The Method An Oligonucleotide Mediated E. Coli Knockout Or Point Mutations

Recombineering (Red/ET, recombination mediated genetic engineering) is a DNA cloning and modification technique that makes use of recombination between short oligonucleotides in Escherichia coli. Recombineering is a great alternative whenever traditional cloning methods are difficult to be accomplished, such as the DNA fragment is too large, the appropriate restriction sites is difficult to find, or gel extraction is inefficient. More over, DNA mutations may be in the process of ultraviolet radiation and ethidium bromide staining. Recombineering is rapidly becoming a routine cloning technique.The homologous recombination function is provided by three Red genes inλphage. Reda (Exo) is a 5'-3'exonuclease, creating a single stranded protruded overhang, redp(Bet) is the single strand binding protein that binds to the protruded DNA end and promotes single stranded DNA annealing, Redy(gam) protects the incoming DNA from being degraded by host's endonuclease, RecBCD. These three genes are the usual sense recombinase gene. RecA gene can increase the efficiency of recombination.Homologous recombination can be realized between homologous regions as short as 36 base pairs, which may be introduced through the synthesized PCR primers.In this paper, a novel recombineering method was presented to achieve the E. coli gene knockout and point mutation through oligonucleotide mediated by recombinant engineering. First the homologous arm flaked sucrose 6 fucosyltransferase gene (sacB) and kanamycin resistance gene (neo) cassette was integrated into the target gene through recombineering. Then the second recombineering with oligonucleotide removed the cassette through sucrose counter-selection. Gene deletion as well as point mutation were performed successfully without any bases redundancy.Oligonucleotide design in gene deletion is the same of both sides of target gene nucleotide sequence,while oligonucleotide design in point mutation introduces the base needs in the mutant oligonucleotides.The experimental methods used to achieve LacZ gene deletion,LacZ gene point mutation, rhaSR gene deletion and pheS gene point mutation.