I am tring for homologous recombination of long fragment in bifidobacteria , i want to replace apart of the pyrimidine production genes with another one to increase production of another substance .For this purpose i am using temperature sensitive plasmid and incubate at 42 degree . and let for homologous recombination . how ever after all that there is no homologous recombination after PCR check .
please help me with the best way for such like purpose .
Have you checked that your vector really is temperature sensitive by growing at 42oC in the absence of selection and then checking for loss of resistance? If it is not temperature sensitive then I would use a suicide plasmid like one based on the R6K replicon plasmids.
Mr. He, Mr. Christopher thanks for your answer
Mr. Christopher I have make sure that my plasmid is temp. sensetive
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