Improvements And Site-directed Mutagenesis Of Chromosome-based Recombination Engineering Systems

Red/ET recombination technology is a high efficient recombination system based on ? phage Red recombination system or Rac prophage Rec E/Rec T.It is very useful for the genetic manipulation of bacterial genome or large plasmid,which helps for the functional genetics and genomics study.Red/ET recombination utilizes exo,bet,gam or recE/recT enzymes and the homologous recombination occurs on the homologous region between target gene and the bacterial chromosome or plasmid.It is inexpensive,principally,no gene mutation will be involved.LS-GR is a chromosome-based recombineering strain into which gam,reda,red? and recA genes are integrated.To improve LS-GR's recombination efficiency,recJ and sbcB genes were deleted,and resulted strain LS2006 has higher recombineering activity than that of LS-GR though DSBR and SSOR activities comparison.LS2006 will be very useful for gene modification experiments,such as gene knockout and gene transfer.Bet codes Beta protein which binds to ssDNA and promotes the annealing of complementary strands.To explore the structure and function relationship of the Beta protein,a series of truncated Beta mutants were constructed and their SSOR activity were determined.Our results show that while length Beta protein is essential for its best recombination activity.In vivo site-directed mutagenesis introduces the specific changes to target DNA(i.e.genomic or plasmid),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 expression of the target protein traits.In this study a Red/ET-mediated recombination technology mediated site-directed mutagenesis method was established.MutS protein in the DNA mismatch repair system corrects the wrongly incorporated DNA.Deletion of mutS gene was also performed to improve the recombination efficiency.