Direct detection and quantification of horizontal gene transfer by using flow cytometry and gfp as a reporter gene.
A new cultivation-independent method for studying conjugal gene transfer between bacteria was evaluated. The method was based on direct detection and enumeration of donor and transconjugant bacterial cells by flow cytometry. Specific detection of transconjugants was obtained by using a conjugative plasmid tagged with a reporter gene (gfp) encoding green fluorescent protein. A chromosomal encoded repressor (lacI(ql)) repressed expression of GFP in the donor bacteria. Enumeration of the donor cells was performed after induction of GFP expression by the addition of inducer isopropyl-thio-beta-D-galactoside (IPTG). The method presented here provided simple and precise quantification of horizontal gene transfer between both Escherichia coli and Pseudomonas putida strains.
D015169:Colony Count, Microbial; D003227:Conjugation, Genetic; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D005434:Flow Cytometry / Q000379:methods*; D022761:Gene Transfer, Horizontal; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D016958:Pseudomonas putida / Q000235:genetics* / Q000254:growth & development
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins