Difference between revisions of "Part:BBa K4759046"
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The P450 enzymes are redox-dependent proteins, through which they source electrons from reducing cofactors to drive their activities. | The P450 enzymes are redox-dependent proteins, through which they source electrons from reducing cofactors to drive their activities. | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
+ | Through extensive reading of the literature, we summarized 11 pairs of redox partners with good results. After that, we screened four pairs of redox partners with good effects by molecular docking and mathematical modeling and then verified them experimentally. | ||
+ | Generally, the method of determining whether the redox partner is suitable is through 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 to successfully construct a sensor to detect redox partners. sfGFP is a superfolder fluorescent protein that emits green light when irradiated with ultraviolet light. What is special about it is that it can be broken into two parts. | ||
+ | We divide sfGFP into sfGFP-1-10 and sfGFP-11, and although these two parts are cut off, there is an interaction force between them, and as long as they are properly folded in space, they will emit light again. Thus, four iron redox proteins are fused to the N-terminus of sfGFP-1-10 and Olep to the C-terminus 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, pRSFDuet-petH-petF-GFP-1-10-GFP-11-oleP | ||
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+ | The above four recombinant plasmids are converted to BL21(DE3) to obtain recombinant strains G2 strain to G5 strain. | ||
+ | |||
+ | https://static.igem.wiki/teams/4759/wiki/4-1.png | ||
+ | |||
+ | Fig1: The self-assembly of Olep and Fdx based on the three-dimensional structure of sfGFP (PDB: 5BT0) | ||
+ | |||
+ | The recombinant strains G2 to G5 are subjected to shaker fermentation experiments. After the fermentation is completed, 200 ul bacteria are 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). Calculate 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) is 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). | ||
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+ | https://static.igem.wiki/teams/4759/wiki/4-2.png | ||
+ | |||
+ | 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, SelFdR_0978/SelFdx_1499, petH/petF) in combination with the P450 enzyme. Four groups of redox partners are 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. and transformed to C41 (DE3) to obtain the recombinant strain R2 strain to R5 strain. The recombinant strains G2 to G5 are subjected to shaker fermentation experiments. The recombinant strain R5 (containing recombinant plasmid pRSFDuet-petH-petF-olep) has the highest conversion rate, significantly increasing from 41.4% to 85.6%. Therefore, the redox companion PetH/PetF derived from Synechocystis is successfully screened as the most suitable redox partner for the P450 enzyme Olep, and the construction of the sfGFP sensor is verified, which could efficiently and accurately screen the redox partner adapted by the P450 enzyme. | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> |
Revision as of 16:07, 10 October 2023
fdr_0978-RBS2-fdx_1499-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
Through extensive reading of the literature, we summarized 11 pairs of redox partners with good results. After that, we screened four pairs of redox partners with good effects by molecular docking and mathematical modeling and then verified them experimentally. Generally, the method of determining whether the redox partner is suitable is through 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 to successfully construct a sensor to detect redox partners. sfGFP is a superfolder fluorescent protein that emits green light when irradiated with ultraviolet light. What is special about it is that it can be broken into two parts. We divide sfGFP into sfGFP-1-10 and sfGFP-11, and although these two parts are cut off, there is an interaction force between them, and as long as they are properly folded in space, they will emit light again. Thus, four iron redox proteins are fused to the N-terminus of sfGFP-1-10 and Olep to the C-terminus 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, pRSFDuet-petH-petF-GFP-1-10-GFP-11-oleP
The above four recombinant plasmids are converted to BL21(DE3) to obtain recombinant strains G2 strain to G5 strain.
Fig1: The self-assembly of Olep and Fdx based on the three-dimensional structure of sfGFP (PDB: 5BT0)
The recombinant strains G2 to G5 are subjected to shaker fermentation experiments. After the fermentation is completed, 200 ul bacteria are 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). Calculate 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) is 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, SelFdR_0978/SelFdx_1499, petH/petF) in combination with the P450 enzyme. Four groups of redox partners are 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. and transformed to C41 (DE3) to obtain the recombinant strain R2 strain to R5 strain. The recombinant strains G2 to G5 are subjected to shaker fermentation experiments. The recombinant strain R5 (containing recombinant plasmid pRSFDuet-petH-petF-olep) has the highest conversion rate, significantly increasing from 41.4% to 85.6%. Therefore, the redox companion PetH/PetF derived from Synechocystis is successfully screened as the most suitable redox partner for the P450 enzyme Olep, and the construction of the sfGFP sensor is verified, which could efficiently and accurately screen the redox partner adapted by the P450 enzyme. Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1219
Illegal PstI site found at 281
Illegal PstI site found at 607
Illegal PstI site found at 922 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1219
Illegal PstI site found at 281
Illegal PstI site found at 607
Illegal PstI site found at 922 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1219
Illegal BglII site found at 1037
Illegal BglII site found at 2208
Illegal BamHI site found at 1213 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1219
Illegal PstI site found at 281
Illegal PstI site found at 607
Illegal PstI site found at 922 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1219
Illegal PstI site found at 281
Illegal PstI site found at 607
Illegal PstI site found at 922
Illegal NgoMIV site found at 625
Illegal AgeI site found at 697 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1342