Computational design and characterization of a temperature-sensitive plasmid replicon for gram positive thermophiles.

Journal of Biological Engineering
Daniel G Olson, Lee R Lynd

Abstract

Temperature-sensitive (Ts) plasmids are useful tools for genetic engineering, but there are currently none compatible with the gram positive, thermophilic, obligate anaerobe, Clostridium thermocellum. Traditional mutagenesis techniques yield Ts mutants at a low frequency, and therefore requires the development of high-throughput screening protocols, which are also not available for this organism. Recently there has been progress in the development of computer algorithms which can predict Ts mutations. Most plasmids currently used for genetic modification of C. thermocellum are based on the replicon of plasmid pNW33N, which replicates using the RepB replication protein. To address this problem, we set out to create a Ts plasmid by mutating the gene coding for the RepB replication protein using an algorithm designed by Varadarajan et al. (1996) for predicting Ts mutants based on the amino-acid sequence of the protein. A library of 34 mutant plasmids was designed, synthesized and screened, resulting in 6 mutants which exhibited a Ts phenotype. Of these 6, the one with the most temperature-sensitive phenotype (M166A) was compared with the original plasmid. It exhibited lower stability at 48°C and was completely unable to replicate ...Continue Reading

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Sep 15, 2010·Proceedings of the National Academy of Sciences of the United States of America·Daniel G OlsonLee R Lynd
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Citations

Oct 15, 2013·FEMS Microbiology Reviews·Sara E Blumer-SchuetteRobert M Kelly
Feb 26, 2013·Applied and Environmental Microbiology·Jilai ZhouLee R Lynd
May 12, 2018·Microorganisms·Eivind B DrejerTrygve Brautaset
Feb 21, 2021·Applied and Environmental Microbiology·Johannes YayoAntonius J A van Maris
Mar 30, 2015·Metabolic Engineering Communications·Daniel G OlsonLee R Lynd
May 23, 2014·ACS Synthetic Biology·Oliver WrightTom Ellis

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Methods Mentioned

BETA
PCR
protein folding

Software Mentioned

PredBur
Bio
Gen5
Rad CFX manager

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