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eluent; 7, 300 mM imidazole eluent) | eluent; 7, 300 mM imidazole eluent) | ||
<br><br> According to the information in the Uniprot database, the ceaS2 protein has 573 amino acids and | <br><br> According to the information in the Uniprot database, the ceaS2 protein has 573 amino acids and | ||
− | the molecular weight of the protein is 62.34 kDa. In Figure | + | the molecular weight of the protein is 62.34 kDa. In Figure, electrophoretic display of recombinant |
protein molecular weight is consistent with the theoretical molecular weight of the protein, which | protein molecular weight is consistent with the theoretical molecular weight of the protein, which | ||
leads to the conclusion that this is the expression of our target protein. The results show that | leads to the conclusion that this is the expression of our target protein. The results show that | ||
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<h4>In order to ensure the reliability and accuracy of the experiment, we first examined the concentration | <h4>In order to ensure the reliability and accuracy of the experiment, we first examined the concentration | ||
of the purified protein. Proteins were quantified using the Thermol Scientific BCA Protein Quantification | of the purified protein. Proteins were quantified using the Thermol Scientific BCA Protein Quantification | ||
− | Kit. The standardized curve of protein concentration of OD562 interval measured | + | Kit. The standardized curve of protein concentration of OD562 interval measured. |
</h4> | </h4> | ||
<center><img src="https://static.igem.org/mediawiki/2017/e/e0/Proof-%E5%9F%BA%E4%BA%8E%E7%94%98%E6%B2%B9%E7%9A%84%E4%B8%99%E7%83%AF%E9%85%B8%E7%BB%86%E8%83%9E%E5%B7%A5%E5%8E%82%E7%9A%84%E5%AE%9E%E7%8E%B0_ENG_%E5%9B%BE%E4%BA%8C.png" | <center><img src="https://static.igem.org/mediawiki/2017/e/e0/Proof-%E5%9F%BA%E4%BA%8E%E7%94%98%E6%B2%B9%E7%9A%84%E4%B8%99%E7%83%AF%E9%85%B8%E7%BB%86%E8%83%9E%E5%B7%A5%E5%8E%82%E7%9A%84%E5%AE%9E%E7%8E%B0_ENG_%E5%9B%BE%E4%BA%8C.png" | ||
class="img-responsive"></center> | class="img-responsive"></center> | ||
<h4> | <h4> | ||
− | Standardized curve of protein concentration | + | <center>Standardized curve of protein concentration</center> |
<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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We used high performance liquid chromatography (HPLC) to determine the reaction solution. Determination conditions and parameters: | We used high performance liquid chromatography (HPLC) to determine the reaction solution. Determination conditions and parameters: | ||
87H chromatographic column, 5 mM H2SO4 mobile phase, flow rate 0.6 mL / min, UV absorbance 210 nm. | 87H chromatographic column, 5 mM H2SO4 mobile phase, flow rate 0.6 mL / min, UV absorbance 210 nm. | ||
− | Figure | + | Figure for the liquid phase determination results. |
</h4> | </h4> | ||
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<h4> | <h4> | ||
− | Identification function of ceaS2 by liquid chromatogram | + | <center>Identification function of ceaS2 by liquid chromatogram</center> |
<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 | + | <center>LC-MS of ceaS2 generated acrylic acid</center> |
<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) | ||
− | <br> According to the liquid chromatogram | + | <br> According to the liquid chromatogram, the control group had no peaks, and the retention time |
and molecular weight of the sample group were consistent with the standard sample of acrylic acid. | and molecular weight of the sample group were consistent with the standard sample of acrylic acid. | ||
Also, the molecular ion peaks and fragment ion peaks in the secondary mass spectrum of the sample | Also, the molecular ion peaks and fragment ion peaks in the secondary mass spectrum of the sample | ||
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must pass through, according to which we designed a new approach to acrylic acid biosynthesis based | must pass through, according to which we designed a new approach to acrylic acid biosynthesis based | ||
on glycerol. | on glycerol. | ||
− | <br> | + | <br> The new pathway, as shown below |
</h4> | </h4> | ||
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<h4> | <h4> | ||
− | The acrylic biosynthetic pathway based on E. coli glycerol aerobic metabolic pathway | + | <center>The acrylic biosynthetic pathway based on E. coli glycerol aerobic metabolic pathway</center> |
<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 | ||
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inducing the expression of ceaS2 enzyme in E. coli, the reaction system was prepared and carried | inducing the expression of ceaS2 enzyme in E. coli, the reaction system was prepared and carried | ||
out. The reaction solution was filtered and the acrylic acid content was determined by high performance | out. The reaction solution was filtered and the acrylic acid content was determined by high performance | ||
− | liquid chromatography (HPLC). The results are as follows: | + | liquid chromatography (HPLC). The results are as follows:<br> |
− | <br> | + | <br> <center>Primitive peak figure of liquid phase, with standard control sample. Shown as follows</center> |
<br> | <br> | ||
Latest revision as of 20:58, 1 November 2017