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</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 functions in the densely coded phage genome | 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 functions in the densely coded phage genome <cite>chan</cite>. 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 choose a phage strain commonly used for cloning cDNA libraries. This phage strain, named Lambda Zap (by Stratagene) <cite>short</cite> is engineered to contain unique restriction sites in nonessential areas of the phage genome. This makes the strain much easier to work with than standard lambda strains, which contain few uniques restriction sites, often in the middle of critical genes. Note that we chose to work with a commercial strain for convenience, and that our method can be easily ported to freely available lambda phage cloning strains. | To satisfy the second constraint, we choose a phage strain commonly used for cloning cDNA libraries. This phage strain, named Lambda Zap (by Stratagene) <cite>short</cite> is engineered to contain unique restriction sites in nonessential areas of the phage genome. This makes the strain much easier to work with than standard lambda strains, which contain few uniques restriction sites, often in the middle of critical genes. Note that we chose to work with a commercial strain for convenience, and that our method can be easily ported to freely available lambda phage cloning strains. |
Revision as of 11:08, 26 October 2007
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