IGEM:Caltech/2007/Project: Difference between revisions
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* [[IGEM:Caltech/2007/Project/Recombineering|Recombineering]] the cro, N, and Q amber mutation genes into lambda zap | * [[IGEM:Caltech/2007/Project/Recombineering|Recombineering]] the cro, N, and Q amber mutation genes into lambda zap | ||
* | * [[IGEM:Caltech/2007/Project/Riboregulator|Riboregulator]] design and testing | ||
*Titering [[IGEM:Caltech/2007/Project/Cro|Cro]], [[IGEM:Caltech/2007/Project/N|N-antiterminator]], and [[IGEM:Caltech/2007/Project/Q|Q-antiterminator]] construct-containing bacterial to test construct-mediated rescue of lytic activity (in amber phage) | *Titering [[IGEM:Caltech/2007/Project/Cro|Cro]], [[IGEM:Caltech/2007/Project/N|N-antiterminator]], and [[IGEM:Caltech/2007/Project/Q|Q-antiterminator]] construct-containing bacterial to test construct-mediated rescue of lytic activity (in amber phage) | ||
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The future of the project lies in confirming trans activation: that is, to prove protein concentration is several times greater in aTc-saturated trans-containing cells as compared to aTc-saturated strains with no trans plasmid. The successful complementary cis-trans pairs can then be incorporated into the N-J23100 and Q-J23116 constructs. Cis repression in the N-construct has yet to be tested, but Q’s results would imply successful repression of lytic action. Based on the presence of plaques on D1210-N and D1210-Q strains even without aTc, even a small amount of trans-activation woudl allow amber phages to successfully enter the lytic cycle. | The future of the project lies in confirming trans activation: that is, to prove protein concentration is several times greater in aTc-saturated trans-containing cells as compared to aTc-saturated strains with no trans plasmid. The successful complementary cis-trans pairs can then be incorporated into the N-J23100 and Q-J23116 constructs. Cis repression in the N-construct has yet to be tested, but Q’s results would imply successful repression of lytic action. Based on the presence of plaques on D1210-N and D1210-Q strains even without aTc, even a small amount of trans-activation woudl allow amber phages to successfully enter the lytic cycle. | ||
Cis repression of the N and Q constructs can be tested by further titering. A quantitative measure of the increase in protein concentration upon trans-activation will be obtained by fluorescence measurements by flow cytometry. So far, trans activation has been shown in YFP Quanta experiments (cis3-trans1 and cis3-trans2 seem the most promising), although combinations remain that need to be tested. The ultimate test, though, lies in titering amber mutant phage into strains containing both cis and trans plasmids. A positive result (no plaques on cis containing strains, plaques on cis-trans strains) would show successful integration of two independent project components (N/Q protein dependent lysis switch, and the “lock and key” riboregulator). Successful integration demonstrates that standardization on multiple levels (BioBrick parts making each construct, and the more abstract merging of riboregulators into viral decision-making) can allow rapid | Cis repression of the N and Q constructs can be tested by further titering. A quantitative measure of the increase in protein concentration upon trans-activation will be obtained by fluorescence measurements by flow cytometry. So far, trans activation has been shown in YFP Quanta experiments (cis3-trans1 and cis3-trans2 seem the most promising), although combinations remain that need to be tested. The ultimate test, though, lies in titering amber mutant phage into strains containing both cis and trans plasmids. A positive result (no plaques on cis containing strains, plaques on cis-trans strains) would show successful integration of two independent project components (N/Q protein dependent lysis switch, and the “lock and key” riboregulator). Successful integration demonstrates that standardization on multiple levels (BioBrick parts making each construct, and the more abstract merging of riboregulators into viral decision-making) can allow rapid construction of complex synthetic biological control pathways. | ||
Our current top priority is now to transform the trans1 and trans2 plasmids into the cis3-Q construct cell, titer and check for the presence of plaques. Once successful, we can clone the construct entirely into the recombineered amber phage. In the final system, infecting amber λ-Zap virus will selectively lyse those cells that express the specific trans RNA in their transcriptional profile. | Our current top priority is now to transform the trans1 and trans2 plasmids into the cis3-Q construct cell, titer and check for the presence of plaques. Once successful, we can clone the construct entirely into the recombineered amber phage. In the final system, infecting amber λ-Zap virus will selectively lyse those cells that express the specific trans RNA in their transcriptional profile. |
Latest revision as of 12:29, 26 October 2007
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