Recombinase-mediated cassette exchange (RMCE) system for functional genomics studies in Mycoplasma mycoides

Biological Procedures Online
Vladimir N NoskovRay-Yuan Chuang

Abstract

We have previously established technologies enabling us to engineer the Mycoplasma mycoides genome while cloned in the yeast Saccharomyces cerevisiae, followed by genome transplantation into Mycoplasma capricolum recipient cells to produce M. mycoides with an altered genome. To expand the toolbox for genomic modifications, we designed a strategy based on the Cre/loxP-based Recombinase-Mediated Cassette Exchange (RMCE) system for functional genomics analyses. In this paper, we demonstrated replacement of an approximately 100 kb DNA segment of the M. mycoides genome with a synthetic DNA counterpart in two orientations. The function of the altered genomes was then validated by genome transplantation and phenotypic characterization of the transplanted cells. This method offers an easy and efficient way to manipulate the M. mycoides genome and will be a valuable tool for functional genomic studies, such as genome organization and minimization.

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Citations

Feb 11, 2016·Critical Reviews in Biotechnology·Mario Juhas, James W Ajioka
Dec 17, 2016·Nucleic Acids Research·David M BrownJohn I Glass
Mar 26, 2016·Science·Clyde A HutchisonJ Craig Venter
Oct 6, 2020·Current Opinion in Systems Biology·Sanjay VasheeCarole Lartigue
Mar 31, 2021·Cell·James F PelletierElizabeth A Strychalski
Oct 6, 2020·ACS Synthetic Biology·Luis Garcia-MoralesCarole Lartigue

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Methods Mentioned

BETA
PCR
electrophoresis

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