Orf60a And Rec A Red Restructuring Efficiency And The Influence Of The Construction Of A New Carrier Pkr And Predig

Recombineering (Recombination Mediated Genetic Engineering) is an efficient method of in vivo genetic engineering applicable to Escherichia coli genome or episomal DNA.It is a modification and DNA cloning technique that makes use of recombination between short oligonucleotides. This method circumvents the need for most standard in vitro cloning techniques.Recombineering allows construction of DNA molecules with precise junctions without constraints being imposed by restriction enzyme site location. Bacteriophage homologous recombination proteins catalyze these recombineering reactions using double-and single-stranded linear DNA substrates, so-called targeting constructs, introduced by electroporation. Gene knockouts, deletions and point mutations are readily made, gene tags can be inserted and regions of BAG or the E. coli genome can be subcloned by gene retrieval using recombineering. Most of these constructs can be made within one weekThe homologous recombination function is provided by three Red genes in λ phage. Reda is a5’-3’exonuclease, creating a single stranded protruded overhang, Redβ is the single strand binding protein that binds to the protruded DNA end and promotes single stranded DNA annealing, Redγ protects the incoming DNA from being degraded by host’s endonuclease, RecBCD. Orf60a and recA gene can increase the efficiency of recombination.For further research of the synergistic effect (or antagonist effect) between orf60a and recA,DSBR (Double Strand Break Repair)and SSOR (Single Strand Oligo Repair) were used in this study.In conclusion, the efficiency of mRed system is much lower than that of the efficiency of system contains Red. Second, in most of the experimental conditions, orf60a and recA have very strong synergistic effect, but sometimes there is also a certain antagonism. Last, we constructed two plasmids by Recombineering. One is the cloning vector pKR with kanamycin reistance marker; and the other is red vector pRedIG which harbors a gentamicin resistance marker.