Difference between revisions of "Part:BBa K4759067:Experience"

(→‎Applications of BBa_K4759067)
 
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===Applications of BBa_K4759067===
 
===Applications of BBa_K4759067===
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After obtaining the best redox partner, 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 show better results.
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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/4-7.png
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https://static.igem.wiki/teams/4759/wiki/p-1.png
  
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Fig1: Fermentation of 23 mutants and control groups
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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.
 
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/4-8.png
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https://static.igem.wiki/teams/4759/wiki/4-7.png
  
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Fig2: 9 mutants + wild-type + negative control, 50 ml/250 ml system fermentation
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Fig2: Fermentation of 23 mutants and control groups
  
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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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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.
  
−
https://static.igem.wiki/teams/4759/wiki/4-9.png
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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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Fig3: A:Fluorescence intensity of wild type with 23 mutants
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https://static.igem.wiki/teams/4759/wiki/p2.png
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B: Conversion of the 9 mutants with the highest fluorescence intensity with wild type
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Next, we analyzed the interaction forces between their redox partners and Olep and made predictions of the Olep structure of mutant D68P.
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Fig3: Conversion of the 9 mutants with the highest fluorescence intensity with wild type
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 15:28, 12 October 2023


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Applications of BBa_K4759067

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

User Reviews

UNIQ99fa64ef62371361-partinfo-00000000-QINU UNIQ99fa64ef62371361-partinfo-00000001-QINU