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<div align="center" style="padding-top:20px"> | <div align="center" style="padding-top:20px"> | ||
− | <h3 | + | <h3>In this part, we validate how ceaS2 works as expected and how we construct GAACF1.0.</h3> |
</div> | </div> | ||
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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/2017/4/44/%28%E5%B0%8F%29%E5%A4%A7%E8%82%A03_pETDuet-NOX-CAT_7451.png" class="img-responsive"> | ||
+ | <h4> </h4> | ||
+ | </div> | ||
+ | <div class="col-md-6"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/9/94/NPU-ceaS2jiaotu.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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we used the affinity chromatography nickel column to separate and purify the ceaS2 protein and identified | we used the affinity chromatography nickel column to separate and purify the ceaS2 protein and identified | ||
it by 12% SDS-PAGE electrophoresis. The results were as follows:</h4> | it by 12% SDS-PAGE electrophoresis. The results were as follows:</h4> | ||
− | <img src="https://static.igem.org/mediawiki/2017/7/7f/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%B8%80.png" | + | <center><img src="https://static.igem.org/mediawiki/2017/7/7f/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%B8%80.png" |
− | class="img-responsive"> | + | 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 | ||
affinity chromatography; 4, 50 mM imidazole eluent; 5, 100 mM imidazole eluent; 6, 200 mM imidazole | affinity chromatography; 4, 50 mM imidazole eluent; 5, 100 mM imidazole eluent; 6, 200 mM imidazole | ||
eluent; 7, 300 mM imidazole eluent) | eluent; 7, 300 mM imidazole eluent) | ||
− | <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> | ||
− | <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"> | + | class="img-responsive"></center> |
<h4> | <h4> | ||
− | + | <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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<h3> 1.4 Determination of Acrylic Acid by Liquid Chromatography</h3> | <h3> 1.4 Determination of Acrylic Acid by Liquid Chromatography</h3> | ||
<h4> | <h4> | ||
− | + | 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> | ||
− | <img src="https://static.igem.org/mediawiki/2017/b/bc/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%B8%89.png" | + | <center><img src="https://static.igem.org/mediawiki/2017/b/bc/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%B8%89.png" |
− | class="img-responsive"> | + | class="img-responsive"></center> |
<h4> | <h4> | ||
− | + | <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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minutes presenting a single and satisfying peak shape. The samples in the control group did not correspond | minutes presenting a single and satisfying peak shape. The samples in the control group did not correspond | ||
with the treatment group. D-G3P and DHAP reaction group samples both had peaks, in line with the | with the treatment group. D-G3P and DHAP reaction group samples both had peaks, in line with the | ||
− | standard sample of acrylic acid. Therefore, we can initially determine that | + | standard sample of acrylic acid. Therefore, we can initially determine that ceas2 is able to catalyze |
D-G3P and DHAP to generate acrylic acid. | D-G3P and DHAP to generate acrylic acid. | ||
</h4> | </h4> | ||
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<br> | <br> | ||
</h4> | </h4> | ||
− | <img src="https://static.igem.org/mediawiki/2017/9/9d/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%E5%9B%9B.png" | + | <center><img src="https://static.igem.org/mediawiki/2017/9/9d/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%E5%9B%9B.png" |
− | class="img-responsive"> | + | class="img-responsive"></center> |
+ | |||
+ | <br> | ||
+ | |||
+ | <center><img src="https://static.igem.org/mediawiki/2017/f/f9/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%94.png" | ||
+ | class="img-responsive"></center> | ||
+ | |||
+ | <br> | ||
− | + | <center> <img src="https://static.igem.org/mediawiki/2017/7/74/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%E5%85%AD.png" | |
− | class="img-responsive"> | + | class="img-responsive"></center> |
− | < | + | <br> |
− | + | ||
− | <img src="https://static.igem.org/mediawiki/2017/2/25/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%B8%83.png" | + | <center><img src="https://static.igem.org/mediawiki/2017/2/25/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%B8%83.png" |
− | class="img-responsive"> | + | class="img-responsive"></center> |
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<h4> | <h4> | ||
− | + | <center>LC-MS of ceaS2 generated acrylic acid</center> | |
− | <br> (From top to bottom is acrylic acid standard, control, | + | <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 | ||
− | also fully demonstrated that | + | also fully demonstrated that ceas2 catalyzed DHAP or ceaS2 to produce the acrylic acid. |
<br> | <br> | ||
</h4> | </h4> | ||
<h2>2. GAACF 1.0: de novo synthesis of acrylic acid</h2> | <h2>2. GAACF 1.0: de novo synthesis of acrylic acid</h2> | ||
<h3>2.1 A New Approach to Acrylic Biosynthesis</h3> | <h3>2.1 A New Approach to Acrylic Biosynthesis</h3> | ||
− | <h4> | + | <h4> G3P are common secondary products of the E. coli central metabolic pathway. By introducing the ceaS2 |
enzyme directly into the chassis cells, we can construct a new pathway of acrylic biosynthesis based | enzyme directly into the chassis cells, we can construct a new pathway of acrylic biosynthesis based | ||
on any carbon source. DHAP and G3P are the carbon flow nodes that E. coli glycerol metabolic pathway | on any carbon source. DHAP and G3P are the carbon flow nodes that E. coli glycerol metabolic pathway | ||
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> | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <center><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"> | + | class="img-responsive"></center> |
<h4> | <h4> | ||
− | + | <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> | ||
− | <img src="https://static.igem.org/mediawiki/2017/d/d4/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%B9%9D.png" | + | <center><img src="https://static.igem.org/mediawiki/2017/d/d4/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%B9%9D.png" |
− | class="img-responsive"> | + | class="img-responsive"> Whole Cell Catalytic Acrylic de novo Synthesis of Liquid Chromatography</center> |
<br> | <br> | ||
<br> From the peak chromatogram, it can be seen that the goal of de novo synthesis of acrylic acid from | <br> From the peak chromatogram, it can be seen that the goal of de novo synthesis of acrylic acid from | ||
the cells have been realized. | the cells have been realized. | ||
<br> We have achieved the prototype of the glycerol-based acrylic cell factory based on the E. coli BL21 | <br> We have achieved the prototype of the glycerol-based acrylic cell factory based on the E. coli BL21 | ||
− | (DE3) strain as the chassis cell | + | (DE3) strain as the chassis cell. |
<br> | <br> | ||
</h4> | </h4> |
Latest revision as of 20:58, 1 November 2017