Construction and characterization of a temperature-sensitive pRC4 replicon for Rhodococcus and Gordonia

Abstract

Temperature-sensitive plasmids are useful for genome engineering and several synthetic biology applications. There are only limited reports on temperature-sensitive plasmids for Rhodococcus and none for Gordonia. Here, we report the construction of a temperature-sensitive pRC4 replicon that is functional in Rhodococcus and Gordonia. The amino acid residues were predicted for the temperature-sensitive phenotype in the pRC4 replicon using in silico methods and molecular simulation of the DNA-binding replication protein with the origin of replication. The amino acid residues were mutated, and the temperature-sensitive phenotype was validated in Gordonia sp. IITR100. Similar results were also observed in Rhodococcus erythropolis, suggesting that the temperature-sensitive phenotype was exhibited across genera.

Keywords: Molecular docking; Molecular dynamics simulation; Non-replicating plasmid; RepB; Shuttle vector; Site directed mutagenesis.