Molecular Cloning of Actinomyces Bacteriophage DNA in E. Coli.
TEXAS UNIV HEALTH SCIENCE CENTER AT SANANTONIO
Pagination or Media Count:
Actinomyces are gram-positive filamentous bacteria that colonize tooth and mucosal surfaces and coaggregate with other bacteria to initiate plaque formation in the oral cavity. Bacteriophages that cause cell lysis in strains of Actinomyces have been isolated from dental plaque samples. The identification of a phage gene encoding host cell lysis activity may provide a means for the design of strategies that inhibit plaque formation. The purpose of this study was to initiate molecular cloning of DNA fragments from an Actinomyces lytic phage, designated phi3, that contained a putative lysin gene. Purified phi3 genomic DNA was digested with EcoRI, and DNA fragments of 10-, 2- and 1.8- kilobasepairs kb were subcloned onto the expression vector, lambdagt11. This vector allows color selection of recombinant clones, which appear as colorless plaques on a medium containing the chromagen, X-gal, versus non-recombinant clones which appear blue. Three libraries consisting of the phi63 DNA fragments in lambdagt11 were obtained and the cloning efficiencies ranged between 10exp 3 and 10exp 6 plaque forming units per milliliter of recombinant phages. Analysis of randomly selected recombinant clones revealed the presence of the expected phi63 DNA fragments that were used in the subcloning and they were stably maintained in E. coli. Further analysis of the inserted DNA fragments will indicate whether they contain a putative lysin gene. Importantly, the results of this study indicate the feasibility of cloning of Actinomyces phage DNA fragments onto an E. coli expression vector.
- Genetic Engineering and Molecular Biology
- Medicine and Medical Research