IGEM:Caltech/2007/Project

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(<center>Project Overview</center>)
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==<center>Current Status</center>==
==<center>Current Status</center>==
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Currently, ''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. The addition of anhydrotetracycline (aTc) 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. Heterologous N and Q have been shown to complement phages with amber mutations in the respective genes. Adding a cis-repressor to the Q construct lowered production of Q and prevented complementation. We were unable to express sufficient cro from a plasmid to rescue a cro mutant phage.  
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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.
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* [[IGEM:Caltech/2007/Project/construct|Explanation of Construct]]
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* [[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 a cro mutant phage.
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* [[IGEM:Caltech/2007/Project/titerResults|Summary of Titering Results]]
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<br>
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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, ''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.
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* [[IGEM:Caltech/2007/Project/cis3Results|Summary cis3 Quanta Data]]s
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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. Phages resulting from the recombineering process are also being screened for successful N and Q amber mutants.
 
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Revision as of 10:10, 26 October 2007


iGEM 2007

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Project Background

Introduction to Bacteriophage λ

Project Details

The project consisted of five individual parts, each assigned to one team member, and categorized into one of three independent principles underlying the project:

Current Status

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.

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 a cro mutant phage.


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.



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