Difference between revisions of "Part:BBa M50499:Design"
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<partinfo>BBa_M50499 short</partinfo>
 
<partinfo>BBa_M50499 short</partinfo>
  
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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.
  
 
<partinfo>BBa_M50499 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_M50499 SequenceAndFeatures</partinfo>
  
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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.  
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For our experiments, we placed this part upstream from GFP in order to assay expression levels through fluorescent intensity. 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 when CAP binds due to steric hindrance.  
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===Design Notes===
 
===Design Notes===
  
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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.
  
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We were unsure whether to put the CRP binding site immediately downstream or with a buffer sequence. We ended up putting it right after the promoter.
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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.  
  
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===Source===
  
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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).
  
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===Source===
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===References===
  
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IGEM part: BBa_S05450 = constitutive promoter
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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.
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IGEM part: BBa_M36547 = CRP binding site
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Paper: www.ncbi.nlm.nih.gov/pmc/articles/PMC338411/
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Lawson, C. L. et al. Catabolite activator protein: DNA binding and transcription activation. Curr. Opin. Struct. Biol. 14, 10–20 (2004).
  
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===References===
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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).
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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).
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 +
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).
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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.
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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).

Latest revision as of 00:00, 12 December 2018


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.


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 1059
    Illegal PstI site found at 688
    Illegal PstI site found at 849
  • 12
    INCOMPATIBLE 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
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 1059
    Illegal PstI site found at 688
    Illegal PstI site found at 849
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 1059
    Illegal PstI site found at 688
    Illegal PstI site found at 849
  • 1000
    COMPATIBLE 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).