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Biosystems. 2007 Jul-Aug;90(1):115-20. Epub 2006 Jul 16.

Dual selection of a genetic switch by a single selection marker.

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1
Department of Biomedical Engineering, University of California, Davis, 451 E. Health Sciences Dr., Davis, CA 95616, USA.

Abstract

Forward engineering of synthetic genetic circuits in living cells is expected to deliver various applications in biotechnology and medicine and to provide valuable insights into the design principles of natural gene networks. However, lack of biochemical data and complexity of biological environment complicate rational design of such circuits based on quantitative simulation. Previously, we have shown that directed evolution can complement our weakness in designing genetic circuits by screening or selecting functional circuits from a large pool of nonfunctional ones. Here we describe a dual selection strategy that allows selection of both ON and OFF states of genetic circuits using tetA as a single selection marker. We also describe a successful demonstration of a genetic switch selection from a 2000-fold excess background of nonfunctional switches in three rounds of iterative selection. The dual selection system is more robust than the previously reported selection system employing three genes, with no observed false positive mutants during the simulated selections.

[Indexed for MEDLINE]

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