Difference between revisions of "Part:BBa K4759213"

Latest revision as of 18:19, 11 October 2023

T7-RBS1-petH-RBS2-petF

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.

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

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
INCOMPATIBLE WITH RFC[12]
Illegal NotI site found at 1109
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1638
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 <partinfo>BBa_K4759213 short</partinfo>
-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.
+===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.
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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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-===Usage and Biology===
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 <span class='h3bb'>Sequence and Features</span>