Difference between revisions of "Part:BBa K4759044"

Revision as of 18:03, 11 October 2023

RBM3-linker-GFP1-10

The P450 enzymes are redox-dependent proteins, through which they source electrons from reducing cofactors to drive their activities.

Usage and Biology

Generally, the method of determining whether the redox partners was suitable required tedious steps such as the construction of plasmids, heterologous expression, construction of catalytic systems, and detection of conversion rate after catalysis. Therefore, we wanted to find a convenient way to do a quick screening. We used the fluorescent protein sfGFP and successfully constructed a sensor to detect redox partners. We divided sfGFP into N-terminal and C-terminal, and although these two parts were cut off, there was an interaction force between them. Thus, four iron redox proteins were fused to the N-terminal of sfGFP-1-10 and Olep to the C-terminal of sfGFP-11, respectively, to obtain the recombinant plasmid pRSFDuet-BM3-GFP-1-10-GFP-11-oleP, pRSFDuet-camA-camB-GFP-1-10-GFP-11-oleP, pRSFDuet-FdR_0978-Fdx_1499-GFP-1-10-GFP-11-oleP, and pRSFDuet-petH-petF-GFP-1-10-GFP-11-oleP. The above four recombinant plasmids were converted to BL21(DE3) to obtain recombinant strains G2 to G5.

Fig1: The self-assembly of Olep and Fdx based on the three-dimensional structure of sfGFP (PDB: 5BT0)

The recombinant strains G2 to G5 were subjected to shaker fermentation experiments. After the fermentation was completed, 200 ul bacteria were added to the 96-well plate with a microplate reader to determine biomass (wavelength 600 nm) and fluorescence value (excitation wavelength 488 nm, emission wavelength 520 nm). Then we calculated the fluorescence intensity (fluorescence value/biomass) of the strain. The fluorescence intensity of the recombinant strain G5 (containing recombinant plasmid pRSFDuet-petH-petF-GFP-1-10-GFP-11-olep) was the highest (1.2×106) and 6 times higher than that of the control strain G2 (containing recombinant plasmid pRSFDuet-camA-camB-GFP-1-10-GFP-11-olep).

Fig2: BL21 morphology diagram seen under excitation light 488nm and emitted light 520nm, green is green fluorescence of sfGFP

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. Sequence and Features