In vivo repackaging of recombinant cosmid molecules for analyses of Salmonella typhimurium, Streptococcus mutans, and mycobacterial genomic libraries

Abstract

Strains of Escherichia coli K-12 were constructed that permitted the amplification of in vitro-packaged recombinant cosmid-transducing particles by in vivo repackaging of recombinant cosmid molecules. Thermal induction of these thermoinducible, excision-defective lysogens containing recombinant cosmid molecules yielded high titers of packaged recombinant cosmids and low levels of PFU. These strains were used to amplify packaged recombinant cosmid libraries of Mycobacterium leprae, Mycobacterium vaccae, Salmonella typhimurium, and Streptococcus mutans DNA. Contiguous and noncontiguous libraries were compared for the successful identification of cloned genes. Construction of noncontiguous libraries allowed the dissociation of desired genes from genes that were deleterious to the survival of a cosmid recombinant and permitted selection for unliked traits that resulted in a selected phenotype. In vivo repackaging of recombinant cosmids permitted (i) amplification of the original in vitro-packaged collection of transducing particles, (ii) storage of cosmid libraries as phage lysates, (iii) facilitation of complementation screening, (iv) expression analysis of repackaged recombinant cosmids after UV-irradiated cells were infected, (v) in situ enzyme or immunological screening, and (vi) facilitation of recovery of recombinant cosmid molecules containing transposon inserts.