Difference between revisions of "Part:BBa K4759220"
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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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Generally, the method of determining whether the redox partner is suitable is through tedious steps such as the construction of plasmids, heterologous expression, construction of catalytic systems, and detection of conversion rate after catalysis. Therefore, we wanted to find a convenient way to do a quick screening. We used the fluorescent protein sfGFP to successfully construct a sensor to detect redox partners. sfGFP is a superfolder fluorescent protein that emits green light when irradiated with ultraviolet light. What is special about it is that it can be broken into two parts. 
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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 divide sfGFP into sfGFP-1-10 and sfGFP-11, and although these two parts are cut off, there is an interaction force between them, and as long as they are properly folded in space, they will emit light again. Thus, four iron redox proteins are fused to the N-terminus of sfGFP-1-10 and Olep to the C-terminus of sfGFP-11, respectively, to obtain the recombinant plasmid pRSFDuet-BM3-GFP-1-10-GFP-11-oleP, pRSFDuet-camA-camB-GFP-1-10-GFP-11-oleP, pRSFDuet-FdR_0978-Fdx_1499-GFP-1-10-GFP-11-oleP, pRSFDuet-petH-petF-GFP-1-10-GFP-11-oleP
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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
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Fig. 1: The fusion expression and different expressional ratio between Olep and PetH/PetF
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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/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>

Revision as of 03:16, 12 October 2023


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 1109
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1638
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1696
    Illegal AgeI site found at 1794
  • 1000
    COMPATIBLE WITH RFC[1000]