Coding

Part:BBa_K1197014

Designed by: Alişan Kayabölen   Group: iGEM13_METU_Turkey   (2013-10-02)

House fly cytochrome P450 reductase (fCPR)

fCPR is a cytochrome P450 reductase found originally in Musca domestica (house fly). It is necessary for functional working of CYP6G1 as well as many other cytochrome P450 enzymes. It makes CYP6G1 enzyme catalytically active.


Reference: Andersen, J.F., Utermohlen, J.G., Feyereisen, R., 1994. Expression of house fly CYP6A1 and NADPH-cytochrome P450 reductase in Escherichia coli and reconstitution of an insecticide-metabolizing P450 system. Biochemistry 33, 2171e2177.

• P450 Monooxgenase Production and its Activity:


CYP6G1 gene , which will produce P450, is synthesized by using B. subtilis specific constituve promoter Pveg. fCPR gene, used for activation of CYP6G1 in B. subtilis, is also transcribed with same promoter. These two genes has transcribed with SacB genes, after translation which helps to export these proteins through outside of the cell, into the bee gut. [1] To be able know, how much enzyme is produced in a given time and how much imidacloprid is converted into nontoxic components by P450, model of this circuit was required.


Enzyme Kinetics for P450 Monooxygenase:

To be able to estimate the rate of the P450 reaction with imidacloprid, we used Michaelis-Menten Kinetics. The harmful imidacloprid concentration for honey bees is reported as 1200 µg/l [2], so in our model we assumed that the imidacloprid concentration in bee gut as 1200 µg/l. And P450 monooxgenase will be produced from our circuit and directly secreted into bee gut where imidacloprid is found.


• So according to these parameters, we have calculated our enzyme activity according to our enzyme production rate. In 3500 s , 4E10 molecule of imidocloprid is degraded according to our model.

References:

1- http://www.ncbi.nlm.nih.gov/pubmed/23470655 2- http://www.bioone.org/doi/abs/10.1603/0022-0493-101.6.1743 3- http://www.sciencedirect.com/science/article/pii/S0048357512000685

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1247
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1769


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