A new cloning system using a mutant esterase containing MCS as an indicator for gene cloning

A new cloning vector, pTcp216M-MCS, containing an engineered esterase ESTL120P as an indicator was constructed for the screening of positive clones harboring recombinant plasmids. After cloning the mutant esterase gene into pTrc99A, a synthetic multiple cloning site (MCS) was inserted into the predicted gaps of the esterase ESTL120P. These gaps were selected based on the results of comparative analysis of a family of carboxylesterases that was conducted using multiple sequence alignments and a secondary structure prediction tool. The reliability and reproducibility of the resulting vector with the novel indicator to discriminate positive clones from negative ones were compared with those of pTGFP and pBluescript II SK+, which utilized a green fluorescent protein and beta-galactosidase as an indicator, respectively. The results demonstrated that pTcp216M-MCS had a higher fidelity and indicating ability than other vectors, which suggested that the esterase could be used as a new indicator for gene cloning.