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<a href="https://2017.igem.org/Team:NPU-China/Description">Description</a> | <a href="https://2017.igem.org/Team:NPU-China/Description">Description</a> | ||
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<a href="https://2017.igem.org/Team:NPU-China/Design">Design</a> | <a href="https://2017.igem.org/Team:NPU-China/Design">Design</a> | ||
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− | + | <h3>Introduction</h3> | |
− | + | <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> 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 | |
+ | <br> 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> (图) | ||
+ | <br> Cell factory of acrylic acid (GAACF) 1.0: | ||
+ | <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. | ||
+ | With ceaS2 enzyme as the core part, it is possible to create a new pathway to synthesize acrylic | ||
+ | acid from glycerol metabolic pathway in organisms and construct a cell factory with high yield of | ||
+ | acrylic acid. | ||
+ | <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 is a more flexible and has more development prospects. | ||
+ | <br> 【E.coli图+路径图+质粒图】 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> 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. | ||
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− | Cell factory of acrylic acid (GAACF) | + | <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! | |
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− | + | </h4> | |
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− | + | <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> 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> 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) (Learn more about HPLC!)and HTS (High throughput screening) | ||
+ | (Learn more about HTS!). | ||
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− | + | </h4> | |
− | + | 【ceaS2酶结构图+5埃范围内活性中心示意图】 | |
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− | + | <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. | |
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− | <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> | + | <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> 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. | ||
+ | ) (质粒图注释) | ||
+ | <br> | ||
+ | </h4> | ||
+ | 【E.coli新路径图(含旧路径部分),区别主要途径和还原力模块+质粒图】 | ||
+ | |||
+ | <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> 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 URA defect screening marker as the backbone and used the pTDH3 constitutive | ||
+ | promoter and tPFK1 constitutive terminator to construct ceaS2 plasmid. | ||
+ | <br> 【S.C图+路径图+质粒图】 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. | ||
+ | <br> 【新途径+质粒图】 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. | ||
+ | (质粒图注释) | ||
+ | <br> | ||
+ | </h4> | ||
+ | |||
+ | <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> 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> | ||
+ | 【筛选条件组合表,分为E.coli和S.C的】 | ||
+ | <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> | ||
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</div> | </div> | ||
Revision as of 15:12, 30 October 2017