Cloning of Haloacid Dehalogenase Gene from Bacillus Cereus Local Strain with the Addition of Restriction Sites

Previous studies had successfully isolated and characterized the haloacid dehalogenase gene from Bacillus cereus local strain, namely bcfd1 gene. In the further research, this gene would be sub cloned into expression vector in order to analyse its expression. However, this gene could not be directly sub cloned because it doesn’t have suitable restriction sites that facilitate correct orientation of cloning. Therefore, the addition of suitable restriction sites at both end of the gene was necessary. The research is started by designing specific pair of primers to amplify the bcfd1 gene from Bacillus cereus chromosome by adding EcoRI on forward primer and HindIII on reverse primer. The 870 bp of bcfd1 gene code for haloacid dehalogenase with suitable restiction sites has been successfully cloned into the pGEM-T Easy cloning vector. The recombinant clone that was obtained from screened by ampicillin resistant and ß-galactosidase activity was confirmed by size screening, restriction analysis, and re-PCR. This clone already to be performed for the further sub cloning process in order to get the right cloning direction.