CLONING OF LIPASE GENE ISOLATED FROM BACILLUS SUBTILIS 168 INTO THE E.COLI DH5Α HOST

Lipase gene was aimed to be cloned from Bacillus subtilis 168 into the E.coli DH5α host. The strain of Baciilus subtilis 168 is ordered from the ATCC . primer designing was done by obtaining a sequence of the lipase gene from NCBI website, A fragment of lipase gene 212 bp in size was obtained using a pair of highly degenerate primers (estA FP and estA RP). the primers are designed to amplify the lipase gene from Bacillus subtilis 168 The used primers have the restriction sites on both ends for restriction enzymes ( BamHI and EcoRI). The targeted gene (lipase) was carried on a plasmid (PUC18) . PUC 18 plasmid was isolated from E.coli DH5 Alpha which has a poly linker sequence located within the lacZα provides several (10 in case of pUC18/pUC19) unique restriction sites for DNA insertion. The unique restriction sites used for integration of lipase gene insertion into pUC18 vectors interrupt the lacZα fragment so that appropriate E. coli cells possessing recombinant pUC DNA are β-galactosidase deficient and, as a result, produce white colonies on X-gal medium. The PCR product was successfully amplified by using optimizes PCR cycle. Competent cell preparation was done by using calcium chloride method, Positive transformants shows white colonies with associated antibiotic agar after overnight incubation at 37 ?C. The positive clones were streaked onto X-gal medium agar to screen for true lipase producers and were incubated overnight at 37 ?C. The recombinant clones formed an intense white color on the X-gal medium agar plate. The aim of the project is to ensure a successful transformation and expression of lipase gene that is used various applications ex: detergent industry and medical purposes etc. as the E. coli generation time has a faster growth rate and more stable than Bacillus spp.