Abstract
BackgroundAntibiotic resistance genes are widely used for selection of recombinant bacteria, but their use risks contributing to the spread of antibiotic resistance. In particular, the practice is inappropriate for some intrinsically resistant bacteria and in vaccine production, and costly for industrial scale production. Non-antibiotic systems are available, but require mutant host strains, defined media or expensive reagents. An unexplored concept is over-expression of a host essential gene to enable selection in the presence of a chemical inhibitor of the gene product. To test this idea in E. coli, we used the growth essential target gene fabI as the plasmid-borne marker and the biocide triclosan as the selective agent.ResultsThe new cloning vector, pFab, enabled selection by triclosan at 1 μM. Interestingly, pFab out-performed the parent pUC19-ampicillin system in cell growth, plasmid stability and plasmid yield. Also, pFab was toxic to host cells in a way that was reversed by triclosan. Therefore, pFab and triclosan are toxic when used alone but in combination they enhance growth and plasmid production through a gene-inhibitor interaction.ConclusionThe fabI-triclosan model system provides an alternative plasmid selection method based on essential gene over-expression, without the use of antibiotic-resistance genes and conventional antibiotics.
Highlights
Antibiotic resistance genes are widely used for selection of recombinant bacteria, but their use risks contributing to the spread of antibiotic resistance
All plasmids are available from the authors. (B) Growth of pFab and pUCFA clones on LB plates containing triclosan (LBT) and LBA plates. (C) Plasmids propagated in different E. coli hosts were digested with BamHI and analyzed by gel electrophoresis
This study provides an example of how essential genes can be used in combination with non-antibiotic inhibitors to select and maintain recombinant bacteria
Summary
Antibiotic resistance genes are widely used for selection of recombinant bacteria, but their use risks contributing to the spread of antibiotic resistance. An unexplored concept is over-expression of a host essential gene to enable selection in the presence of a chemical inhibitor of the gene product To test this idea in E. coli, we used the growth essential target gene fabI as the plasmid-borne marker and the biocide triclosan as the selective agent. The first reported non-antibiotic system involves an auxotrophic bacterial strain and complementation using a plasmid-encoded biosynthesis gene, such that only transformants grow on defined media lacking the nutrient [5]. Another system is termed repressor-titration, where the lac (page number not for citation purposes)
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