Part:BBa_M50499:Design
Catabolite Activator Protein (CAP) Repressed Promoter
We placed the constitutive promoter (BBa_S05450, iGEM) immediately upstream from a catabolite activator protein (CAP) binding site (BBa_M36547, iGEM). When CAP binds to the binding site, expression will be repressed.
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 1059
Illegal PstI site found at 688
Illegal PstI site found at 849 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 144
Illegal NheI site found at 167
Illegal PstI site found at 688
Illegal PstI site found at 849 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 1059
Illegal PstI site found at 688
Illegal PstI site found at 849 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 1059
Illegal PstI site found at 688
Illegal PstI site found at 849 - 1000COMPATIBLE WITH RFC[1000]
This construct is an adaption of the natural lac operon, which is positively regulated by CAP, since the CAP binding site is naturally upstream of the RNA polymerase binding site. In this construct, we put the CAP binding site downstream in order to repress transcription due to steric hindrance when CAP binds.
Design Notes
This construct is an adaption of the natural lac operon, which is positively regulated by CAP, since the CAP binding site is naturally upstream of the RNA polymerase binding site. In this construct, we put the CAP binding site downstream in order to repress transcription due to steric hindrance when CAP binds.
Since CAP binding is inversely correlated to glucose concentrations (and positively correlated with cyclic AMP (cAMP) levels), this promoter exhibits glucose-inducibility and cAMP-repressibility. We employed this construct as a potential DNA-based glucose biosensor in E coli.
Source
We used only pre-existing iGEM parts: the constitutive promoter (IGEM part: BBa_S05450) and the CAP binding site (IGEM part: BBa_M36547). The CAP binding site was actually from a previous BioE44 project! We confirmed the CAP sequence from a paper as well (www.ncbi.nlm.nih.gov/pmc/articles/PMC338411).
References
Keizer J, Magnus G. ATP-sensitive potassium channel and bursting in the pancreatic beta cell. A theoretical study. Biophys J. 1989;56(2):229–42.
Lawson, C. L. et al. Catabolite activator protein: DNA binding and transcription activation. Curr. Opin. Struct. Biol. 14, 10–20 (2004).
Notley-McRobb, L., Death, A. & Ferenci, T. The relationship between external glucose concentration and cAMP levels inside Escherichia coli: Implications for models of phosphotransferase-mediated regulation of adenylate cyclase. Microbiology (1997).
Morita, T., Shigesada, K., Kimizuka, F. & Aiba, H. Regulatory effect of a synthetic CRP recognition sequence placed downstream of a promoter. Nucleic Acids Res. (1988).
Cuero, R., Navia, J., Agudelo, D. & Medina, P. Construct of DNA glucose sensor yeast plasmid for early detection of diabetes. Integr. Obes. Diabetes 3, 1–9 (2017).
Czarniecki, D., Noel, R. J. & Reznikoff, W. S. The -45 region of the Escherichia coli lac promoter: CAP-dependent and CAP-independent transcription. J. Bacteriol. (1997).
Xie M, Ye H, Wang H, Charpin-El Hamri G, Lormeau C, Saxena P, et al. Β-Cell-Mimetic Designer Cells Provide Closed-Loop Glycemic Control. Science. 2016;354(6317):1296–301.
Duan, F. F., Liu, J. H. & March, J. C. Engineered commensal bacteria reprogram intestinal cells into glucose-responsive insulin-secreting cells for the treatment of diabetes. Diabetes 64, 1794–1803 (2015).