Line 188: | Line 188: | ||
<br> | <br> | ||
</h4> | </h4> | ||
− | + | <div id="COREPART" style="padding-top:50px;margin-top:-50px;"> | |
− | <h2 style="text-align:center">CORE PART</h2> | + | <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!). | |
− | + | <br> | |
− | + | </h4> | |
+ | </div> | ||
【ceaS2酶结构图+5埃范围内活性中心示意图】 | 【ceaS2酶结构图+5埃范围内活性中心示意图】 | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | <h2 style="text-align:center"> | + | <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> 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】 | |
− | + | </div> | |
− | + | ||
− | </ | + | |
− | <h2 style="text-align:center"> | + | <div id="SYSTEM" style="padding-top:50px;margin-top:-50px;"> |
− | <h4>To make the engineering bacteria produce acrylic acid, it takes two stages. First, bacteria must | + | <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> | ||
+ | </div> | ||
+ | |||
+ | <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> 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> | <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> |
Revision as of 15:22, 30 October 2017