Composite

Part:BBa_K1197015

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

Catalytically active CYP6G1 coding device

This device was constructed to produce catalytically active CYP6G1 in B. subtilis and secrete it outside. Secretion is needed since CYP6G1 enzyme should degrade imidacloprid in bee midgut. To provide constitutively production and secretion of this enzyme, we used Pveg constitutive promoter, SpoVG RBS with SacB signal peptide (K541501) which is used by Fatih_Turkey Team in 2011.

Also fCPR production is necessary to make CYP6G1 enzyme catalytically active. Thus, SpoVG RBS and SacB signal peptide was used before fCPR too.

• 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. 

According to Michaelis-Menten Kinetics;


• 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 BglII site found at 767
    Illegal BamHI site found at 3178
    Illegal XhoI site found at 1435
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1168
    Illegal BsaI.rc site found at 3700


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