Composite

Part:BBa_K4759218

Designed by: Jianhua Chen   Group: iGEM23_Jiangnan-China   (2023-10-07)
Revision as of 18:18, 11 October 2023 by JianghuaChen (Talk | contribs) (→‎Usage and Biology)

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T7-RBS1-RBM3

Design Notes

(BM3 Redox domain)：a gene coding for Reducatase domain from P450BM3(the selfsufficient P450 enzyme). it has an excellent electron transfer system and the fastest substrate oxidation rate among the P450s owing to the proximity and specific positioning of its domains . Researcher has found that fusing P450s to reductase genes can improve the biocatalysis, We use this part to construct electron transfer pathway to Olep

We selected four conventional redox partners (BM3, CamA/CamB, SelFdR0978/SelFdx1499, PetH/PetF) in combination with the P450 enzyme. Four groups of redox partners were constructed on the high-copy plasmid pRSFDuet to obtain recombinant plasmids: pRSFDuet-BM3-olep, pRSFDuet-camA-camB-olep, pRSFDuet-FdR0978-Fdx1499-olep, and pRSFDuet-petH-petF-olep. Then they were transformed to C41 (DE3) to obtain the recombinant strain R2 to R5. The recombinant strains R2 to R5 were subjected to shaker fermentation experiments. The recombinant strain R5 (containing recombinant plasmid pRSFDuet-petH-petF-olep) had the highest conversion rate, which significantly increased from 41.4% to 85.6%. Therefore, the redox partners PetH/PetF derived from Synechocystis was successfully screened as the most suitable redox partners for the P450 enzyme Olep, and the construction of the sfGFP sensor was verified, which could efficiently and accurately screen the redox partner adapted by the P450 enzyme.

4-3.png Fig1: (A) Screening proper redox partners for Olep from different sources. The G1 strain that contains the empty pRSFDuet-1 plasmid was used as a control. The fluorescent intensities were calculated and the color of cells and fluorescent images were presented for G2-G5 strains that express different redox partners-sfGFP-1-10 and sfGFP-11-Olep, respectively. (B) The conversion rates were calculated for R2-R5 strains that express different redox partners and Olep, respectively. The R1 strain that contains the empty pRSFDuet-1 plasmid was used as a control. The blue-filled triangle represents the fluorescent intensity/OD600. The red hollow triangle represents the conversion rate (%). Values and triangles represent the means and standard deviations of biological triplicates

4-4.png Fig2: The structures and interactions between Olep and Fdxs are presented. The key interacting residues in Olep-Fdx complexes are depicted as sticks and highlighted in yellow. Heme and substrates are displayed as sticks, colored in red and wheat, respectively. The Fe2S2 cluster is visualized as spheres. The distances (Å) between the iron–sulfur cluster and heme-iron are measured and indicated by dashed red lines. The interaction areas of Olep-Fdx are calculated by NovoPro (https://www.novopro.cn/). The numbers of hydrogen bonds and salt bridges are predicted by PDBePISA (https://www.ebi.ac.uk/pdbe/)


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1426
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 472
    Illegal BsaI.rc site found at 1396
    Illegal SapI.rc site found at 952
    Illegal SapI.rc site found at 1552


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