Difference between revisions of "Part:BBa K4759233"
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K4759233 short</partinfo>
 
<partinfo>BBa_K4759233 short</partinfo>
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===Design===
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Redox proteins and redox proteases are linked by RBS2, joining the two proteins together. The original base of the amino acid encoding PetF at position 61 is GAT, we mutated it to CGT, where the amino acid in ferredoxin is mutated from Asp to Arg.
  
 
===Usage and Biology===
 
===Usage and Biology===
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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.  
+
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 selecting 23 of them to get better results.  
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(Site-directed Mutagenesis parts can be search from BBa_K4759053 to BBa_K4759075, and constructed modeling screening for redox partners can be search from BBa_K4759076 to BBa_K4759099)
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(Site-directed Mutagenesis parts can be searched from BBa_K4759053 to BBa_K4759075, and constructed modeling screening for redox partners can be searched from BBa_K4759076 to BBa_K4759099)
  
 
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https://static.igem.wiki/teams/4759/wiki/4-7.png
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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.  
+
We conducted control tests with the positive control group, negative control group, and wild-type strains. Finally, we 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/4-8.png
 
https://static.igem.wiki/teams/4759/wiki/4-8.png
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Fig. 3: A:Fluorescence intensity of wild type with 23 mutants
 
Fig. 3: A:Fluorescence intensity of wild type with 23 mutants
 
B: Conversion of the 9 mutants with the highest fluorescence intensity with wild type
 
B: Conversion of the 9 mutants with the highest fluorescence intensity with wild type
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<!-- Add more about the biology of this part here
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===Usage and Biology===
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===References===
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[1] Li, S.; Du, L.; Bernhardt, R. Redox Partners: Function Modulators of Bacterial P450 Enzymes. Trends Microbiol 2020, 28, 445-454. DOI: 10.1016/j.tim.2020.02.012
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 +
[2] Lou, D.; Tan, J.; Zhu, L.; Ji, S.; Tang, S.; Yao, K.; Han, J.; Wang, B. Engineering Clostridium absonum 7alpha-hydroxysteroid Dehydrogenase for Enhancing Thermostability Based on Flexible Site and DeltaDeltaG Prediction. Protein Pept Lett 2018, 25, 230-235. DOI: 10.2174/0929866524666171113113100
 +
 
 +
[3] Sagadin, T.; Riehm, J.; Putkaradze, N.; Hutter, M. C.; Bernhardt, R. Novel approach to improve progesterone hydroxylation selectivity by CYP106A2 via rational design of adrenodoxin binding. FEBS J 2019, 286, 1240-1249. DOI: 10.1111/febs.14722
 +
 
 +
[4] Meng, S.; Li, Z.; Ji, Y.; Ruff, A. J.; Liu, L.; Davari, M. D.; Schwaneberg, U. Introduction of aromatic amino acids in electron transfer pathways yielded improved catalytic performance of cytochrome P450s. Chinese Journal of Catalysis 2023, 49, 81-90. DOI: 10.1016/s1872-2067(23)64445-6
 +
 
 +
[5] Tovchigrechko, A.; Vakser, I. A. GRAMM-X public web server for protein-protein docking. Nucleic Acids Res 2006, 34, W310-314. DOI: 10.1093/nar/gkl206
  
 
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Latest revision as of 06:43, 12 October 2023


T7-RBS1-petH-RBS2-petF(D61R)

Design

Redox proteins and redox proteases are linked by RBS2, joining the two proteins together. The original base of the amino acid encoding PetF at position 61 is GAT, we mutated it to CGT, where the amino acid in ferredoxin is mutated from Asp to Arg.

Usage and Biology

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 selecting 23 of them to get better results. (Site-directed Mutagenesis parts can be searched from BBa_K4759053 to BBa_K4759075, and constructed modeling screening for redox partners can be searched from BBa_K4759076 to BBa_K4759099)

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. Finally, we selected 9 mutants with the highest fluorescence intensity for subsequent catalytic verification by detecting their green fluorescence intensity.

4-8.png

Fig. 2: 9 mutants + wild-type + negative control, 50 ml/250 ml system fermentation

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%.

4-9.png

Fig. 3: A:Fluorescence intensity of wild type with 23 mutants B: Conversion of the 9 mutants with the highest fluorescence intensity with wild type

References

[1] Li, S.; Du, L.; Bernhardt, R. Redox Partners: Function Modulators of Bacterial P450 Enzymes. Trends Microbiol 2020, 28, 445-454. DOI: 10.1016/j.tim.2020.02.012

[2] Lou, D.; Tan, J.; Zhu, L.; Ji, S.; Tang, S.; Yao, K.; Han, J.; Wang, B. Engineering Clostridium absonum 7alpha-hydroxysteroid Dehydrogenase for Enhancing Thermostability Based on Flexible Site and DeltaDeltaG Prediction. Protein Pept Lett 2018, 25, 230-235. DOI: 10.2174/0929866524666171113113100

[3] Sagadin, T.; Riehm, J.; Putkaradze, N.; Hutter, M. C.; Bernhardt, R. Novel approach to improve progesterone hydroxylation selectivity by CYP106A2 via rational design of adrenodoxin binding. FEBS J 2019, 286, 1240-1249. DOI: 10.1111/febs.14722

[4] Meng, S.; Li, Z.; Ji, Y.; Ruff, A. J.; Liu, L.; Davari, M. D.; Schwaneberg, U. Introduction of aromatic amino acids in electron transfer pathways yielded improved catalytic performance of cytochrome P450s. Chinese Journal of Catalysis 2023, 49, 81-90. DOI: 10.1016/s1872-2067(23)64445-6

[5] Tovchigrechko, A.; Vakser, I. A. GRAMM-X public web server for protein-protein docking. Nucleic Acids Res 2006, 34, W310-314. DOI: 10.1093/nar/gkl206

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1266
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1266
    Illegal NotI site found at 1039
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1266
    Illegal BglII site found at 1577
    Illegal BamHI site found at 1260
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1266
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1266
  • 1000
    COMPATIBLE WITH RFC[1000]