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Generating molecular diversity by homologous recombination in Escherichia coli

Posted by on in 2005
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Wang PL, Lo BK, Winter G.
Protein Eng Des Sel. 2005 Aug;18(8):397-404. Epub 2005 Jun 27.
Centre for Protein Engineering, University of Cambridge, Hills Road, Cambridge CB2 2QH, UK. plwang@stanford.edu.

Abstract

We explored the use of recE-mediated homologous recombination to generate molecular diversity in Escherichia coli.

Two homologous genes were placed on different phagemid vectors each comprising multiple EcoRI restriction sites and overlapping N- and C-terminal portions of beta-lactamase. By co-infection of these phage into RecE+ EcoRI+ E.coli, we were able to introduce double-strand breaks into these vectors, allowing efficient homologous recombination (in up to 10% of bacteria) by the recE pathway and selection of the recombinants by resistance to ampicillin. Recombination gave single crossovers; these were more frequent near the EcoRI sites and the recombination frequency increased with the target length and degree of homology.

The system was used to create a large combinatorial chicken antibody library (10(10)) for display on filamentous phage and to isolate several antibody fragments with binding affinities in the 10-100 nM range.

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