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Genetics and Molecular Biology |
Department of Chemical Engineering, University of California, Berkeley, CA 94720-1462, USA1
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA2
Author for correspondence: Jay D. Keasling. Tel: +1 510 642 4862. Fax: +1 510 643 1228. e-mail: keasling{at}socrates.berkeley.edu
Genes placed under the control of the arabinose-inducible araBAD promoter (PBAD) of Escherichia coli are expressed in an all-or-none fashion, in which the percentage of induced cells in the population, rather than the degree of induction in individual cells, varies with the concentration of arabinose in the culture medium. Previous work showed that all-or-none gene expression from PBAD was due to the arabinose-dependent expression of the gene encoding the low-affinity high-capacity transporter (araE), and that expression of heterologous genes from PBAD in individual cells could be regulated by placing the araE gene under control of an arabinose-independent promoter. Based on these results, two expression systems were developed to allow regulatable control of genes under control of PBAD. In one system, the native araE promoter on the chromosome was replaced by constitutive promoters of different strengths. In the second system, the araE gene under control of the same constitutive promoters was placed on a medium-copy plasmid. Both systems allow regulatable expression of a plasmid-borne PBAD-controlled heterologous gene and a homogeneous population of cells over a wide range of arabinose concentrations. While the degree of induction varied slightly with the strength of the constitutive promoter, expression was affected most by the arabinose concentration.
Keywords: arabinose transport system, regulatable gene expression, Red recombination, GFP fusions, FACS analysis
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