome in our bacteria and prevent them from being lysed.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To avoid that our probiotic bacteria, which would contain and transfer the phage to the target pathogenic bacteria, would also be killed by the phage, we used the natural way to make them immune. Therefore we took the lambda cI gene and put it behind a constitutive promoter. The cI protein will then prevent the expression of the lambda genome in our bacteria and prevent them from being lysed.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[https://2008.igem.org/Team:Heidelberg/Project/Killing_I back]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[https://2008.igem.org/Team:Heidelberg/Project/Killing_I back]]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Cloning strategy===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Cloning strategy===</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Our goal to clone a modified bacteriophage λ which can be transported by conjugation, allows the selection of infected cells through a chloamphenicol resistance and harbours GFP for visualization was designed in two strategies whereas strategy two builds on strategy one. A detailed description of the two strategies can be seen below (see 1.1 and 1.2). The used λ cI protein generator was synthesised by geneart and just transferred in a standard plasmid as it can be seen in 1.3.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Our goal to clone a modified bacteriophage λ which can be transported by conjugation, allows the selection of infected cells through a chloamphenicol resistance and harbours GFP for visualization was designed in two strategies whereas strategy two builds on strategy one. A detailed description of the two strategies can be seen below (see 1.1 and 1.2). The used λ cI protein generator was synthesised by geneart and just transferred in a standard plasmid as it </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:HD_phage2_klein.png|center|thumb|500px|'''Figure 1.7:''' Overview of the phage cloning strategy two.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:HD_phage2_klein.png|center|thumb|500px|'''Figure 1.7:''' Overview of the phage cloning strategy two.]]</div></td></tr>
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</table>Maximilian.hoernerhttp://2008.igem.org/wiki/index.php?title=Team:Heidelberg/Project/Killing_I&diff=91147&oldid=prevBdv: /* Cloning strategy */2008-10-29T16:16:15Z<p><span class="autocomment">Cloning strategy</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>==== <del class="diffchange diffchange-inline">phage </del>cloning strategy one====</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>==== <ins class="diffchange diffchange-inline">Phage </ins>cloning strategy one====</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:HD_phage1_klein.gif|center|thumb|500px|'''Figure 1.6:''' Overview of the phage cloning strategy one.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:HD_phage1_klein.gif|center|thumb|500px|'''Figure 1.6:''' Overview of the phage cloning strategy one.]]</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>==== <del class="diffchange diffchange-inline">phage </del>cloning strategy two====</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>==== <ins class="diffchange diffchange-inline">Phage </ins>cloning strategy two====</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:HD_phage2_klein.png|center|thumb|500px|'''Figure 1.7:''' Overview of the phage cloning strategy two.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:HD_phage2_klein.png|center|thumb|500px|'''Figure 1.7:''' Overview of the phage cloning strategy two.]]</div></td></tr>
</table>Bdvhttp://2008.igem.org/wiki/index.php?title=Team:Heidelberg/Project/Killing_I&diff=91133&oldid=prevMaximilian.hoerner: /* Phages - Project Description */2008-10-29T16:15:28Z<p><span class="autocomment">Phages - Project Description</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:Phage_killing.jpg|right|thumb|400px|Figure 1.1 <del class="diffchange diffchange-inline"> </del>Project overview]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:Phage_killing.jpg|right|thumb|400px|<ins class="diffchange diffchange-inline">'''</ins>Figure 1.1<ins class="diffchange diffchange-inline">:''' </ins>Project overview]]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Antibiotics are a very powerful way to cure bacterial infections, but their power withers as more and more bacterial strains become resistant to different antibiotics. The great power of evolution leads to chromosomal mutations that not only make bacteria resistant to natural antibiotics, but also to newly developed synthetic ones [[Team:Heidelberg/Project/Killing_I#References|[1]]]. Bacteria also have a very effective way to share those resistances with each other, not limited by species boundaries: the transfer of plasmids by conjugation (which will be investigated in more detail in the second part of the project). Multiresistant bacterial strains like ''Pseudomonas aeruginosa'', ''Staphylococcus aureus'' and ''Enterococcus faecalis'' hinder the usage of antibiotics against infectious diseases, because they cannot be killed by ordinary antibiotics and can give their plasmids to other pathogens, which then become resistant themselves. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Antibiotics are a very powerful way to cure bacterial infections, but their power withers as more and more bacterial strains become resistant to different antibiotics. The great power of evolution leads to chromosomal mutations that not only make bacteria resistant to natural antibiotics, but also to newly developed synthetic ones [[Team:Heidelberg/Project/Killing_I#References|[1]]]. Bacteria also have a very effective way to share those resistances with each other, not limited by species boundaries: the transfer of plasmids by conjugation (which will be investigated in more detail in the second part of the project). Multiresistant bacterial strains like ''Pseudomonas aeruginosa'', ''Staphylococcus aureus'' and ''Enterococcus faecalis'' hinder the usage of antibiotics against infectious diseases, because they cannot be killed by ordinary antibiotics and can give their plasmids to other pathogens, which then become resistant themselves. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>3. An accumulation of Q makes the RNA polymerase resistant to the tR’ terminator and thereby allows expression of the late genes, which are essential for the lytic cycle. They encode for the viral coat proteins and for lysis proteins. These late genes are expressed with a delay after the delayed early genes, which results from the threshold concentration of Q that has to be accumulated to overcome the tR’ terminator.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>3. An accumulation of Q makes the RNA polymerase resistant to the tR’ terminator and thereby allows expression of the late genes, which are essential for the lytic cycle. They encode for the viral coat proteins and for lysis proteins. These late genes are expressed with a delay after the delayed early genes, which results from the threshold concentration of Q that has to be accumulated to overcome the tR’ terminator.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:Schema_regulatorgene.png|thumb|600px|Figure 1.2 <del class="diffchange diffchange-inline"> </del>Regulatory genes of the lambda phage]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:Schema_regulatorgene.png|thumb|600px|<ins class="diffchange diffchange-inline">'''</ins>Figure 1.2<ins class="diffchange diffchange-inline">:''' </ins>Regulatory genes of the lambda phage]]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In contrast, the lysogenic state is also initiated during the transcription of the early delayed genes:</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In contrast, the lysogenic state is also initiated during the transcription of the early delayed genes:</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A conjugative plasmid of gram negative bacteria contains a characteristic set of genes and sequences. The tra locus includes the genes for the sex pili and for proteins that carry out synthesis, replication and transfer of the DNA during mating. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A conjugative plasmid of gram negative bacteria contains a characteristic set of genes and sequences. The tra locus includes the genes for the sex pili and for proteins that carry out synthesis, replication and transfer of the DNA during mating. </div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:Schema_conjugation.png|thumb|400px|Figure 1.5 <del class="diffchange diffchange-inline"> </del>Bacterial conjugation]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:Schema_conjugation.png|thumb|400px|<ins class="diffchange diffchange-inline">'''</ins>Figure 1.5<ins class="diffchange diffchange-inline">:''' </ins>Bacterial conjugation]]</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 sex pili are formed by pilin and regulatory proteins. These are polymeric structures that extend into the periphery from the surface of the donor cell and can attach to the surface of other bacteria and initiate conjugation. They do make the physical contact between the cells by retracting, but they do not form the channel, through which the DNA is shuttled.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The sex pili are formed by pilin and regulatory proteins. These are polymeric structures that extend into the periphery from the surface of the donor cell and can attach to the surface of other bacteria and initiate conjugation. They do make the physical contact between the cells by retracting, but they do not form the channel, through which the DNA is shuttled.</div></td></tr>
</table>Maximilian.hoernerhttp://2008.igem.org/wiki/index.php?title=Team:Heidelberg/Project/Killing_I&diff=91130&oldid=prevBdv: /* Phages - Project Description */2008-10-29T16:15:17Z<p><span class="autocomment">Phages - Project Description</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Whether to undergo a lytic or a lysogenic mode depends on the multiplicity of infection and the physiological conditions of the bacterial cell. Many phages infecting a cell favour lysogeny. Rich nutrition of the bacterium favours the lytic state, because it increases N expression levels. Since lytic and lysogenic genes are under the control of the same promoters, the critical point for the decision of a state are the RNA and protein levels, influenced by the activity of degrading enzymes [[Team:Heidelberg/Project/Killing_I#References|[4][5][6][7][8][9]]]. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Whether to undergo a lytic or a lysogenic mode depends on the multiplicity of infection and the physiological conditions of the bacterial cell. Many phages infecting a cell favour lysogeny. Rich nutrition of the bacterium favours the lytic state, because it increases N expression levels. Since lytic and lysogenic genes are under the control of the same promoters, the critical point for the decision of a state are the RNA and protein levels, influenced by the activity of degrading enzymes [[Team:Heidelberg/Project/Killing_I#References|[4][5][6][7][8][9]]]. </div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Our work <del class="diffchange diffchange-inline">focussed </del>on engineering a non-lysogenic phage, because we wanted to produce an efficient killing module for the probiotic bacterium. In preventing the phage from undergoing a lysogenic cycle, we assure that it will kill the target bacteria as fast as possible and without extern triggers. For this task we aimed at discarding the int gene from the phage genome, so that it would be incapable of integrating into the host genome. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Our work <ins class="diffchange diffchange-inline">focused </ins>on engineering a non-lysogenic phage, because we wanted to produce an efficient killing module for the probiotic bacterium. In preventing the phage from undergoing a lysogenic cycle, we assure that it will kill the target bacteria as fast as possible and without extern triggers. For this task we aimed at discarding the int gene from the phage genome, so that it would be incapable of integrating into the host genome. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Cloning work with the lambda phage genome is tricky, because it contains few single-cutter restriction sites. Therefore we had to cut out one fragment containing the int, the xis and another gene, gam. Xis and int can and shall be discarded, but the gam gene codes for a protein that prevents the overhanging single strains at the cos sites from being degraded by host enzymes. So we would need the gam gene in our engineered phage. This is why we designed an insert for the phage to put in the remaining genome after cutting out the fragment containing xis, int and gam. This insert contains gam, and an antibiotic selection marker to be able to select those bacterial clones that contain the lambda genome. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Cloning work with the lambda phage genome is tricky, because it contains few single-cutter restriction sites. Therefore we had to cut out one fragment containing the int, the xis and another gene, gam. Xis and int can and shall be discarded, but the gam gene codes for a protein that prevents the overhanging single strains at the cos sites from being degraded by host enzymes. So we would need the gam gene in our engineered phage. This is why we designed an insert for the phage to put in the remaining genome after cutting out the fragment containing xis, int and gam. This insert contains gam, and an antibiotic selection marker to be able to select those bacterial clones that contain the lambda genome. </div></td></tr>
</table>Bdv