Construction and Evaluation of Self-Directing Expression System Using Regulatory Elements of Cry Gene of Bacillus Thuringiensis
Abstract An expression system based on the cry gene regulatory elements was constructed. The Terminator region of cry gene from B. thuringiensis subsp. kurstaki HD-1 was cloned in pSG1151 plasmid downstream to gfpmut1. The promoter region of the cry gene was amplified to give three different reading frames. The Promoter region of cry gene was cloned in pSG1151T plasmid upstream to gfpmut1. The expression of GFP under the promoter/terminator expression system was evaluated by checking the expression of gfpmut1 under the same promoter. The GFP content of pSG1151 and three constructs; pDSA1, pDSA2 and pDSA3 were compared by fluorescence spectroscopy. The fluorescent intensity of pSG1151 and pDSA1 were compared at time interval of 6 hours upto 72 hours. Both the samples showed detectable fluorescence that increased with time up to 12 hours, but the increase in the fluorescence of pDSA1 was 3 times higher as compared to pSG1151. A cold peptidase gene was cloned under the control of the cry promoter. The transformed E.coli DH5α colonies were patched on skim milk agar plates and the clones of pSG1151CP and pDSA1CP were compared on the basis of zone of clearance. The zone of clearance of pDSA1CP was much higher as compared to that of pSG1151CP. The cell-free supernatant of Bacillus sp. S1DI 10 and recombinant pDSA1CP collected at different time points was assayed for the specific activity of the extracellular protease. At 72 hours the protease activity in pDSA1CP was 2.7 fold higher compared to that of wild Bacillus sp. S1DI 10.