Recombinant Engineering-mediated Gene Mutations, And The Construction Of A New Tolerance-bearing Gene CcFI Strain

Recombineering (recombination-mediated genetic engineering) is an efficient method of in vivo genetic engineering applicable to chromosomal as well as episomal replicons in E. coli. Recombineering renders the construction of DNA molecule at any sites without restricted by restriction enzymes site location, and gene deletions, point mutations and insertion of gene tags. Recombineering has the advantages of simple operation, short cycle and high recombination efficiency, and it helps for the functional genetics and genomics study greatly.In vivo site-directed mutagenesis introduces the specific changes to target DNA, including base additions or deletions, point mutations, etc. Being a very useful tool in genetic engineering and protein engineering studies, it is able to quickly and efficiently improve the expression of the target protein traits. PCR mediated site-directed mutagenesis, oligonucleotide-directed mutagenesis and cassette mutagenesis have some problems like too many cycles or high cost.In this study a recombineering technology mediated site-directed mutagenesis method was established, with the S208G, E192N point mutations and E192 point saturation mutation experiment on the nanA gene in plasmid pLS122. MutH and MutL protein in the DNA mismatch repair system corrects the wrongly incorporated DNA. MutT, Ung and UvrB protein also may correct the wrongly incorporated DNA in E. coli. Deletions of mutH, mutL, mutT, uvrB, ung gene were performed to improve the recombination efficiency but the recombination efficiency was kept at 50%. It was found that DNA phosphorothioation modification has DNA degradation phenotypecan in vivo, moreover, homologous recombination efficiency was associated with the homologous arm length. In this study phosphorothioation modification in mutations segments 5’end and extending left side homologous arm length of mutation segments were performed to improve the recombination efficiency. Based on the results, DNA phosphorothioation modification caused a 20% reduction of the mutation efficiency, and long length homologous arm of mutation segments co-transformate with pLS122 with the molar ratio of 1:1 got the mutation efficiency up to 75%. However, point saturated mutation experiment only obtained 15 kinds of amino acid mutations. Recombineering mediated site-directed mutagenesis method has the advantages of short cycles, simple operation, which can be widely used in the field of point mutations if the mutation efficiency can be more improved.CcdB encoded by ccdB gene has strong lethality to E. coli, which makes the ccdB gene can be used to be the ideal counterselection markers of cloning. Toxic gene ccdB can exist in E. coli DB3.1 Xpir, it is also suitable for the propagation of R6K replicon plasmis.The disadvantages of using E. coli DB3.1 Xpir include low transformation efficiency, when low copy number for R6K plasmid. To obtain a high transformation efficiency ccdB tolerant strain,in this study, the 462th arginine of E. coli BUN20 GyrA protein was mutated to cysteine. The engineered strain was named as E. coli LS027. E. coli LS027 can effectively resist the ccdB toxicity, and shows an transformation efficiency of 6.9×108/μg which is about 100 folder higher than E. coli DB3.1 Xpir; and the R6K plasmid shows high copy number which is about 200 copies per cell as it harbors a.pir116 gene at the same time. This new ccdB tolerant strain will find general applications in gene manipulation.