Cloning and Expression of Pseudomonas aeruginosa AlkB Gene in E. coli

Pre identified hydrocarbons degrading bacteria were used in this study, specific primer was conducted to amplification of AlkB gene, approximately 1206bp band size of this gene for Pseudomonas aeruginosa was detected and proofed by sequence and alignment analysis with NCBI database. The AlkB gene was inserted in PET-21a(+) plasmid vector as expression vector, then transformed in BL21(DE3) competent E. coli and confirmed by colony PCR technique using the T7 promoter and T7 terminator primers. The expression of the inserted gene was checked by determined the concentration of AlkB protein for multiple periods by Bradford assay method and the SDS-polyacrylamide gel electrophoresis method was revealed band of similar to 46 KD molecular weight of the concerned protein. The gene amplification and cloning strategy was lay out before the practical part of the study by SnapGene software, this study was conducted to introduce cloned bacteria which facilitate the first step (key step) of alkane's biodegradation and propose an appropriate strategy to construct genetically engineered microorganisms with multiple recombinant plasmid for enhance the degradation of the aliphatic fraction of hydrocarbon