IGEM:Caltech/2007/Project/Recombineering: Difference between revisions
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Our project requires phage strains with two main characteristics. First, our phage strains must be defective in expression of the <i>N, Q,</i> or <i>cro</i> developmental genes, while still being easy to propagate. Second, the strains must allow easy cloning of heterologous constructs -- our riboregulated <i>N, Q,</i> and <i>cro</i> expression constructs -- into them. | Our project requires phage strains with two main characteristics. First, our phage strains must be defective in expression of the <i>N, Q,</i> or <i>cro</i> developmental genes, while still being easy to propagate. Second, the strains must allow easy cloning of heterologous constructs -- our riboregulated <i>N, Q,</i> and <i>cro</i> expression constructs -- into them. | ||
Recombineering allows us to satisfy the first constraint in an elegant way. Specifically, we will use recombineering to insert in-frame amber stop mutations into the coding sequences of <i>N, Q | Recombineering allows us to satisfy the first constraint in an elegant way. Specifically, we will use recombineering to insert in-frame amber stop mutations into the coding sequences of <i>N, & Q</i> or <i>cro</i>. The amber stop mutation prevents successful translation of these genes in most <i>E. coli</i> strains, crippling the phages. By stopping translation with only a single point mutation, we minimize the mutation's impact on other regulatory and coding sequences. Finally, phages with amber stop mutations can be easily propagated in special <i>amber suppressor</i> E. coli strains, a standard decades-old technique in classical lambda genetics. | ||
To satisfy the second constraint, we can carefully choose | |||
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. | 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. |
Revision as of 15:27, 25 October 2007
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