Plasmid selection in Escherichia coli using an endogenous essential gene marker
Antibiotic resistance genes are widely used for selection of recombinant bacteria, but their use risks contributing to 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 the 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.
Results: The new cloning vector, pFab, enabled selection by triclosan at 1microM. 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.
Conclusions: The 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.
Author: Shan Goh and Liam Good
Credits/Source: BMC Biotechnology 2008, 8:61
Published on: 2008-08-11
Copyright by the authors listed above - made available via BioMedCentral (Open Access). Please
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