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

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 at 55°C.The plasmid described in this work could be useful in future efforts to genetically engineer C. thermocellum, and the method used to generate this plasmid may be useful for others trying to make Ts plasmids.