The Mutant Library Construction Of Legionella Pneumophila Serum 10 By Signature-Tagged Mutagenesis.

ObjectsThe DNA signature tags were obtained by PCR from the single stands DNA tags. The signature tags(STs) was cloned into pC6 to construct recombinant plasmid pC6RS .Then it was transformed into E.coli CC118 to product multiple recombinant plasmid. pC6RS extracted from CC118 was subsequently transformed into E.coli S17-1. By plate conjugation mating, recombinant pC6RS, which contained STs, shuttled from E.coli S17-1 into Legionnaire pneumophila serum 10 so that the signature tags could randomly inserted into Legionnaire pneumophila serum 10 genome. A library of 2,043 Legionnaire pneumophila serum 10 signature tagged transpose mutants was obtained with identified by L-cysteine dependent and Ampicillin-sensitive screening.MethodExperiment 1: The shuttle plasmid construction of Signature-Tagged Mutagenesis technology. The DNA signature tags were obtained by PCR from the single stands DNA tags. The signature tags (STs) was cloned into pC6 to construct recombinant plasmid pC6RS. Then it was transformed into E.coli CC118 to product multiple recombinant plasmid. pC6RS extracted from CC118 was subsequently transformed into E.coli S17-1.Experiment 2: The mutant library construction of Legionella pneumophila...