In vivo plasmid construction by integrative recombination within a 156 bp sequence homology |
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Institution: | 1. Institute of Systems and Synthetic Biology, Genopole, CNRS, UEVE, Université Paris-Saclay, 5 rue Henri Desbruères, 91030 Évry, France;2. Chemical and Synthetic Biology Group, School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India |
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Abstract: | pHG165, a pBR322 copy number derivative of pUC8, has a 38-bp polylinker multiple cloning site located at the 5′ end of lacα. A further 156 bp, 3′ to the multiple cloning site, completes the coding sequence for the production of the β galactosidase α peptide. We describe the use of in vivo plasmid-chromosone cointegrates as a construction method that in this instance has enabled us to cross out the pHG165 multiple cloning site to obtain a wild-type β-galactosidase sequence for the α peptide. Because the DNA sequence available for homologous recombination was only 156 bp in length, the frequency of crosses that removed the multiple cloning site was less than 1 × 10−9. These crosses were easily obtained, however, after amplification by ampicillin selection. |
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