Difference between revisions of "Part:BBa K4759069"

 
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<partinfo>BBa_K4759069 short</partinfo>
 
<partinfo>BBa_K4759069 short</partinfo>
  
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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(F64I)-olep to express PetH, PetF and Olep in E. coli C43.  
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Site-directed mutagenesis is a PCR-based method to mutate specified nucleotides of a sequence within a plasmid vector. This technique allows one to study the relative importance of a particular amino acid for protein structure and function.
  
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===Usage and Biology===
 
===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.
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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/4-7.png
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Fig. 1: 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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Revision as of 18:36, 11 October 2023


petH-RBS2-petF(F64I)

Site-directed mutagenesis is a PCR-based method to mutate specified nucleotides of a sequence within a plasmid vector. This technique allows one to study the relative importance of a particular amino acid for protein structure and function.

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.

4-7.png

Fig. 1: 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.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1249
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1249
    Illegal NotI site found at 1022
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1249
    Illegal BglII site found at 1560
    Illegal BamHI site found at 1243
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1249
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1249
  • 1000
    COMPATIBLE WITH RFC[1000]