Difference between revisions of "Part:BBa K4759052"

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<partinfo>BBa_K4759052 short</partinfo>
 
<partinfo>BBa_K4759052 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  and ferredoxin PetF from the algae (Synechocystis PCC 6803) as the redox chaperones of Olep.  
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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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To better improve the interaction of PetH and PetF, we use linker to link PetH and PetF.
  
 
===Usage and Biology===
 
===Usage and Biology===
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The fusion combination strategy of redox partners helps to improve the catalytic activity of Olep. To further screen the optimal redox partner, we decided to make a fusion combination of petF\petH. We designed to take different linkages, and combinations between petF\petH and OleP.  
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The fusion combination strategy of redox partners can help to improve the catalytic activity of Olep. To further screen the optimal redox partner, we decided to make a fusion combination of PetF\PetH.
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We adopted the following strategies and all recombinant plasmid were transformed to C41 (DE3), respectively: recombinant strains R6 (petH and petF was connected by linker, and was connected to Olep through Linker); recombinant strains R7 (petH and petF was connected by linker); recombinant strains R8 (petH\petF was constructed on pACYCDuet, while oleP expression gene was constructed on pRSFDuet). recombinant strains R9 (petH and petF were connected by a linker, and were constructed on pACYCDuet, while the oleP expression gene was constructed on pRSFDuet).
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We adopted the following strategies and all recombinant plasmid were transformed to C41 (DE3), respectively: recombinant strains R6 (PetH, PetF, and Olep were fused by two linkers); recombinant strains R7 (petH and petF were fused by linker); recombinant strains R8 (PetH and PetF was constructed on pACYCDuet, while oleP expression gene was constructed on pRSFDuet); recombinant strains R9 (<i>petH</i> and <i>petF</i> were fused by a linker, and were constructed on pACYCDuet, while the <i>oleP</i> expression gene was constructed on pRSFDuet).
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https://static.igem.wiki/teams/4759/wiki/4-5.png
 
https://static.igem.wiki/teams/4759/wiki/4-5.png
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Fig1: The fusion expression and different expressional ratio between Olep and PetH/PetF
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Fig. 1: The fusion expression and different expressional ratio between Olep and PetH/PetF
  
 
Recombinant strains R6 to R9 were subjected to shake flask fermentation. HPLC assay for product generation. The transformation rate of recombinant strains R6 to R9 were lower than that of the recombinant strain R5.
 
Recombinant strains R6 to R9 were subjected to shake flask fermentation. HPLC assay for product generation. The transformation rate of recombinant strains R6 to R9 were lower than that of the recombinant strain R5.
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https://static.igem.wiki/teams/4759/wiki/4-6.png
 
  
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Fig2: The conversion rate of DCA for R5-R9 strains. The blue-filled triangle represents the biomass (OD600).  
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https://static.igem.wiki/teams/4759/wiki/r5.png
  
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Fig. 2: The conversion rate of DCA for R5-R9 strains. The blue-filled triangle represents the biomass (OD600).
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 03:15, 12 October 2023


petH-linker-petF

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. To better improve the interaction of PetH and PetF, we use linker to link PetH and PetF.

Usage and Biology

The fusion combination strategy of redox partners can help to improve the catalytic activity of Olep. To further screen the optimal redox partner, we decided to make a fusion combination of PetF\PetH. We adopted the following strategies and all recombinant plasmid were transformed to C41 (DE3), respectively: recombinant strains R6 (PetH, PetF, and Olep were fused by two linkers); recombinant strains R7 (petH and petF were fused by linker); recombinant strains R8 (PetH and PetF was constructed on pACYCDuet, while oleP expression gene was constructed on pRSFDuet); recombinant strains R9 (petH and petF were fused by a linker, and were constructed on pACYCDuet, while the oleP expression gene was constructed on pRSFDuet).

4-5.png

Fig. 1: The fusion expression and different expressional ratio between Olep and PetH/PetF

Recombinant strains R6 to R9 were subjected to shake flask fermentation. HPLC assay for product generation. The transformation rate of recombinant strains R6 to R9 were lower than that of the recombinant strain R5.

r5.png

Fig. 2: The conversion rate of DCA for R5-R9 strains. The blue-filled triangle represents the biomass (OD600). Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 1022
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1551
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]