Molecular characterization of cry4B gene from local isolates of Bacillus thuringiensis encoding 70kDa crystal protein

The toxic region (2.0 kb) of cry4b gene amplified from six different local isolates of bacillus thuringiensis (dab bt 1-6) was cloned in ptz57r/t. e. coli dh5a were transformed with this recombinant plasmid. the toxic region was restricted with ecorl and hindlll,and ligated in the expression vector pt7-7. e. coli bl21c were transformed with the recombinant dna. the expression profile of recombinant organism containing cry4b gene was studied. the expression conditions were optimised with respect to iptg concentration,time of induction and incubation temperature. it was found that the high level of expression occurred at 0.5mm of iptg,at 37°c for 3 hours. the toxicity of genetically modified organisms and crude recombinant cry4b proteins was determined against third instar larvae of mosquito anopheles stephensi. the lc50 of e. coli transformed with cry4b gene isolated form six bt isolates against 3 rd instar larvae of anopheles stephensi ranged between 175±3.34-288±3.02 μg/ml,whereas that of crude preparation of cry4b ranged between 363±2.67-566±2.96 ng/ml. of all the isolates dab bt 5 was found to be the most toxic isolate. copyright 2013 zoological society of pakistan.