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Modification of RSF1010-Based Broad Host-Range Plasmids for Improved Conjugation and Cyanobacterial Bioprospecting

30 Pages Posted: 9 Aug 2019 Publication Status: Published

See all articles by Bryan Bishé

Bryan Bishé

University of California, San Diego (UCSD) - Division of Biological Sciences

Arnaud Taton

University of California, San Diego (UCSD) - Division of Biological Sciences

James W. Golden

University of California, San Diego (UCSD) - Division of Biological Sciences

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Abstract

To facilitate the genetic engineering of diverse cyanobacterial strains, we have modified broad host-range RSF1010-based plasmids to improve transmissibility, increase copy number, and facilitate cloning. RSF1010-based plasmids replicate in diverse bacterial strains but produce low amounts of useable DNA for cloning. We previously engineered a mobAY25F mutation in RSF1010-based plasmids that improved cloning but decreased conjugation efficiency. Here, we engineered RSF1010-based plasmids to restore conjugation efficiency, which was demonstrated in three diverse laboratory strains of cyanobacteria. We then used an improved RSF1010-based plasmid in mating experiments with cultured samples of wild cyanobacteria. This plasmid, which confers antibiotic resistance and carries a yfp reporter gene, allowed selection of exconjugant cyanobacteria and facilitated the isolation of genetically tractable strains from mixed wild cultures. Improved RSF1010 vectors can be used for bioprospecting genetically tractable strains and are compatible with the CYANO-VECTOR cloning system, a versatile toolbox for constructing plasmids for cyanobacterial genetic engineering.

Suggested Citation

Bishé, Bryan and Taton, Arnaud and Golden, James W., Modification of RSF1010-Based Broad Host-Range Plasmids for Improved Conjugation and Cyanobacterial Bioprospecting (August 9, 2019). Available at SSRN: https://ssrn.com/abstract=3434724 or http://dx.doi.org/10.2139/ssrn.3434724
This is a paper under consideration at Cell Press and has not been peer-reviewed.

Bryan Bishé

University of California, San Diego (UCSD) - Division of Biological Sciences

San Diego, CA
United States

Arnaud Taton

University of California, San Diego (UCSD) - Division of Biological Sciences

San Diego, CA
United States

James W. Golden (Contact Author)

University of California, San Diego (UCSD) - Division of Biological Sciences ( email )

San Diego, CA
United States

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