Difference between revisions of "Part:BBa K4759201"

 
 
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<partinfo>BBa_K4759201 short</partinfo>
 
<partinfo>BBa_K4759201 short</partinfo>
  
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Since the CYP107 family is a typical three-component P450 enzyme that requires a pair of redox partner to transfer electrons from the electron donor NAD(P)H to the P450 enzyme activity center, ferredoxin reductase CamA and ferredoxin CamB are selected as redox partner. The codon-optimized camA gene and camB gene are synthesized, and they are subcloned to the plasmid pACYCDuet to obtain the recombinant plasmid pACYCDuet-camA-camB
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ferredoxin reductase CamA and ferredoxin CamB are selected as redox partner.  
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===Design ===
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codon-optimized
  
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
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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.
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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.
  
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https://static.igem.wiki/teams/4759/wiki/4-3.png
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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
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https://static.igem.wiki/teams/4759/wiki/4-4.png
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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/)
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 18:20, 11 October 2023


T7-RBS1-camA-RBS2-camB

ferredoxin reductase CamA and ferredoxin CamB are selected as redox partner.

Design

codon-optimized

Usage and Biology

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
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1366
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1366
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1366
    Illegal BamHI site found at 1360
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1366
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1366
    Illegal AgeI site found at 124
    Illegal AgeI site found at 418
  • 1000
    COMPATIBLE WITH RFC[1000]