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 | 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 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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Titering experiments where cro, N, and Q amber phages were allowed to infect D1210 cells containing the built construct show that heterologous N and Q can complement phages with amber mutations in the respective genes. Adding a cis-repressor to the Q construct lowered production of Q even further, as it eliminated lysis completely. We were unable to express sufficient cro from a plasmid to rescue | Titering experiments where cro, N, and Q amber phages were allowed to infect D1210 cells containing the built construct show that heterologous N and Q can complement phages with amber mutations in the respective genes. Adding a cis-repressor to the Q construct lowered production of Q even further, as it eliminated lysis completely. We were unable to express sufficient cro from a plasmid to rescue lytic behavior of the amber cro mutant phage. | ||
* [[IGEM:Caltech/2007/Project/titerResults|Summary of Titering Results]] | * [[IGEM:Caltech/2007/Project/titerResults|Summary of Titering Results]] | ||
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Revision as of 09:28, 26 October 2007
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