Abstract

With the ever-growing demand for designing proteins and enzymes with better sensitivity, selectivity, stability and affinity, oligo-based site-directed mutagenesis has become instrumental and indispensable in genetic engineering. The conventional method is considered cumbersome, for it relies on replica-plating to screen the mutants based on the reversal in resistance and sensitivity to two antibiotics: Tetracycline and Ampicillin respectively. It also necessitates sub-cloning the mutated gene in an expression vector to ultimately express the mutant-protein. Our orthogonal system facilitates fluorescence-based screening of mutants, using a novel ‘Red-Green’ Dual-Fluorescent GFP-mutant. While point-mutating the gene-of-interest, introducing a single point-mutation in the coding sequence of this GFP-mutant codes for its ‘Green-Only’ isoform. The loss of red fluorescence in the transformed colonies shall indicate successful mutagenesis. Apart from simplifying the screening method, this system facilitates the mutagenesis of the target-gene and expression of the mutated-gene using a single plasmid, thus eliminating the need for sub-cloning.