Coding

Part:BBa_K3594014

Designed by: Runhan Luo   Group: iGEM20_SHSBNU_China   (2020-10-23)
Revision as of 15:24, 23 October 2020 by GraceLuo (Talk | contribs)


Cytochrome P450 gene (CYP)

CYP is a large protein complex responsible for the degrading of guaiacol, CYP enters researchers' field of vision mainly as a decomposing product of lignin. Past studies have found that there are several G+ bacteria that can use guaiacol as a carbon source. The first step of their metabolism is mainly catalyzed by cytochrome P450 (CYP), which generates catechol through demethylation. This step of demethylation usually requires some redox chaperone proteins to assist.

In an article from AMB Express in 2019, the authors did a very detailed study on the demethylation process of guaiacol. Their data shows that only two enzymes from the G+ bacteria Rhodococcus rhodochrous need to be prepared, namely Cytochrome P450 (WP_085469912 from R. rhodochrous J3) and Ferredoxin reductase (WP_085469913 from R. rhodochrous J3), and transfer them to G-bacteria Pseudomonas After putida EM42, with or without additional carbon source supplementation, the effective degradation of guaiacol can be achieved.

Therefore, we hope to transfer these two enzymes into Escherichia coli to express and decompose the guaiacol in the intestines of locusts.

References: J. García‑Hidalgo et al., AMB Express. 9 (34) (2019) https://amb-express.springeropen.com/articles/10.1186/s13568-019-0759-8#Tab1

The degradation pathway of guaiacol requires CYP and Ferredoxin Reductase to transform guaiacol to formaldehyde and catechol. T--SHSBNU China--protein concentration-gene expression.png

Characterization

We used SDS-PAGE to verify whether our engineered bacteria could express CYP. From the image below, column 3 and 4 are plasmid without induction; column 5 and 6 are plasmid with induction. As a result, plasmid with induction could express CYP T--SHSBNU China--SDS-PAGE.png style="height:370px;width:550px;"

we assume that the change of protein concentration in the external environment is related to the amount of gene expression, the rate of bacterial lysis, the rate of protein degradation, and the rate of locust intestinal peristalsis. [[File:T--SHSBNU China--SHSBNU China parts33.jpeg] And got the following results: The concentration of the protein changes over time. The abscissa represents time, and the ordinate represents the protein concentration. It can be seen from the image that the protein increases rapidly after the bacterial self-lysis. After reaching a certain concentration, due to the degradation rate of the protein itself and the locust intestinal peristalsis. The rate decreases slightly, and the concentration increases with the next bacterial self-lysis, which forms a stable cycle that can release enzymes efficiently. T--SHSBNU China--concentration of enzymes.jpg

The predicted concentration of guaiacol accord with our goal. T--SHSBNU China--guaiacol goes down.jpeg


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 315
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 163
    Illegal NgoMIV site found at 288
    Illegal NgoMIV site found at 763
    Illegal NgoMIV site found at 1039
    Illegal NgoMIV site found at 1057
    Illegal AgeI site found at 399
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
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