http://2008.igem.org/wiki/index.php?title=Team:Caltech/Project/Phage_Pathogen_Defense&feed=atom&action=historyTeam:Caltech/Project/Phage Pathogen Defense - Revision history2024-03-28T17:11:44ZRevision history for this page on the wikiMediaWiki 1.16.5http://2008.igem.org/wiki/index.php?title=Team:Caltech/Project/Phage_Pathogen_Defense&diff=64417&oldid=prevJkm at 17:00, 25 October 20082008-10-25T17:00:16Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Overexpression of ''rcsA'' led to induction of λ lysogens. ''rcsA'' was placed downstream of an AHL inducible promoter and overexpressed through induction with 10 nM AHL. Following induction, phage concentration in the supernatant was recorded through titering. This concentration was compared to the phage concentration in the supernatant without ''rcsA'' overexpression, given by a construct where ''rcsA'' is not driven by a promoter. The data show a 40 fold increase in phage concentration between the promoter-less ''rcsA'' construct and the strongest AHL induced ''rcsA'' construct (Figure 5, columns 1 and 4). The promoter-less ''rcsA'' construct should accurately represent background phage levels for the lysogen absent of any ''rcsA'' expression. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Overexpression of ''rcsA'' led to induction of λ lysogens. ''rcsA'' was placed downstream of an AHL inducible promoter and overexpressed through induction with 10 nM AHL. Following induction, phage concentration in the supernatant was recorded through titering. This concentration was compared to the phage concentration in the supernatant without ''rcsA'' overexpression, given by a construct where ''rcsA'' is not driven by a promoter. The data show a 40 fold increase in phage concentration between the promoter-less ''rcsA'' construct and the strongest AHL induced ''rcsA'' construct (Figure 5, columns 1 and 4). The promoter-less ''rcsA'' construct should accurately represent background phage levels for the lysogen absent of any ''rcsA'' expression. </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Further work was done to characterize the response of the AHL inducible switch under three different RBSs. The data show induction efficiency is directly correlated with strength of ''rcsA'' overexpression (Figure 5, columns 2-4). In each of the constructs, there is a 3 to 5 fold increase in phage levels after induction with 10 <del class="diffchange diffchange-inline">nm </del>AHL. This increase does not accurately reflect the dynamic range of the AHL inducible promoter, because it does not take into account the background phage levels without the promoter. In the weaker RBSs (B0032 and B0033), the phage concentration from the uninduced supernatant is comparable to the background level from the promoter-less construct (Figure 5, columns 1-3). Thus, in these inducible promoter systems, there is high dynamic range and negligible background compared to basal phage concentrations. However, in the strongest RBS (B0034), the uninduced phage level is an order of magnitude higher than the promoter-less background level. This high basal level expression suggests that the higher expression levels due to the strong RBS is leading to leaky ''rcsA'' activation of the phage lytic cycle. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Further work was done to characterize the response of the AHL inducible switch under three different RBSs. The data show induction efficiency is directly correlated with strength of ''rcsA'' overexpression (Figure 5, columns 2-4). In each of the constructs, there is a 3 to 5 fold increase in phage levels after induction with 10 <ins class="diffchange diffchange-inline">nM </ins>AHL. This increase does not accurately reflect the dynamic range of the AHL inducible promoter, because it does not take into account the background phage levels without the promoter. In the weaker RBSs (B0032 and B0033), the phage concentration from the uninduced supernatant is comparable to the background level from the promoter-less construct (Figure 5, columns 1-3). Thus, in these inducible promoter systems, there is high dynamic range and negligible background compared to basal phage concentrations. However, in the strongest RBS (B0034), the uninduced phage level is an order of magnitude higher than the promoter-less background level. This high basal level expression suggests that the higher expression levels due to the strong RBS is leading to leaky ''rcsA'' activation of the phage lytic cycle. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Rcsainduction.png|600px|thumb|center|Figure 5 - ''rscA'' induction of phage production. In this figure, each construct was infected with λ phage and lysogens were selected. Lysogens were grown to OD600=0.1 and ''rcsA'' was induced with 10 nM of AHL. 1.5 hours after induction, the supernatant was used to titer wildtype stock of ''E. coli''. Plaques were counted to calculate the phage concentration inside the supernatant after induction. For more information see [[Team:Caltech/Protocols/rcsA_Lysogen_Induction|<font style="color:#BB4400">induction protocol.</font>]]]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Rcsainduction.png|600px|thumb|center|Figure 5 - ''rscA'' induction of phage production. In this figure, each construct was infected with λ phage and lysogens were selected. Lysogens were grown to OD600=0.1 and ''rcsA'' was induced with 10 nM of AHL. 1.5 hours after induction, the supernatant was used to titer wildtype stock of ''E. coli''. Plaques were counted to calculate the phage concentration inside the supernatant after induction. For more information see [[Team:Caltech/Protocols/rcsA_Lysogen_Induction|<font style="color:#BB4400">induction protocol.</font>]]]]</div></td></tr>
</table>Jkmhttp://2008.igem.org/wiki/index.php?title=Team:Caltech/Project/Phage_Pathogen_Defense&diff=63528&oldid=prevCaltechigem at 07:39, 25 October 20082008-10-25T07:39:44Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>An obvious delivery mechanism would be the adaptation of natural lysogens, a phase of the phage life cycle in which the phage integrates its DNA into the genome of the host organism. Lysogens lie dormant until disturbed by some external stimuli, but can be stimulated to enter the lytic cycle and release phage. There is one major limitation to this method: phage released will be specific to the lysogenic hosts, ''E. coli''. We can avoid this limitation by incorporating as a lysogen a phage that is not normally infectious to ''E. coli'', such as the P22 phage from ''Salmonella''. This phage could potentially target pathogenic bacteria besides ''E. coli''. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>An obvious delivery mechanism would be the adaptation of natural lysogens, a phase of the phage life cycle in which the phage integrates its DNA into the genome of the host organism. Lysogens lie dormant until disturbed by some external stimuli, but can be stimulated to enter the lytic cycle and release phage. There is one major limitation to this method: phage released will be specific to the lysogenic hosts, ''E. coli''. We can avoid this limitation by incorporating as a lysogen a phage that is not normally infectious to ''E. coli'', such as the P22 phage from ''Salmonella''. This phage could potentially target pathogenic bacteria besides ''E. coli''. </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>To demonstrate the feasibility of this approach, a simple model system was developed using λ phage and JW3996-1, a strain of ''E. coli'' immune to λ infection due to a ''lamB'' deletion[5]. In this model, the host is immune to infection unless LamB is expressed from a plasmid. After infection and selection for lysogens, the ''lamB'' plasmid is counterselected against to regain immunity. Extending this model to a more realistic situation, we envision using a phage that targets a pathogenic bacterial species, constitutively expressing the phage receptor within ''E. coli'' to create lysogens, and inducing the lysogens to infect non-''E. coli'' pathogenic bacteria. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>To demonstrate the feasibility of this approach, a simple model system was developed using λ phage and JW3996-1, a strain of ''E. coli'' immune to λ infection due to a ''lamB'' deletion [5]. In this model, the host is immune to infection unless LamB is expressed from a plasmid. After infection and selection for lysogens, the ''lamB'' plasmid is counterselected against to regain immunity. Extending this model to a more realistic situation, we envision using a phage that targets a pathogenic bacterial species, constitutively expressing the phage receptor within ''E. coli'' to create lysogens, and inducing the lysogens to infect non-''E. coli'' pathogenic bacteria. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We require one other plasmid which controls the induction of the lysogens to release phage into the environment. Control of this aspect of the system is vital for integration with the overall iGEM project. The induction of λ phage lysogens has been explored since 1950 [6], and serves as a model system for the study of other temperate phages. The most famous induction mechanism is ultraviolet radiation (UV). In UV induction of λ lysogens, UV induced DNA damage activates the cellular SOS mechanism, which activates the expression of RecA, a protein involved in SOS response. RecA, in turn, cleaves the λ repressor and leads to phage activation. In this study, activation of the ''recA'' pathway was not readily feasible, so an alternative effector was used to elicit induction. A secondary plasmid expressing the ''E. coli'' regulatory gene, ''rcsA'', is used as the trigger for λ phage induction. ''rcsA'' is one of several regulatory genes which provide a ''recA'' independent pathway for λ induction [7]. Overexpression of RcsA leads to λ induction, and, furthermore, RcsA appears to directly compete with the λ repressor cI [6]. In this project, ''rcsA'' is used to trigger phage production. The effectiveness and dynamic range of ''rcsA'' induction is explored.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We require one other plasmid which controls the induction of the lysogens to release phage into the environment. Control of this aspect of the system is vital for integration with the overall iGEM project. The induction of λ phage lysogens has been explored since 1950 [6], and serves as a model system for the study of other temperate phages. The most famous induction mechanism is ultraviolet radiation (UV). In UV induction of λ lysogens, UV induced DNA damage activates the cellular SOS mechanism, which activates the expression of RecA, a protein involved in SOS response. RecA, in turn, cleaves the λ repressor and leads to phage activation. In this study, activation of the ''recA'' pathway was not readily feasible, so an alternative effector was used to elicit induction. A secondary plasmid expressing the ''E. coli'' regulatory gene, ''rcsA'', is used as the trigger for λ phage induction. ''rcsA'' is one of several regulatory genes which provide a ''recA'' independent pathway for λ induction [7]. Overexpression of RcsA leads to λ induction, and, furthermore, RcsA appears to directly compete with the λ repressor cI [6]. In this project, ''rcsA'' is used to trigger phage production. The effectiveness and dynamic range of ''rcsA'' induction is explored.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:lambteta.png|thumb|right|300px|Figure 1 - Constructs involved in expression of the ''lamB'' receptor and the TetA cassette ]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:lambteta.png|thumb|right|300px|Figure 1 - Constructs involved in expression of the ''lamB'' receptor and the TetA cassette ]]</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>All constructs were made with standard assembly methods. ''lamB'' and ''rcsA'' were cloned out of DH10B genomic DNA. Two families of plasmids were constructed for this project. The first consists of plasmids that express the ''lamB'' receptor and the tetracycline resistance cassette. The level of ''lamB'' expression required for λ phage infection is unknown, so the strength of the promoter/RBS combination upstream of ''lamB'' was varied between the four constructs of this family.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>All constructs were made with standard assembly methods. ''lamB'' and ''rcsA'' were cloned out of DH10B genomic DNA. Two families of plasmids were constructed for this project. The first consists of plasmids that express the ''lamB'' receptor and the tetracycline resistance cassette. The level of ''lamB'' expression required for λ phage infection is unknown, so the strength of the promoter/<ins class="diffchange diffchange-inline">ribosome binding site (</ins>RBS<ins class="diffchange diffchange-inline">) </ins>combination upstream of ''lamB'' was varied between the four constructs of this family.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{clear}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{clear}}</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:rcsa.png|thumb|left|300px|Figure 2 - Constructs for characterization of rcsA induction ]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:rcsa.png|thumb|left|300px|Figure 2 - Constructs for characterization of rcsA induction ]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The second family consists of constructs used to characterize ''rcsA'' induction of λ phage. ''rcsA'' was placed downstream of an acyl-homoserine lactone (AHL) inducible promoter, LuxR. This promoter was extensively characterized by Doug for the [[Team:Caltech/Project/Population_Variation|<font style="color:#BB4400">oxidative burst project</font>]]. Using flow cytometry data, the peak response for this promoter was determined to be 10 nM AHL. Plasmids with <del class="diffchange diffchange-inline">3 </del>different RBSs in front of ''rcsA'' were constructed to characterize the response of induction. Furthermore, a promoterless ''rcsA'' construct was built as a negative control, and a construct with constitutively active λ repressor ''cI'' driven by J23106 was also assembled.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The second family consists of constructs used to characterize ''rcsA'' induction of λ phage. ''rcsA'' was placed downstream of an acyl-homoserine lactone (AHL) inducible promoter, LuxR. This promoter was extensively characterized by Doug for the [[Team:Caltech/Project/Population_Variation|<font style="color:#BB4400">oxidative burst project</font>]]. Using flow cytometry data, the peak response for this promoter was determined to be 10 nM AHL. Plasmids with <ins class="diffchange diffchange-inline">three </ins>different RBSs in front of ''rcsA'' were constructed to characterize the response of induction. Furthermore, a promoterless ''rcsA'' construct was built as a negative control, and a construct with constitutively active λ repressor ''cI'' driven by J23106 was also assembled.</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>{{Clear}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{Clear}}</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Overexpression of ''rcsA'' led to induction of λ lysogens. ''rcsA'' was placed downstream of an AHL inducible promoter and overexpressed through induction with 10 nM AHL. Following induction, phage concentration in the supernatant was recorded through titering. This concentration was compared to the phage concentration in the supernatant without ''rcsA'' overexpression, given by a construct where ''rcsA'' is not driven by a promoter. The data show a 40 fold increase in phage concentration between the promoter-less ''rcsA'' construct and the strongest AHL induced ''rcsA'' construct (Figure 5, columns 1 and 4). The promoter-less ''rcsA'' construct should accurately represent background phage levels for the lysogen absent of any ''rcsA'' expression. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Overexpression of ''rcsA'' led to induction of λ lysogens. ''rcsA'' was placed downstream of an AHL inducible promoter and overexpressed through induction with 10 nM AHL. Following induction, phage concentration in the supernatant was recorded through titering. This concentration was compared to the phage concentration in the supernatant without ''rcsA'' overexpression, given by a construct where ''rcsA'' is not driven by a promoter. The data show a 40 fold increase in phage concentration between the promoter-less ''rcsA'' construct and the strongest AHL induced ''rcsA'' construct (Figure 5, columns 1 and 4). The promoter-less ''rcsA'' construct should accurately represent background phage levels for the lysogen absent of any ''rcsA'' expression. </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Further work was done to characterize the response of the AHL inducible switch under three different <del class="diffchange diffchange-inline">ribosome binding sites (</del>RBSs<del class="diffchange diffchange-inline">)</del>. The data show induction efficiency is directly correlated with strength of ''rcsA'' overexpression (Figure 5, columns 2-4). In each of the constructs, there is a 3 to 5 fold increase in phage levels after induction with 10 nm AHL. This increase does not accurately reflect the dynamic range of the AHL inducible promoter, because it does not take into account the background phage levels without the promoter. In the weaker RBSs (B0032 and B0033), the phage concentration from the uninduced supernatant is comparable to the background level from the promoter-less construct (Figure 5, columns 1-3). Thus, in these inducible promoter systems, there is high dynamic range and negligible background compared to basal phage concentrations. However, in the strongest RBS (B0034), the uninduced phage level is an order of magnitude higher than the promoter-less background level. This high basal level expression suggests that the higher expression levels due to the strong RBS is leading to leaky ''rcsA'' activation of the phage lytic cycle. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Further work was done to characterize the response of the AHL inducible switch under three different RBSs. The data show induction efficiency is directly correlated with strength of ''rcsA'' overexpression (Figure 5, columns 2-4). In each of the constructs, there is a 3 to 5 fold increase in phage levels after induction with 10 nm AHL. This increase does not accurately reflect the dynamic range of the AHL inducible promoter, because it does not take into account the background phage levels without the promoter. In the weaker RBSs (B0032 and B0033), the phage concentration from the uninduced supernatant is comparable to the background level from the promoter-less construct (Figure 5, columns 1-3). Thus, in these inducible promoter systems, there is high dynamic range and negligible background compared to basal phage concentrations. However, in the strongest RBS (B0034), the uninduced phage level is an order of magnitude higher than the promoter-less background level. This high basal level expression suggests that the higher expression levels due to the strong RBS is leading to leaky ''rcsA'' activation of the phage lytic cycle. </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:Rcsainduction.png|600px|thumb|center|Figure 5 - ''rscA'' induction of phage production. In this figure, each construct was infected with λ phage and lysogens were selected. Lysogens were grown to OD600=0.1 and ''rcsA'' was induced with 10 nM of AHL. 1.5 hours after induction, the supernatant was used to titer wildtype stock of ''E. coli''. Plaques were counted to calculate the phage concentration inside the supernatant after induction. For more information see [[Team:Caltech/Protocols/rcsA_Lysogen_Induction|<font style="color:#BB4400">induction protocol.</font>]]]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Rcsainduction.png|600px|thumb|center|Figure 5 - ''rscA'' induction of phage production. In this figure, each construct was infected with λ phage and lysogens were selected. Lysogens were grown to OD600=0.1 and ''rcsA'' was induced with 10 nM of AHL. 1.5 hours after induction, the supernatant was used to titer wildtype stock of ''E. coli''. Plaques were counted to calculate the phage concentration inside the supernatant after induction. For more information see [[Team:Caltech/Protocols/rcsA_Lysogen_Induction|<font style="color:#BB4400">induction protocol.</font>]]]]</div></td></tr>
</table>Caltechigemhttp://2008.igem.org/wiki/index.php?title=Team:Caltech/Project/Phage_Pathogen_Defense&diff=63527&oldid=prevCaltechigem at 07:36, 25 October 20082008-10-25T07:36:16Z<p></p>
<a href="http://2008.igem.org/wiki/index.php?title=Team:Caltech/Project/Phage_Pathogen_Defense&diff=63527&oldid=61510">Show changes</a>Caltechigemhttp://2008.igem.org/wiki/index.php?title=Team:Caltech/Project/Phage_Pathogen_Defense&diff=61510&oldid=prevJkm at 19:59, 23 October 20082008-10-23T19:59:36Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Image:lamb.png|Figure 3 - λ Infection of Constitutively Active lamB Constructs. Constitutively active ''lamB'' (Table 1) was used to augment the ''lamB''- phenotype in JW3996-1 cells. Cells were titered with a known high concentration stock of phage 1x10<del class="diffchange diffchange-inline">></del>sup>10</sup> phage/mL. The efficiency of infection of ''lamB'' constructs can be seen through the observed phage concentration after titering. Zero plaques were observed for the J23113+33 construct.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Image:lamb.png|Figure 3 - λ Infection of Constitutively Active lamB Constructs. Constitutively active ''lamB'' (Table 1) was used to augment the ''lamB''- phenotype in JW3996-1 cells. Cells were titered with a known high concentration stock of phage 1x10<ins class="diffchange diffchange-inline"><</ins>sup>10</sup> phage/mL. The efficiency of infection of ''lamB'' constructs can be seen through the observed phage concentration after titering. Zero plaques were observed for the J23113+33 construct.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Image:LamBExpression.png|Figure 4 - ''lamB'' Infection vs ''lamB'' expression level. Infection levels for the constructs in Figure 3 plotted against the expression levels recorded in the registry for the Promoter/RBSs used.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Image:LamBExpression.png|Figure 4 - ''lamB'' Infection vs ''lamB'' expression level. Infection levels for the constructs in Figure 3 plotted against the expression levels recorded in the registry for the Promoter/RBSs used.</div></td></tr>
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</table>Jkmhttp://2008.igem.org/wiki/index.php?title=Team:Caltech/Project/Phage_Pathogen_Defense&diff=61445&oldid=prevJkm at 18:43, 23 October 20082008-10-23T18:43:00Z<p></p>
<a href="http://2008.igem.org/wiki/index.php?title=Team:Caltech/Project/Phage_Pathogen_Defense&diff=61445&oldid=60969">Show changes</a>Jkmhttp://2008.igem.org/wiki/index.php?title=Team:Caltech/Project/Phage_Pathogen_Defense&diff=60969&oldid=prevFeiChen at 09:59, 23 October 20082008-10-23T09:59:19Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Conclusion==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Conclusion==</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Future work should be focused on broadening the host range of the system. Thus, a key aspect needs to be adapting other temperate phages the model system. A significant result would be the incorporation and production of a non-E. coli bacteriophage by E. coli. A promising candidate for this is the Salmonella phage P22; P22 has been shown to be viably produced in E. coli when the prophage is expressed. Incorporation of the P22 prophage should be feasible following the framework of the current model. Furthermore, co-culture experiments with the current constructs and wild-type E. coli could be used to validate the current system's effectiveness at eliminating pathogens. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Future work should be focused on broadening the host range of the system. Thus, a key aspect needs to be adapting other temperate phages the model system. A significant result would be the incorporation and production of a non-E. coli bacteriophage by E. coli. A promising candidate for this is the Salmonella phage P22; P22 has been shown to be viably produced in E. coli when the prophage is expressed. Incorporation of the P22 prophage should be feasible following the framework of the current model. Furthermore, co-culture experiments with the current constructs and wild-type E. coli could be used to validate the current system's effectiveness at eliminating pathogens. </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>FeiChenhttp://2008.igem.org/wiki/index.php?title=Team:Caltech/Project/Phage_Pathogen_Defense&diff=60968&oldid=prevFeiChen at 09:57, 23 October 20082008-10-23T09:57:46Z<p></p>
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</table>FeiChenhttp://2008.igem.org/wiki/index.php?title=Team:Caltech/Project/Phage_Pathogen_Defense&diff=60965&oldid=prevFeiChen at 09:56, 23 October 20082008-10-23T09:56:01Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>__NOTOC__</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>__NOTOC__</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Introduction==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Introduction==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:PathogenicEcoli.jpg|thumb|left|Pathogenic ''E. coli''.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:PathogenicEcoli.jpg|thumb|left|Pathogenic ''E. coli''.]]</div></td></tr>
</table>FeiChenhttp://2008.igem.org/wiki/index.php?title=Team:Caltech/Project/Phage_Pathogen_Defense&diff=60964&oldid=prevFeiChen: /* Conclusion */2008-10-23T09:54:55Z<p><span class="autocomment">Conclusion</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Conclusion==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Conclusion==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We have successfully developed a model system in which bacteriophages not specific to E. coli can be integrated as a lysogen within an E. coli host. We successfully demonstrated this model with bacteriophage λ and the JW3996-1 strain of E. coli which is immune to λ infection. A system for inducing the release of phage was constructed around the E. coli regulatory protein RcsA. Work was done to optimize a switch using an AHL inducible promoter for ''rcsA'' induction. <del class="diffchange diffchange-inline">'</del>''rcsA<del class="diffchange diffchange-inline">'</del>'' overexpression led to significant phage induction, producing approximately 1x108 plaque forming units (pfu)/mL. This value is 4 orders of magnitude greater than the phage induction reported by Rozanov et al. However, direct comparisons cannot be readily made between the data, because the method of overexpression of ''rcsA'' differs between the two papers, and the level at which ''rcsA'' was overexpressed in Rozanov’s study is unknown. The background phage concentration in uninduced lysogens was also much higher than reported in literature [7]. At present, there is no clear cause for this increase in basal induction levels within the λ lysogens. Phage induction via ''rcsA'' can be reduced through lambda repressor overexpression. The mechanism by which RcsA activates the lytic cycle of the dormant prophage is largely unknown; however, there is evidence that RscA acts through inactivation of the phage repressor, ''cI''. [7] Therefore, increased production of the repressor ''cI'' led to a decrease in ''rcsA'' mediated phage induction. The addition of constitutively active ''cI'' repressor led to the construction of an ''rcsA'' phage induction system with a high dynamic range and relatively low background. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We have successfully developed a model system in which bacteriophages not specific to E. coli can be integrated as a lysogen within an E. coli host. We successfully demonstrated this model with bacteriophage λ and the JW3996-1 strain of E. coli which is immune to λ infection. A system for inducing the release of phage was constructed around the E. coli regulatory protein RcsA. Work was done to optimize a switch using an AHL inducible promoter for ''rcsA'' induction. ''rcsA'' overexpression led to significant phage induction, producing approximately 1x108 plaque forming units (pfu)/mL. This value is 4 orders of magnitude greater than the phage induction reported by Rozanov et al. However, direct comparisons cannot be readily made between the data, because the method of overexpression of ''rcsA'' differs between the two papers, and the level at which ''rcsA'' was overexpressed in Rozanov’s study is unknown. The background phage concentration in uninduced lysogens was also much higher than reported in literature [7]. At present, there is no clear cause for this increase in basal induction levels within the λ lysogens. Phage induction via ''rcsA'' can be reduced through lambda repressor overexpression. The mechanism by which RcsA activates the lytic cycle of the dormant prophage is largely unknown; however, there is evidence that RscA acts through inactivation of the phage repressor, ''cI''. [7] Therefore, increased production of the repressor ''cI'' led to a decrease in ''rcsA'' mediated phage induction. The addition of constitutively active ''cI'' repressor led to the construction of an ''rcsA'' phage induction system with a high dynamic range and relatively low background. </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>Future work should be focused on broadening the host range of the system. Thus, a key aspect needs to be adapting other temperate phages the model system. A significant result would be the incorporation and production of a non-E. coli bacteriophage by E. coli. A promising candidate for this is the Salmonella phage P22; P22 has been shown to be viably produced in E. coli when the prophage is expressed. Incorporation of the P22 prophage should be feasible following the framework of the current model. Furthermore, co-culture experiments with the current constructs and wild-type E. coli could be used to validate the current system's effectiveness at eliminating pathogens. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Future work should be focused on broadening the host range of the system. Thus, a key aspect needs to be adapting other temperate phages the model system. A significant result would be the incorporation and production of a non-E. coli bacteriophage by E. coli. A promising candidate for this is the Salmonella phage P22; P22 has been shown to be viably produced in E. coli when the prophage is expressed. Incorporation of the P22 prophage should be feasible following the framework of the current model. Furthermore, co-culture experiments with the current constructs and wild-type E. coli could be used to validate the current system's effectiveness at eliminating pathogens. </div></td></tr>
</table>FeiChenhttp://2008.igem.org/wiki/index.php?title=Team:Caltech/Project/Phage_Pathogen_Defense&diff=60963&oldid=prevFeiChen at 09:54, 23 October 20082008-10-23T09:54:28Z<p></p>
<a href="http://2008.igem.org/wiki/index.php?title=Team:Caltech/Project/Phage_Pathogen_Defense&diff=60963&oldid=60961">Show changes</a>FeiChen