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We built a new cell factory of acrylic acid through the four part: CO-PART, SYSTEM, PATHWAY, PRODUCTION! <br> | We built a new cell factory of acrylic acid through the four part: CO-PART, SYSTEM, PATHWAY, PRODUCTION! <br> | ||
</h4> | </h4> | ||
− | <h2 style="text-align:center | + | <a id="COREPART"></a> |
+ | <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> | <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> | 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> | ||
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</h4> | </h4> | ||
【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】 | ||
− | + | <a id="PATHWAY"></a> | |
− | <h2 style="text-align:center | + | <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> | <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> | ||
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</h4> | </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】 | 【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】 | ||
− | + | <a id="SYSTEM"></a> | |
− | <h2 style="text-align:center | + | <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> | <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> | 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> | ||
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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> | 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> | </h4> | ||
− | + | <a id="PRODUCTION"></a> | |
− | <h2 style="text-align:center | + | <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> | <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> | 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> |
Revision as of 15:06, 30 October 2017