Construction and characterization of cloning vector and temperature sensitive vectors for Gordonia sp. IITR100

Gordonia spp. are Gram-positive, non-sporulating bacteria which have several industrial as well as environmental applications. In order to enhance their potential, metabolic engineering is required which often involves genome manipulation. For this purpose, temperature sensitive plasmids are useful as the desired genes for integrases or recombinases can be supplied transiently, followed by curing of the plasmid. Here, we report the construction of a cloning vector and temperature sensitive vectors based upon the pKB1 replicon from Gordonia westfalica. The amino acid residues or regions for creating temperature sensitive mutants were predicted based on in silico methods, and the dynamics of these mutant proteins were studied using docking and molecular dynamic simulations. The desired mutations were incorporated in the replication protein by site directed mutagenesis. The results were validated by growth kinetics of the wild type and mutants at permissive and non-permissive temperatures. This is the first report on temperature sensitive vectors based on native replicon from a plasmid originating from Gordonia.