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<h3>1.1 construction of pET-28a-ceaS2 plasmid:</h3> | <h3>1.1 construction of pET-28a-ceaS2 plasmid:</h3> | ||
<h4>First, we constructed pET-28a-ceaS2 plasmid with pET-28a plasmid skeleton as vector. | <h4>First, we constructed pET-28a-ceaS2 plasmid with pET-28a plasmid skeleton as vector. | ||
− | < | + | <div class="col-md-12" style="padding-top:30px"> |
+ | <div class="col-md-6"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/7/7b/NPU-newE.png" class="img-responsive"> | ||
+ | <h4> </h4> | ||
+ | </div> | ||
+ | <div class="col-md-6"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/2/21/%E5%A4%A7%E8%82%A0%E5%8E%9F%E5%A7%8B%E4%BB%A3%E8%B0%A2.png" class="img-responsive"> | ||
+ | <h4> </h4> | ||
+ | |||
+ | <h4> </h4> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | </h4> | ||
+ | |||
+ | |||
<h3>1.2 Expression and Purification of ceaS2 Protein:</h3> | <h3>1.2 Expression and Purification of ceaS2 Protein:</h3> | ||
<h4>We transferred the pET-28a-ceaS2 plasmid constructed into E.coli BL21 (DE3) strain and induced E. coli | <h4>We transferred the pET-28a-ceaS2 plasmid constructed into E.coli BL21 (DE3) strain and induced E. coli | ||
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class="img-responsive"></center> | class="img-responsive"></center> | ||
− | <h4> | + | <h4> Purified ceaS2 after Ni-NTA affinity chromatography |
<br> (M, protein marker (from top to bottom is 25、35、48、63、75、100、135、180 kDa);Lane1, precipitation samples | <br> (M, protein marker (from top to bottom is 25、35、48、63、75、100、135、180 kDa);Lane1, precipitation samples | ||
in the cell lysates; 2, supernatant samples in the cell lysates; 3, supernatant flow through Ni-NTA | in the cell lysates; 2, supernatant samples in the cell lysates; 3, supernatant flow through Ni-NTA | ||
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class="img-responsive"></center> | class="img-responsive"></center> | ||
<h4> | <h4> | ||
− | + | Standardized curve of protein concentration | |
<br> The purified and quantified protein was used for enzymatic activity reaction. The control group | <br> The purified and quantified protein was used for enzymatic activity reaction. The control group | ||
was not add ceaS2 enzyme, and the protein buffer was used to make up the volume. The reaction system | was not add ceaS2 enzyme, and the protein buffer was used to make up the volume. The reaction system | ||
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<h4> | <h4> | ||
− | + | Identification function of ceaS2 by liquid chromatogram | |
<br> (The black line is acrylic acid standard, green line is control, blue line is reaction of D-G3P | <br> (The black line is acrylic acid standard, green line is control, blue line is reaction of D-G3P | ||
as substrate, red line is reaction DHAP as substrate) | as substrate, red line is reaction DHAP as substrate) | ||
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<h4> | <h4> | ||
− | + | LC-MS of ceaS2 generated acrylic acid | |
<br> (From top to bottom is acrylic acid standard, control, ceas2 reaction by DHAP as substrate, secondary | <br> (From top to bottom is acrylic acid standard, control, ceas2 reaction by DHAP as substrate, secondary | ||
mass spectrum of acrylic acid) | mass spectrum of acrylic acid) | ||
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<h4> | <h4> | ||
− | + | The acrylic biosynthetic pathway based on E. coli glycerol aerobic metabolic pathway | |
<br> This new approach is the shortest compared to other known acrylic pathways, where only three enzymes | <br> This new approach is the shortest compared to other known acrylic pathways, where only three enzymes | ||
are needed to achieve the synthesis of acrylic acid from glycerol, so this pathway not merely has | are needed to achieve the synthesis of acrylic acid from glycerol, so this pathway not merely has |
Revision as of 20:07, 1 November 2017