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Team:UC Berkeley/GatewayGenomic
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==Introduction== | ==Introduction== | ||
| - | The ''in vivo'' plasmid based gateway scheme was successful and produce the desired product, but it also yielded a considerable amount of background. Closer analysis of the side products revealed that the background resulted from ccdB mutations that arose when the gene replicated and recombined ''in vivo''. In an effort to circumvent background resulting from mutations in a negative selection gene, we decided to use positive selection. This way, we would select for the desired products rather than selecting against unwanted side products and unreacted starting materials. | + | The ''in vivo'' plasmid based gateway scheme was successful and produce the desired product, but it also yielded a considerable amount of background. Closer analysis of the side products revealed that the background resulted from ccdB mutations that arose when the gene replicated and recombined ''in vivo''. In an effort to circumvent background resulting from mutations in a negative selection gene, we decided to use positive selection. This way, we would be able to select for the desired products rather than selecting against unwanted side products and unreacted starting materials. |
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| + | In order to implement positive selection, we used inducible origins of replication on our plasmids. These origins of replication will only be functional and allow plasmid replication if a specific protein is expressed in the cell. In our design, the assembly vector with an R6K origin (induced by the protein Pir) is integrated into the genome. The gene of interest alongside an OriV origin (induced by the protein TrfA) can then recombine with the genome. In this manner, the gene of interest can be moved to an assembly vector by inserting the entry vector into the genome. We could allow selective replication of the plasmids in our scheme by preforming the reaction in a cell expressing TrfA and then transforming the resulting mixture of plasmids into a cell expressing Pir. | ||
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| + | We also incorporated our lysis device into our new entry vector in order to simplify the experimental protocol for this scheme by eliminating minipreps. | ||
Revision as of 21:25, 29 October 2008
Introduction
The in vivo plasmid based gateway scheme was successful and produce the desired product, but it also yielded a considerable amount of background. Closer analysis of the side products revealed that the background resulted from ccdB mutations that arose when the gene replicated and recombined in vivo. In an effort to circumvent background resulting from mutations in a negative selection gene, we decided to use positive selection. This way, we would be able to select for the desired products rather than selecting against unwanted side products and unreacted starting materials.
In order to implement positive selection, we used inducible origins of replication on our plasmids. These origins of replication will only be functional and allow plasmid replication if a specific protein is expressed in the cell. In our design, the assembly vector with an R6K origin (induced by the protein Pir) is integrated into the genome. The gene of interest alongside an OriV origin (induced by the protein TrfA) can then recombine with the genome. In this manner, the gene of interest can be moved to an assembly vector by inserting the entry vector into the genome. We could allow selective replication of the plasmids in our scheme by preforming the reaction in a cell expressing TrfA and then transforming the resulting mixture of plasmids into a cell expressing Pir.
We also incorporated our lysis device into our new entry vector in order to simplify the experimental protocol for this scheme by eliminating minipreps.
