High frequency plasmid recombination mediated by 28 bp direct repeats

Molecular Biotechnology
Sofia C RibeiroGabriel A Monteiro

Abstract

The stability in Escherichia coli of a mammalian expression vector (pCIneo) and its derivative candidate DNA vaccine (pGPV-PV) is described. These multicopy pMB1-type plasmids were found to recombine in several recA E. coli strains due to the presence of two 28 bp direct repeats flanking intervening sequences of 1.6 kb (pCIneo) and 3.2 kb (pGPV-PV). In this recombination event, one of the direct repeats and the intervening sequence were deleted or duplicated, originating monomeric or/and hetero-dimeric plasmid forms, respectively. Additionally, the plasmid rearrangement led to the acquisition of a kanamycin resistance phenotype. Recombination frequencies between 7.8 x 10(-7) and 3.1 x 10(-5) were determined for DH5alpha and JM109(DE3) strains, respectively. Higher recombination frequencies were found in cells previously grown up to stationary growth phase being the monomeric plasmid form the prevalent one. Real-time PCR quantification revealed the presence of approximately 1.5 x 10(4) recombined molecules per 2 x 10(5 )cells pre-kanamycin exposure. Under selective pressure of this antibiotic, the number of recombined molecules increased approximately 2,000-fold in a 48-h period replacing the original plasmid form. The high freq...Continue Reading

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Citations

Feb 12, 2009·Applied Microbiology and Biotechnology·Diana M Bower, Kristala L J Prather
Nov 26, 2009·Applied Microbiology and Biotechnology·Sofia C RibeiroGabriel A Monteiro
May 25, 2010·Applied Microbiology and Biotechnology·Pedro H OliveiraGabriel A Monteiro
Jul 9, 2013·Trends in Biotechnology·Pedro H Oliveira, Juergen Mairhofer
Feb 24, 2009·Biotechnology Advances·James A WilliamsClague P Hodgson
Feb 18, 2011·Biotechnology Journal·Pedro H OliveiraGabriel A Monteiro
Aug 21, 2012·FEMS Microbiology Letters·Lívia FecskeováPeter Pristaš
May 11, 2016·Biotechnology & Genetic Engineering Reviews·Michaela ŠimčíkováGabriel A Monteiro
Aug 7, 2009·Trends in Biotechnology·Pedro H OliveiraGabriel A Monteiro
Sep 14, 2018·Applied Microbiology and Biotechnology·Zhixin LuoXin Wang
Mar 18, 2017·International Journal of Molecular Sciences·Suneetha Amara, Venkataswarup Tiriveedhi
May 12, 2021·Microbial Cell Factories·Carina PrellVolker F Wendisch
Jun 25, 2015·Microbiology Spectrum·Duarte Miguel F Prazeres, Gabriel A Monteiro

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