Development of a broad-host-range sacB-based vector for unmarked allelic exchange.

BMC Research Notes
C J Marx

Abstract

Although genome sequences are available for an ever-increasing number of bacterial species, the availability of facile genetic tools for physiological analysis have generally lagged substantially behind traditional genetic models. Here I describe the development of an improved, broad-host-range "in-out" allelic exchange vector, pCM433, which permits the generation of clean, marker-free genetic manipulations. Wild-type and mutant alleles were reciprocally exchanged at three loci in Methylobacterium extorquens AM1 in order to demonstrate the utility of pCM433. The broad-host-range vector for marker-free allelic exchange described here, pCM433, has the advantages of a high copy, general Escherichia coli replicon for easy cloning, an IncP oriT enabling conjugal transfer, an extensive set of restriction sites in its polylinker, three antibiotic markers, and sacB (encoding levansucrase) for negative selection upon sucrose plates. These traits should permit pCM433 to be broadly applied across many bacterial taxa for marker-free allelic exchange, which is particularly important if multiple manipulations or more subtle genetic manipulations such as point mutations are desired.

References

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Citations

Feb 17, 2009·Proceedings of the National Academy of Sciences of the United States of America·Anne Francez-CharlotJulia A Vorholt
Mar 28, 2009·Health Education Research·Sarah F GriffinCarol Rheaume
May 8, 2013·BMC Research Notes·Lon M ChubizChistopher J Marx
May 17, 2012·PLoS Genetics·Ming-Chun Lee, Christopher J Marx
Dec 4, 2013·Applied Microbiology and Biotechnology·Shengbiao HuYouming Zhang
Jan 24, 2014·Nucleic Acids Research·Christopher J GreggGeorge M Church
Sep 10, 2015·Proceedings of the National Academy of Sciences of the United States of America·James P SáenzKai Simons
Oct 30, 2013·Evolution; International Journal of Organic Evolution·Sean Michael CarrollChristopher J Marx
Jan 27, 2015·Metabolic Engineering·Christopher W Johnson, Gregg T Beckham
Jun 24, 2009·Evolution; International Journal of Organic Evolution·Ming-Chun LeeChristopher J Marx
Jul 27, 2012·Cell Reports·Hsin-Hung Chou, Christopher J Marx
Nov 30, 2014·Applied Microbiology and Biotechnology·Andrea M OchsnerJulia A Vorholt
Feb 26, 2016·Molecular Biology and Evolution·Deepa AgasheChristopher J Marx
Jun 9, 2016·Evolution; International Journal of Organic Evolution·William R HarcombeChristopher J Marx
Dec 10, 2013·The Australian and New Zealand Journal of Psychiatry·Theresa M FlemingMathijs Lucassen
Dec 16, 2017·Applied Biochemistry and Biotechnology·Wei-Fan LiangXin-Hui Xing
May 12, 2018·Biotechnology and Bioengineering·Emily FreedCarrie A Eckert
Mar 16, 2017·Journal of Bacteriology·Lon M Chubiz, Christopher J Marx
Apr 24, 2012·Applied and Environmental Microbiology·Paul A BeareRobert A Heinzen
Aug 21, 2013·Applied and Environmental Microbiology·Muhammad Farhan Ul HaqueStéphane Vuilleumier
Mar 20, 2012·Applied and Environmental Microbiology·Andreas KaczmarczykAnne Francez-Charlot
Jun 28, 2011·Applied and Environmental Microbiology·Snaedis H BjornsdottirGudmundur Eggertsson
Apr 1, 2014·Applied and Environmental Microbiology·Christelle GruffazFrançoise Bringel

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Datasets Mentioned

BETA
EU118176

Methods Mentioned

BETA
PCR

Software Mentioned

Clone Manager

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