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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 has stronger malleability and broader development prospects. | So this pathway has stronger malleability and broader development prospects. | ||
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− | + | <img src="https://static.igem.org/mediawiki/2017/7/7b/NPU-newE.png" class="img-responsive"> | |
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− | + | <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"> | |
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− | + | <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"> | |
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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> | ||
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+ | <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> | ||
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+ | <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