IGEM:Caltech/2007/Project: Difference between revisions
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==<center>Current Status</center>== | ==<center>Current Status</center>== | ||
As the recombineering, testing of riboregulators, and titering processes will take place concurrently, we needed to find a simpler way to regulate viral protein concentrations in the cells. To this end, ''E. coli'' strains have been constructed that contain a low-copy plasmid construct (the pSB2K3 plasmid, low-copy until induced by IPTG, and built into D1210 bacterial strains, which contain a further mutation that results in one plasmid per cell) where one of three key developmental viral genes - coding for the Cro, N, or Q proteins - is regulated by a tetracycline-dependent promoter. A constitutive promoter (J23100, the stronger promoter, or J23116, the weaker one) produces a steady stream of tetracycline repressor (tetR), which substitutes for the | As the recombineering, testing of riboregulators, and titering processes will take place concurrently, we needed to find a simpler way to regulate viral protein concentrations in the cells. To this end, ''E. coli'' strains have been constructed that contain a low-copy plasmid construct (the pSB2K3 plasmid, low-copy until induced by IPTG, and built into D1210 bacterial strains, which contain a further mutation that results in one plasmid per cell) where one of three key developmental viral genes - coding for the Cro, N, or Q proteins - is regulated by a tetracycline-dependent promoter. A constitutive promoter (J23100, the stronger promoter, or J23116, the weaker one) produces a steady stream of tetracycline repressor (tetR), which substitutes for the cis repressor in repressing protein levels. The addition of anhydrotetracycline (aTc, acting as the trans activator) inactivates the tetracycline repressor and leads to the production of the respective viral protein in the ''E. coli'' cells. This allows us to control the concentration of viral protein produced in the cells by adding varying amounts of aTc to the bacterial growth media. | ||
* [[IGEM:Caltech/2007/Project/construct|Explanation of Construct]] | * [[IGEM:Caltech/2007/Project/construct|Explanation of Construct]] | ||
* [[IGEM:Caltech/2007/Project/constructList|List of Cloned Constructs]] | * [[IGEM:Caltech/2007/Project/constructList|List of Cloned Constructs]] | ||
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Multiple riboregulator designs are being tested (for both activation and repression levels), and successful designs will be cloned into the plasmid constructs. So far, | Multiple riboregulator designs are being tested (for both activation and repression levels), and successful designs will be cloned into the plasmid constructs. So far, cis construct number 3 and its accompanying trans combinations (cis3trans1 and cis3trans2) seem the most promising. Phages resulting from the recombineering process are also being screened for successful N and Q amber mutants. | ||
* [[IGEM:Caltech/2007/Project/cis3Results|Summary cis3 Quanta Data]]s | * [[IGEM:Caltech/2007/Project/cis3Results|Summary cis3 Quanta Data]]s | ||
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Revision as of 08:22, 26 October 2007
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