METHOD FOR HOMOLOGOUS RECOMBINATION USING THE GENES BET AND EXO, BUT NOT GAM, OF THE LAMBDA RECOMBINATION SYSTEM

摘要

A method for replacing a target nucleotide/polynucleotide sequence of a bacterial chromosome with a different nucleotide/polynucleotide sequence, which method comprises: (a) providing a bacterium which is capable of expressing: (1) the gamma genes exo or a functional equivalent thereof and bet or a functional equivalent thereof, but not the gamma gene gam or a functional equivalent thereof; or (2) the gamma gene bet or a functional equivalent thereof and gam or a functional equivalent thereof; (b) providing a polynucleotide construct which comprises: (i) a sequence which corresponds to a first sequence flanking the left hand side of the target sequence; (ii) a second sequence corresponding to a sequence flanking the right hand side of the target sequence; and (iii) positioned between (i) and (ii), the donor sequence; and (c) introducing the polynucleotide construct into the bacterium, thereby to replace the target nucleotide sequence with the sequence different from that of the target nucleotide sequences. The method can be used in the preparation of attenuated bacteria and in the identification of essential genes.