Iraq is one of the main petroleum oil producers. 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. 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 ̴46 KD molecular weight of the concerned protein. coli and confirmed by colony PCR technique using the T7 promoter and T7 terminator primers. The AlkB gene was inserted in PET-21a(+) plasmid vector as expression vector, then transformed in BL21(DE3) competent E. Sticky ends from different BsoBI sites may not be compatible.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. Sticky ends from different AvaI sites may not be compatible. Sticky ends from different BanII sites may not be compatible. Sticky ends from different BstAPI sites may not be compatible.Įfficient cleavage requires at least two copies of the NarI recognition sequence.Įfficient cleavage requires at least two copies of the PluTI recognition sequence.ĪpoI is typically used at 50☌, but is 50% active at 37☌. G C A N N N N N T G C C G T N N N N N A C G Prolonged incubation with NdeI may lead to removal of additional nucleotides. Sticky ends from different PfoI sites may not be compatible. Sticky ends from different AhdI sites may not be compatible.
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