Assembly of six synthetic fragments of cry 1 F gene for Bt crops to generate pest resistance in plants

The insecticidal protein Cry I F of Bacillus thuringiensisis highly toxic to H. armigera in a synergistic manner with Cry 1 Ac. These crystalline proteins were found to be highly toxic to agriculturally important pests at very low concentrations. However, native Bt gene expression in plant cells is sub-optimal to achieve insect protection in transgenic plants because of the presence of many mRNA destabilizing features. Cloning of fragment 6^th of synthetic cry 1 F was done with Vector (pGEM7Zf) while 4^th and 5^th fragments were cloned with pET vector after ligation (T4 DNA Ligase enzyme). These fragments were cloned with vector pGEM carrying 6^th fragment. Now fragment 1+2+3 were assembled with pGEM7Zf carrying 4^th, 5^th and 6^th fragments. A very good band of around 1900 bp was observed which is an indication of a positive clone. Now this pGEM 7Zf vector has all the six Cry 1 F synthetic fragments in between Xba 1 and Bam H1 sites. A modified Cry I F gene was designed according to higher plant codon usage and avoiding the mRNA destabilizing features. The modified gene has 45.5% of GC content.