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new biochemical reactions is an important research direction of synthetic biology. | new biochemical reactions is an important research direction of synthetic biology. | ||
<br><br> ceaS2, whose full name is N2-(2-carboxyethyl)arginine synthase2, is a kind of enzyme in Streptomyces | <br><br> ceaS2, whose full name is N2-(2-carboxyethyl)arginine synthase2, is a kind of enzyme in Streptomyces | ||
− | clavuligerus. The mentor of our team, Jiang Huifeng, has confirmed the new functions of ceaS2 with the help of TPP (Thiamine pyrophosphate) and magnesium ions | + | clavuligerus. The mentor of our team, Jiang Huifeng, has confirmed the new functions of ceaS2 with the help of TPP (Thiamine pyrophosphate) and magnesium ions. ceaS2 enzyme can catalyze the production of acrylic acid with DHAP (dihydroxy acetone phosphate) or G3P (glyceraldehyde 3-phosphate) as substrate. |
− | + | <br><br> Cell factory of acrylic acid (GAACF) 1.0: | |
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− | <br><br> Cell factory of acrylic acid (GAACF) 1.0: | + | |
<br><br> DHAP and G3P are the central metabolic secondary products which can be easily found in various organisms. | <br><br> DHAP and G3P are the central metabolic secondary products which can be easily found in various organisms. | ||
They are the carbon flow nodes that must be passed in the glycerol metabolic pathway in most organisms. | They are the carbon flow nodes that must be passed in the glycerol metabolic pathway in most organisms. | ||
− | + | ceaS2 enzyme being the core part, it is possible to create a new pathway to synthesize acrylic acid based on glycerol metabolic pathway in organisms and construct a cell factory with a high yield of acrylic acid. | |
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<br><br> First, we took E. coli BL21 (DE3) as the chassis cells and constructed engineering bacteria carrying | <br><br> First, we took E. coli BL21 (DE3) as the chassis cells and constructed engineering bacteria carrying | ||
the gene of ceaS2 enzyme with pET-28a plasmid as the vector. We constructed a new pathway to synthesize | the gene of ceaS2 enzyme with pET-28a plasmid as the vector. We constructed a new pathway to synthesize | ||
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approach is the shortest compared to other pathways. Take the glycerol metabolic pathway of E. coli | approach is the shortest compared to other pathways. Take the glycerol metabolic pathway of E. coli | ||
as an example, we only need three enzymes to achieve the synthesis of acrylic acid from glycerol. | as an example, we only need three enzymes to achieve the synthesis of acrylic acid from glycerol. | ||
− | So this pathway | + | So this pathway has stronger malleability and broader development prospects. |
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− | + | <div class="col-md-12" style="padding-top:30px"> | |
− | + | <div class="col-md-3"> | |
− | + | <img src="https://static.igem.org/mediawiki/2017/7/7b/NPU-newE.png" class="img-responsive"> | |
− | + | <h4> </h4> | |
− | < | + | </div> |
− | <img | + | <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> | |
− | + | </div> | |
− | + | <div class="col-md-3"> | |
− | + | <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> | |
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the results show that the engineering bacteria can use glycerol as carbon source to produce acrylic | the results show that the engineering bacteria can use glycerol as carbon source to produce acrylic | ||
acid. However, the yield of the cell factory 1.0 is not high, only about 1mg / L. | acid. However, the yield of the cell factory 1.0 is not high, only about 1mg / L. | ||
− | <br><br> It is known that acrylic acid can not be metabolized in the cell, so we analyzed the possible reasons | + | |
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+ | <br> | ||
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+ | <br> It is known that acrylic acid can not be metabolized in the cell, so we analyzed the possible reasons | ||
as the following: | as the following: | ||
<br> 1. The activity and the catalytic efficiency of wild type ceaS2 is low. | <br> 1. The activity and the catalytic efficiency of wild type ceaS2 is low. | ||
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reduction force for GLY DH through the two layers of substrate level cycle. At last, we construct | reduction force for GLY DH through the two layers of substrate level cycle. At last, we construct | ||
a new pathway for acrylic acid synthesis- GNCDC(GlyDH-NOX-CAT-DAK-ceaS2) | a new pathway for acrylic acid synthesis- GNCDC(GlyDH-NOX-CAT-DAK-ceaS2) | ||
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+ | <br> | ||
+ | <center><img src="https://static.igem.org/mediawiki/2017/1/10/%E5%A4%A7%E8%82%A0%E8%B7%AF%E5%BE%84%E5%9B%BE.png" class="img-responsive"></center> | ||
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<br><br> The genes of GlyDH and DAK were constructed on two MCS (multiple cloning sites) on the backbone | <br><br> The genes of GlyDH and DAK were constructed on two MCS (multiple cloning sites) on the backbone | ||
of pCDFDuet-1 plasmid. NOX and CAT were constructed on two MCSs on the backbone of pETDuet-1 | of pCDFDuet-1 plasmid. NOX and CAT were constructed on two MCSs on the backbone of pETDuet-1 | ||
− | plasmid. | + | plasmid. |
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− | </h4> | + | <div class="col-md-12" style="padding-top:30px"> |
− | + | <div class="col-md-4"> | |
+ | <img src="https://static.igem.org/mediawiki/2017/5/5c/%E5%A4%A7%E8%82%A01_pCDFDuet-gld-DAK_6550.png" class="img-responsive"> | ||
+ | <h4> </h4> | ||
+ | </div> | ||
+ | <div class="col-md-4"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/c/c9/%E5%A4%A7%E8%82%A02_pET-28a-ceas2_7015.png" class="img-responsive"> | ||
+ | <h4> </h4> | ||
+ | </div> | ||
+ | <div class="col-md-4"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/7/70/%E5%A4%A7%E8%82%A03_pETDuet-NOX-CAT_7451.png" class="img-responsive"> | ||
+ | <h4> </h4> | ||
+ | </div> | ||
+ | </div> | ||
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</div> | </div> | ||
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<br><br> Therefore, in the choice of the chassis organism, we tested two organisms, E. coli MG1655 and | <br><br> Therefore, in the choice of the chassis organism, we tested two organisms, E. coli MG1655 and | ||
Saccharomyces cerevisiae BY4741. BY4741 has a great ability to metabolize glycerol. According | Saccharomyces cerevisiae BY4741. BY4741 has a great ability to metabolize glycerol. According | ||
− | to GAACF1.0, we used the YCPlac33 plasmid with | + | to GAACF1.0, we used the YCPlac33 plasmid with LEU defect screening marker as the backbone and |
− | used the pTDH3 constitutive promoter and tPFK1 constitutive terminator to construct ceaS2 plasmid. | + | used the pTDH3 constitutive promoter and tPFK1 constitutive terminator to construct ceaS2 plasmid.<br> |
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+ | <div class="col-md-12" style="padding-top:30px"> | ||
+ | <div class="col-md-3"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/5/50/NPU-newSC.png" class="img-responsive"> | ||
+ | <h4> </h4> | ||
+ | </div> | ||
+ | <div class="col-md-6"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/7/7b/%E9%85%B5%E6%AF%8D%E5%8E%9F%E5%A7%8B%E4%BB%A3%E8%B0%A2%E5%9B%BE.png" class="img-responsive"> | ||
+ | <h4> </h4> | ||
+ | </div> | ||
+ | <div class="col-md-3"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/4/47/%E9%85%B5%E6%AF%8D1_Y33-Leu-ceas2_9033.png" class="img-responsive"> | ||
+ | <h4> </h4> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <br> We confirmed the proposal can make S.cerevisiae produce acrylic acid, but the | ||
yield is low, so we decided to optimize it. | yield is low, so we decided to optimize it. | ||
<br> First, according to GNCDC(GlyDH-NOX-CAT-DAK-ceaS2) in E.coli, we added NOX to the pathway(the | <br> First, according to GNCDC(GlyDH-NOX-CAT-DAK-ceaS2) in E.coli, we added NOX to the pathway(the | ||
CAT enzyme is active in S.cerevisiae). So we designed a pathway, GNDC(GlyDH-NOX -DAK-ceaS2), | CAT enzyme is active in S.cerevisiae). So we designed a pathway, GNDC(GlyDH-NOX -DAK-ceaS2), | ||
for S.cerevisiae. | for S.cerevisiae. | ||
− | <br><br> | + | |
+ | <div class="col-md-12" style="padding-top:30px"> | ||
+ | <div class="col-md-3"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/2/23/%E9%85%B5%E6%AF%8D3_Y33-URA-gld-DAK_10763.png" class="img-responsive"> | ||
+ | <h4> </h4> | ||
+ | </div> | ||
+ | <div class="col-md-6"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/b/b0/%E9%85%B5%E6%AF%8D%E8%B7%AF%E5%BE%84%E5%9B%BE.png" class="img-responsive"> | ||
+ | <h4> </h4> | ||
+ | </div> | ||
+ | <div class="col-md-3"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/2/25/%E9%85%B5%E6%AF%8D2_Y33-leu-ceas2-NOX_10513.png" class="img-responsive"> | ||
+ | <h4> </h4> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <br><br> The genes of GlyDH and DAK were constructed on the backbone of YCPlac33 plasmid with | ||
URA marker. We used the ADH1 promoter and tGPD1 terminator for GlyDH, the PGK1 promoter and the | URA marker. We used the ADH1 promoter and tGPD1 terminator for GlyDH, the PGK1 promoter and the | ||
tPFK1 terminator for DAK. NOX and ceaS2 were constructed on the backbone of the other YCPlac33 | tPFK1 terminator for DAK. NOX and ceaS2 were constructed on the backbone of the other YCPlac33 | ||
plasmid. We replaced URA marker with Leu marker to screen for two plasmids easily. We used the | plasmid. We replaced URA marker with Leu marker to screen for two plasmids easily. We used the | ||
TEF2 promoter and tRPS2 terminator for GlyDH, the same promoter and terminator as the original | TEF2 promoter and tRPS2 terminator for GlyDH, the same promoter and terminator as the original | ||
− | pathway for ceaS2. | + | pathway for ceaS2. |
<br> | <br> | ||
</h4> | </h4> | ||
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<br> | <br> | ||
</h4> | </h4> | ||
− | + | <center><img src="https://static.igem.org/mediawiki/2017/2/2a/%E7%AD%9B%E9%80%89%E7%BB%84%E5%90%88%E8%A1%A8.png" class="img-responsive"></center> | |
<br> | <br> | ||
<h3>PS. We also made Hardware | <h3>PS. We also made Hardware |
Latest revision as of 19:35, 1 November 2017