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In vivo elimination of parental clones in general and site-directed mutagenesis

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posted on 14.01.2018 by E.G. Holland, F.E. Acca, K.M. Belanger, M.E. Bylo, B.K. Kay, M.P. Weiner, M.M. Kiss
The Eco29k I restriction endonuclease is a Sac II isoschizomer that recognizes the sequence 5'-CCGCGG-3' and is encoded, along with the Eco29k I methylase, in the Escherichia coli strain 29k. We have expressed the Eco29k I restriction-methylation system (RM2) in E. coli strain TG1 to produce the strain AXE688. We have developed a directed molecular evolution (DME) mutagenesis method that uses Eco29k I to restrict incoming parental DNA in transformed cells. Using our DME method, we have demonstrated that our AXE688 strain results in mutated directed molecular evolution libraries with diversity greater than 10(7) from a single transformation and with greater than 90% recombinant clones

Funding

This work was supported by the National Institutes of Health [1 U54 DK093444-01].

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Publisher Statement

This is the author’s version of a work that was accepted for publication in Journal of Immunological Methods. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Journal of Immunological Methods. 2015. 417: 67-75. 10.1016/j.jim.2014.12.008.

Publisher

Elsevier Inc.

issn

0022-1759

Issue date

15/12/2014

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