Cloning And Expression Of Vitreoscilla Hemoglobin Gene In Bacillus Thuringiensis

There is sensitive system controlling and responding to the level of dissolved oxygen in the microorganisms. The system can maintain the steady level of dissolved oxygen by changing the growth state of microorganism. The obligate aerobic bacterium, Vitreoscilla, produce elevated quantities of a homodimeric hemoglobin(VHb) in order to survive under hypoxic growth conditions. Resently, the gene(vgfr) of VHb has been cloned into heterologous hosts and promoted their capacity of growth and protein production under hypoxic growth conditions. In this study, the vgb gene is cloned into Bacillus thuringiensis HBF-l.We study how the gene promotes the growth of Bacillus thuringiensis HBF-1.In this study, ,we take the pMK4, a shuttle vector between Gram negative and positive,as a vector and sacB as a promoter to construct the pMK-SV for efficient expression of vgb gene in Bacillus thuringiensis.The pMK-SV is transformated into Bacillus thuringiensis by the method of spherplast electroporation. After the recombinants are obtained by blotting method and induced. The density of recombinants increases 20.2% more than those of original stain(HBF-l).Firstly, to clone the vgb gene with natural promoter into pMK4, based on vgb from Genebank, the primers are designed. Secondly, the aim gene is obtained by PCR. Thirdly, the fragment is cloned into the expression vector pBV221 to construct the recombinant plasmid pBV-V. Lastly, VHb protein of 15.7kD is expressed by the temperature induction.We construct the plasmid pUC-LV by adding synthesized natural promoter of the vgb gene into the vgb gene. After induction under hypoxic conditions, the density of recombinants increases 157% more than those of original stain and the VHb protein with the physiological activity is expressed.