Regulatable arabinose-inducible gene expression system with consistent control in all cells of a culture.
The arabinose-inducible promoter P(BAD) is subject to all-or-none induction, in which intermediate concentrations of arabinose give rise to subpopulations of cells that are fully induced and uninduced. To construct a host-vector expression system with regulatable control in a homogeneous population of cells, the araE gene of Escherichia coli was cloned into an RSF1010-derived plasmid under control of the isopropyl-beta-D-thiogalactopyranoside-inducible P(tac) and P(taclac) promoters. This gene encodes the low-affinity, high-capacity arabinose transport protein and is controlled natively by an arabinose-inducible promoter. To detect the effect of arabinose-independent araE expression on population homogeneity and cell-specific expression, the gfpuv gene was placed under control of the arabinose-inducible araBAD promoter (P(BAD)) on the pMB1-derived plasmid pBAD24. The transporter and reporter plasmids were transformed into E. coli strains with native arabinose transport systems and strains deficient in one or both of the arabinose transport systems (araE and/or araFGH). The effects of the arabinose concentration and arabinose-independent transport control on population homogeneity were investigated in these strains using flow cytometry. The araE, and araE araFGH mutant strains harboring the transporter and reporter plasmids were uniformly induced across the population at all inducer concentrations, and the level of gene expression in individual cells varied with arabinose concentration. In contrast, the parent strain, which expressed the native araE and araFGH genes and harbored the transporter and reporter plasmids, exhibited all-or-none behavior. This work demonstrates the importance of including a transport gene that is controlled independently of the inducer to achieve regulatable and consistent induction in all cells of the culture.
Khlebnikov
A
A
Department of Chemical Engineering, University of California, Berkeley, California 94720-1462, USA.
Risa
O
O
Skaug
T
T
Carrier
T A
TA
Keasling
J D
JD
D051777:AraC Transcription Factor; D001089:Arabinose / Q000378:metabolism / Q000494:pharmacology; D001426:Bacterial Proteins; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins; D005434:Flow Cytometry; D015964:Gene Expression Regulation, Bacterial; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors
D051777:AraC Transcription Factor; C497371:AraC protein, E coli; C109577:AraE protein, bacteria; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C491627:araE protein, E coli; D001089:Arabinose