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+ | <a href="#" class="dropdown-toggle" data-toggle="dropdown">Team | ||
+ | <b class="caret"></b> | ||
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+ | <ul class="dropdown-menu"> | ||
+ | <li> | ||
+ | <a href="https://2017.igem.org/Team:NPU-China/Aboutus">About us</a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="https://2017.igem.org/Team:NPU-China/Attributions">Attributions</a> | ||
+ | </li> | ||
+ | </ul> | ||
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+ | <a href="#" class="dropdown-toggle" data-toggle="dropdown">Project | ||
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+ | <a href="https://2017.igem.org/Team:NPU-China/Background">Background</a> | ||
+ | </li> | ||
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+ | <a href="https://2017.igem.org/Team:NPU-China/Description">Description</a> | ||
+ | </li> | ||
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+ | <a href="https://2017.igem.org/Team:NPU-China/Design">Design</a> | ||
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+ | <a href="#" class="dropdown-toggle" data-toggle="dropdown">Parts | ||
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+ | <a href="https://2017.igem.org/Team:NPU-China/BasicParts">Basic Parts</a> | ||
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+ | <a href="https://2017.igem.org/Team:NPU-China/CompositeParts">Composite Parts</a> | ||
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+ | <a href="https://2017.igem.org/Team:NPU-China/Collaborations">Collaborations</a> | ||
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+ | <a href="#" class="dropdown-toggle" data-toggle="dropdown">Notebook | ||
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+ | <a href="https://2017.igem.org/Team:NPU-China/Labnotes">Labnotes</a> | ||
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+ | <a href="https://2017.igem.org/Team:NPU-China/Protocols">Protocols</a> | ||
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+ | </ul> | ||
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+ | </div> | ||
+ | </div> | ||
+ | </nav> | ||
+ | <!-- Page Content --> | ||
+ | <div class="batu" style="background: url('https://static.igem.org/mediawiki/2017/f/fe/Npu-background.png') no-repeat fixed; overflow: hidden;"> | ||
+ | <img class="img-responsive" src="https://static.igem.org/mediawiki/2017/4/46/%E9%A2%98%E7%9B%AE%E5%B0%8F%E9%80%9A%E6%A0%8Fdesign.jpg"> | ||
+ | <div class="container" style="padding-top:70px"> | ||
+ | <div class="row"> | ||
+ | <div class="col-md-12"> | ||
+ | <h2 style="text-align:center">Introduction</h2> | ||
+ | <h4>The essence of biochemical synthesis is the catalytic reaction with enzyme as the catalyst. Creating | ||
+ | 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 | ||
+ | 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: | ||
+ | <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. | ||
+ | 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. | ||
+ | |||
+ | <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 | ||
+ | acrylic acid from any carbon source by transforming ceaS2 directly into the chassis cells. This new | ||
+ | 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. | ||
+ | So this pathway has stronger malleability and broader development prospects. | ||
+ | |||
+ | |||
+ | |||
+ | <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> | ||
+ | <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> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <br>Through the whole cell catalysis and HPLC (High Performance Liquid Chromatography), | ||
+ | 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. | ||
+ | |||
+ | |||
+ | <br> | ||
+ | |||
+ | |||
+ | |||
+ | <br> It is known that acrylic acid can not be metabolized in the cell, so we analyzed the possible reasons | ||
+ | as the following: | ||
+ | <br> 1. The activity and the catalytic efficiency of wild type ceaS2 is low. | ||
+ | <br> 2. The low carbon flow rate of glycerol metabolic pathway in E. coli leads to the low concentration | ||
+ | of DHAP and G3P. | ||
+ | <br> 3. Acrylic acid is toxic to the chassis cells. | ||
+ | <br> 4. The reaction conditions such as carbon source, pH, temperature and reaction time are not suitable. | ||
+ | <br> Based on the analyzing results, we have made improvements and built a new cell factory. | ||
+ | <br> | ||
+ | <br> Cell factory of acrylic acid (GAACF) 2.0: | ||
+ | <br> We built a new cell factory of acrylic acid through the four part: CO-PART, SYSTEM, PATHWAY, PRODUCTION! | ||
+ | <br> | ||
+ | </h4> | ||
+ | <div class="container" style="padding-top:50px"> | ||
+ | <div id="COREPART" style="padding-top:50px;margin-top:-50px;"> | ||
+ | <h2 style="text-align:center">Core Part</h2> | ||
+ | <h4>Acrylic acid is a byproduct of ceaS2 enzyme, the catalytic effect of wild type ceaS2 enzyme is very | ||
+ | weak, and acrylic acid production is only 1mg / L. So it is necessary to improve the catalytic | ||
+ | effect of this core factor, ceaS2 enzyme. | ||
+ | <br><br> The gene of ceaS2 enzyme consists of 1719 deoxynucleotides and the protein sequence consists | ||
+ | of 573 amino acids. We need to use bioinformatics to analyze and simulate, in order to help us | ||
+ | decide the correct proposal. | ||
+ | <br> | ||
+ | <center><img src="https://static.igem.org/mediawiki/2017/a/ac/Ceas2.png" class="img-responsive"></center> | ||
+ | <br> We constructed ceaS2 enzyme mutants using the AEMD (Auto Enzyme Mutation Design) platform. We | ||
+ | constructed the ceaS2 wild-type sequence on pET-28a plasmid. We used pET-28a-ceaS2 plasmid as | ||
+ | a template to create point mutation, and then transformed the plasmid into BL21. Then, we did | ||
+ | the whole cell catalysis to get the products. Finally, we screened for ceaS2 mutants with high | ||
+ | catalytic efficiency by HPLC (High Performance Liquid Chromatography) and | ||
+ | HTS (High throughput screening) . | ||
+ | <br> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <center><img src="https://static.igem.org/mediawiki/2017/b/bf/5A_ceaS2.gif"></center> | ||
+ | |||
+ | |||
+ | <div class="container" style="padding-top:50px"> | ||
+ | |||
+ | <div id="Pathway" style="padding-top:50px;margin-top:-50px;"> | ||
+ | <h2 style="text-align:center">Pathway</h2> | ||
+ | <h4>The carbon flow rate of the glycerol metabolic pathway is low. In order to solve the problem, we | ||
+ | need reconstruction and optimization of the original metabolic pathway. | ||
+ | <br> | ||
+ | <br> RE-Construction:We designed the GDC (GlyDH-DAK-ceaS2) pathway to produce acrylic acid from glycerol. | ||
+ | In this pathway, GlyDH(Glycerol dehydrogenase) can efficiently convert Glycerol into DHA(1,3-Dihydroxyacetone). | ||
+ | Then DAK (Dihydroxyacetone kinase) converts DHA into DHAP. Finally, ceaS2 converts DHAP into | ||
+ | acrylic acid. In addition, because GlyDH depends on NAD+, we added two reduction models, NOX | ||
+ | (NADH dehydrogenase )and CAT(Catalase), to the pathway, with the purpose of providing the required | ||
+ | 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) | ||
+ | |||
+ | |||
+ | <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> | ||
+ | |||
+ | |||
+ | |||
+ | <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 | ||
+ | plasmid. | ||
+ | |||
+ | <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> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | <div class="container" style="padding-top:50px"> | ||
+ | |||
+ | <div id="Syetem" style="padding-top:50px;margin-top:-50px;"> | ||
+ | <h2 style="text-align:center">System</h2> | ||
+ | <h4>The choice of the chassis organism is vital to the efficiency of the cell factory. Acrylic acid may | ||
+ | do damage to the cell membrane. So we need to choose an organism which has high tolerance of | ||
+ | acrylic acid. Escherichia coli and Saccharomyces cerevisiae are two model organisms which can | ||
+ | be easily modified in the prokaryotic and eukaryotic. | ||
+ | <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 | ||
+ | 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.<br> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <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. | ||
+ | <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), | ||
+ | for S.cerevisiae. | ||
+ | |||
+ | <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 | ||
+ | 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 | ||
+ | TEF2 promoter and tRPS2 terminator for GlyDH, the same promoter and terminator as the original | ||
+ | pathway for ceaS2. | ||
+ | <br> | ||
+ | </h4> | ||
+ | </div> | ||
+ | |||
+ | <div class="container" style="padding-top:50px"> | ||
+ | |||
+ | <div id="Production" style="padding-top:50px;margin-top:-50px;"> | ||
+ | <h2 style="text-align:center">Production</h2> | ||
+ | <h4>To make the engineering bacteria produce acrylic acid, it takes two stages. First, bacteria must | ||
+ | grow and express the enzyme, then use carbon source to synthesize acrylic acid. To screen for | ||
+ | engineering bacteria, it is a waste of time and reagents to use the traditional fermentation | ||
+ | method. We used whole cell catalysis to carry out the reaction for acrylic acid production | ||
+ | <br><br> After the enzyme is expressed, the bacteria solution will be centrifuged and concentrated 10 | ||
+ | times with buffer before the reaction. Therefore, we optimized the reaction process, selected | ||
+ | the carbon source, Buffer, temperature, pH, reaction time and other conditions to optimize the | ||
+ | production process of the cell factory. | ||
+ | <br> | ||
+ | </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> | ||
+ | <h3>PS. We also made Hardware | ||
+ | <a href="https://2017.igem.org/Team:NPU-China/Hardware">(Click Here)</a> to simulate the industrial production process of acrylic acid!</h3> | ||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | <!-- Blog Post Row --> | ||
+ | |||
+ | |||
+ | </div> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/0/0c/Jz.png" class="img-responsive"> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | </body> | ||
</html> | </html> |
Latest revision as of 19:35, 1 November 2017