IGEM:Caltech/2007/Project/Recombineering: Difference between revisions
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strains must simultaneously be defective in the relevant N, Q or cro developmental genes while still being relatively easy to propagate. | strains must simultaneously be defective in the relevant N, Q or cro developmental genes while still being relatively easy to propagate. | ||
Choosing a phage strain which facilitates insertion of heterologous sequences and tolerates their presence requires some care. The process is somewhat different than, for example, cloning heterologous DNA into a bacterial plasmid. Two features make this task slightly more involved. First, wild type lambda phage does not contain many unique restriction sites, making standard cloning techniques difficult. Second, since evolution has optimized lambda's small genome to have a high density of regulation, meaning that many stretches of DNA serve multiple functions. | |||
Recombineering allows us to accomplish these goals in a single step, by modifying commonly used lambda cloning vector strains to be defective in the genes N, Q, or cro. For convenience, we initially chose to work with the Lambda Zap cloning vector (insert paper reference, JM Short et al, NAR 1988), available from Stratagene. This vector is a standard lambda phage strain engineered to contain multiple, unique, restriction sites in an unessential central portion of the lambda genome. (insert figure from NAR paper detailing MCS) | |||
Recombineering allows us to accomplish these goals in a single step, by modifying commonly used lambda cloning vector strains to be defective in the genes N, Q, or cro. For convenience, we initially chose to work with the Lambda Zap cloning vector (insert paper reference, JM Short et al, NAR 1988), available from Stratagene. This vector is a standard lambda phage strain engineered to contain multiple, unique, restriction sites in an unessential central portion of the lambda genome. (insert figure from NAR paper detailing MCS) Although we initially used this commercial strain for convenience, our method can easily be applied to freely available lambda cloning vectors. | |||
==Status and Future Plans== | ==Status and Future Plans== |
Revision as of 14:53, 25 October 2007
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