Difference between revisions of "Part:BBa K4759043:Experience"

 
(→‎Applications of BBa_K4759043)
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===Applications of BBa_K4759043===
 
===Applications of BBa_K4759043===
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The P450 enzyme requires electrons (NADH, NADPH) to function. Electrons are first transferred to FDR (ferroxyreductase), to FDX
 +
 +
(ferro-reducing protein) under the action of ferroxyreductase, and then to P450 enzyme under the action of redox partner, and
 +
 +
finally to the heme center, after which the enzyme can react with the substrate and undergo hydroxylation.
 +
 +
Through extensive reading of the literature, we summarized 11 pairs of redox partners with good results. After that, we screened
 +
 +
four pairs of redox partners with good effects by molecular docking and mathematical modeling and then verified them
 +
 +
experimentally.
 +
 +
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. 
 +
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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The above four recombinant plasmids are converted to BL21(DE3) to obtain recombinant strains G2 strain to G5 strain.
  
 
===User Reviews===
 
===User Reviews===

Revision as of 17:01, 7 October 2023


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K4759043

The P450 enzyme requires electrons (NADH, NADPH) to function. Electrons are first transferred to FDR (ferroxyreductase), to FDX

(ferro-reducing protein) under the action of ferroxyreductase, and then to P450 enzyme under the action of redox partner, and

finally to the heme center, after which the enzyme can react with the substrate and undergo hydroxylation.

Through extensive reading of the literature, we summarized 11 pairs of redox partners with good results. After that, we screened

four pairs of redox partners with good effects by molecular docking and mathematical modeling and then verified them

experimentally. 

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.

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 The above four recombinant plasmids are converted to BL21(DE3) to obtain recombinant strains G2 strain to G5 strain.

User Reviews

UNIQ0cd2cd7d099ef320-partinfo-00000000-QINU UNIQ0cd2cd7d099ef320-partinfo-00000001-QINU