Explore The BL21 (DE3) Recombinant Engineering Systems Established And Self-cutting Systems

With the development of molecular biology, homologous recombination engineering has become a commonly experiment technology used in genetic engineering, widely used in the Escherichia coli genome modification, such as point mutation, and gene knockout experiments. Compared with traditional the system of RecA homologous, Red homologous recombination technology needs short homologous sequence (40to60bp) is highly efficient, so has wide application in DNA cloning and modifications.Firstly, we verified the feasibility-knockout the recJ gene in protein expression host BL21(DE3), and showed that homologous sequence of50bp and500bp were both able to knocked into BL21(DE3) genome successfully and replaced the recJ gene. Endonuclease related gene hsdR codes type I restriction enzyme EcoK (K12strains) or EcoB (B strain). In E. coli cells HsdR acts as a "antibody" to restrict external DNA. Mutation of hsdR is favorable to a foreign plasmid transformation. By the means of homologous recombination hsdR was knocked out of from BL21(DE3), and testing to get the correct strain BL21(DE3)AhsdR, then verify the efficiency of the correct strain. The transformation efficiency of the BL21(DE3)△hsdR mutant is3-5times higher than that of the wild type.TEV Protease derived from the Nla Protease (Nuclear inclusion a protein) of the tobacco etch virus is often used to remove the affinity tag from fusion protein. We designed the enzyme digestion site between affinity tags and the target protein, and use TEV protease to separate tag and target protein. The purification of the target proteins must first purify the fusion protein which is cleaved by TEV proteases before protein purification. After two-step protein purification, a large portion of protein may be lost. The TEV protease purified in our lab meets the requirements of enzyme digestion conditions, so we set up to construct a system to integrate the TEV protease gene into the BL21(DE3) genome, after induced expression, the TEV expressed will cleave the fusion protein intracelluarly, thus releasing the target protein.Same method was used to construct the fusion strain LS1911contained TEV protease gene.We found that only recombinant fragments containing500bp homologous region could be knocked into the genom, the possible reason may be that the deleted region is much longer than that of reaJ. Unexpectedly, the protein expression experiments failed to get the expected result, TEV protease expression did not happen. We also tried to use cat-tetR-ptetA cassette for mTEV induction, and further conditions optimization is going on.