Synthetic Biology - Parts - Promoters
From Keaslingwiki
Researchers
Project Description
In a Post-Genomics Era, gene expression systems are essential molecular tools for production of recombinant proteins in cells and for synthesis and degradation of small molecules catalyzed by the enzymes expressed from the expression system. Particularly, fine-tuning gene expression is a need for optimizing metabolic pathways and for examining dose dependence of genes or proteins in cells. We are developing novel genetic control systems that will allow us to better control metabolism and other cellular processes.
Unfortunately, many of the carbohydrate-inducible promoters, such as the arabinose-inducible promoter PBAD, are subject to all-or-none induction, in which intermediate concentrations of the inducer (arabinose) give rise to subpopulations of cells that are fully induced and uninduced. In metabolic engineering, these culture heterogeneities can lead to heterogeneities in the final product. Our simulation studies of this phenomenon indicated that we could alleviate the all-or-none response by placing the gene encoding the protein responsible for transporting the inducer into the cell under control of a promoter that was not responsive to the inducer itself. To construct a host/vector expression system with regulatable promoter control in a homogeneous population of cells, the gene that encodes the arabinose transport system (araE) of E. coli was cloned onto an RSF1010-derived plasmid under control of the IPTG inducible Ptac and Ptaclac promoters. This gene encodes the low-affinity, high-capacity arabinose transport protein and is controlled natively by an arabinose-inducible promoter. The effects of the arabinose concentration and arabinose-independent transport control on population homogeneity were investigated in these strains using flow cytometry. Transport-deficient 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. This work demonstrates the importance of a transport gene that is controlled independently of the inducer to achieve regulatable and consistent induction of gene expression in all cells of the culture.
Recently, we have constructed a new expression system (pPro) providing homogenous expression in individual cells, highly regulatable expression over a wide range of inducer concentrations, and strong expression at high inducer concentrations.A series of new expression vectors (pPro) have been constructed for the regulated expression of genes in Escherichia coli. The pPro vectors contain the prpBCDE promoter (P(prpB)) responsible for expression of the propionate catabolic genes (prpBCDE) and prpR encoding the positive regulator of this promoter. The efficiency and regulatory properties of the prpR-P(prpB) system were measured by placing the gene encoding the green fluorescent protein (gfp) under the control of the inducible P(prpB) of E. coli. This system provides homogenous expression in individual cells, highly regulatable expression over a wide range of propionate concentrations, and strong expression (maximal 1,500-fold induction) at high propionate concentrations. Since the prpBCDE promoter has CAP-dependent activation, the prpR-P(prpB) system exhibited negligible basal expression by addition of glucose to the medium.
Relevant Publications
1. P. Wong, S. Gladney, and J. D. Keasling. 1996. “A mathematical model of the lac operon: inducer exclusion, catabolite repression, and diauxic growth on glucose and lactose.” Biotechnol. Prog. 13:132-143.
2. S. J. Van Dien and J. D. Keasling. 1998. “Optimization of polyphosphate degradation and phosphate secretion using hybrid metabolic pathways and engineered host strains.” Biotechnol. Bioeng. 59:754-761.
3. S. J. Van Dien and J. D. Keasling. 1998. “A dynamic model of the Escherichia coli phosphate-starvation response.” J. Theor. Biol. 190:37-49.
4. S. J. Van Dien and J. D. Keasling. 1999. “Effect of polyphosphate metabolism on the Escherichia coli phosphate-starvation response.” Biotechnol. Prog. 15(4):587-593.
5. T. A. Carrier and J. D. Keasling. 1999. “Investigating autocatalytic gene expression systems through mechanistic modeling.” J. Theor. Biol. 201:25-36.
6. A. Khlebnikov, O. Risa, T. Skaug, T. A. Carrier, and J. D. Keasling. 2000. “Regulatable arabinose-inducible gene expression system with consistent control in all cells of a culture.” J. Bacteriol. 182:7029-7034.
7. A. Khlebnikov, K. A. Datsenko, T. Skaug, B. L. Wanner, and J. D. Keasling. 2001. “Homogeneous expression of the PBAD promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter.” Microbiology 147:3241-3247.
8. C. D. Smolke, A. Khlebnikov, and J. D. Keasling. 2001. “Effects of transcription induction homogeneity and transcript stability on expression of two genes in a constructed operon.” Appl. Microbiol. Biotechnol. 57:689-696.
9. A. Khlebnikov, T. Skaug, and J. D. Keasling. 2002. “Modulation of gene expression from the arabinose-inducible araBAD promoter.” J. Ind. Microbiol. Biotechnol. 29:34-37.
10. A. Khlebnikov and J. D. Keasling. 2002. “Effect of lacY expression on homogeneity of induction from the Ptac and Ptrc promoters by natural and synthetic inducers.” Biotechnol. Prog. 18:672-674.
11. Sung Kuk Lee, Jack D. Newman, and Jay D. Keasling. 2005. Catabolic repression of the propionate catabolic genes in Escherichia coli and Salmonella enterica: Evidence for involvement of the cyclic AMP receptor protein. Journal of Bacteriology 187:2793-2800.
12. Sung Kuk Lee, and Jay D. Keasling. 2005. A propionate-inducible expression system for enteric bacteria. Applied and Environmental Microbiology 71:6856-6862.
13. Sung Kuk Lee, and Jay D. Keasling. 2006. Propionate-regulated high-yield protein production in Escherichia coli. Biotechnology and Bioengineering 93 (5):912-918.
14. Timothy S. Ham, Sung Kuk Lee, Jay D. Keasling, and Adam P. Arkin. 2006. A tightly regulated inducible expression system utilizing the fim inversion recombination switch. Biotechnology and Bioengineering 94:1-4.
15. Sung Kuk Lee, and Jay D. Keasling. 2006. A Salmonella-based, propionate-inducible, expression system for Salmonella enterica. Gene 377: 6-11.
16. Sung Kuk Lee, and Jay D. Keasling. 2006. Effect of glucose or glycerol as the sole carbon on gene expression from the Salmonella prpBCDE promoter in Escherichia coli. Biotechnology Progress, In press.
17. Sung Kuk Lee, Howard D. Chou, Brian Pfleger, Jack D. Newman, Yasuo Yoshikuni, and Jay D. Keasling. 2006. Molecular cross-talk between the lac promoter and the araBAD promoter systems. In preparation.
Funding Resources
Synthetic Biology Engineering Research Center (SynBERC)- A National Science Foundation Engineering Research Center
National Institute of Health Grant no. GM070763-01.
