Difference between revisions of "Part:BBa K4759063"

 
 
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<partinfo>BBa_K4759063 short</partinfo>
 
<partinfo>BBa_K4759063 short</partinfo>
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Aspartic acid at site 61 of the PetF is mutated to arginine.<br>
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===Usage and Biology===
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The P450 enzymes are redox-dependent proteins, through which they source electrons from reducing cofactors to drive their activities. This part is coding for ferredoxin reductase PetH and ferredoxin PetF from the algae (<i>Synechocystis PCC</i> 6803) as the redox chaperones of OleP.<br>
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After obtaining the best redox partners PetH/PetF, we performed alanine scanning on petF to speculate which sites had a greater impact on its electron transport capacity. Finally, we found that after mutations in seven of them, the electron transport effect would change greatly, so we mutated the amino acids of these sites into other 19 amino acids by modeling, and selected 23 of them to get better results.
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https://static.igem.wiki/teams/4759/wiki/p-1.png
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Fig1: Fluorescence intensity of wild type with 23 mutants
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We conducted control tests with the positive control group, negative control group, and wild-type strains, and finally selected 9 mutants with the highest fluorescence intensity for subsequent catalytic verification by detecting their green fluorescence intensity.
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https://static.igem.wiki/teams/4759/wiki/4-7.png
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Fig2: Fermentation of 23 mutants and control groups
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We conducted control tests with the positive control group, negative control group, and wild-type strains, and finally selected 9 mutants with the highest fluorescence intensity for subsequent catalytic verification by detecting their green fluorescence intensity.
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By verifying the catalytic ability, we found that the substrate conversion of D68P was higher than that of the wild type in the nine strains with high fluorescence intensity, reaching 89.2%.
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https://static.igem.wiki/teams/4759/wiki/p2.png
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Fig3: Conversion of the 9 mutants with the highest fluorescence intensity with wild type
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The P450 enzymes are redox-dependent proteins, through which they source electrons from reducing cofactors to drive their activities. This part is coding for ferredoxin reductase PetH (SEQ ID NO.7) and ferredoxin PetF (SEQ ID NO.8) from the algae (Synechocystis PCC 6803) as the redox chaperones of OleP. We construct the plasmid pRSFDuet-petH-petF(D61R)-olep to express PetH, PetF and Olep in E. coli C43.
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 15:41, 12 October 2023


petH-RBS2-petF(D61R) Aspartic acid at site 61 of the PetF is mutated to arginine.

Usage and Biology

The P450 enzymes are redox-dependent proteins, through which they source electrons from reducing cofactors to drive their activities. This part is coding for ferredoxin reductase PetH and ferredoxin PetF from the algae (Synechocystis PCC 6803) as the redox chaperones of OleP.
After obtaining the best redox partners PetH/PetF, we performed alanine scanning on petF to speculate which sites had a greater impact on its electron transport capacity. Finally, we found that after mutations in seven of them, the electron transport effect would change greatly, so we mutated the amino acids of these sites into other 19 amino acids by modeling, and selected 23 of them to get better results.

p-1.png

Fig1: Fluorescence intensity of wild type with 23 mutants

We conducted control tests with the positive control group, negative control group, and wild-type strains, and finally selected 9 mutants with the highest fluorescence intensity for subsequent catalytic verification by detecting their green fluorescence intensity.

4-7.png

Fig2: Fermentation of 23 mutants and control groups

We conducted control tests with the positive control group, negative control group, and wild-type strains, and finally selected 9 mutants with the highest fluorescence intensity for subsequent catalytic verification by detecting their green fluorescence intensity.

By verifying the catalytic ability, we found that the substrate conversion of D68P was higher than that of the wild type in the nine strains with high fluorescence intensity, reaching 89.2%.

p2.png

Fig3: Conversion of the 9 mutants with the highest fluorescence intensity with wild type



Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1179
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1179
    Illegal NotI site found at 952
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1179
    Illegal BglII site found at 1490
    Illegal BamHI site found at 1173
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1179
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1179
  • 1000
    COMPATIBLE WITH RFC[1000]