Difference between revisions of "Team:AHUT China/design"

Line 107: Line 107:
  
 
                   <p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 18px;" >As we have described in the background, traditional CO2 capture technology is still in its early stages and is often characterized by high energy consumption and low efficiency. The goal of the project is to develop a new kind of low energy, high efficiency and environmentally friendly CO2 capture method. Based on this goal, we intend to use human carbonic anhydrase 2 (CA2) as the research object, because CA2 can efficiently catalyze CO2 hydration to produce HCO3- (Fig. 1), which can achieve efficient capture of CO2, however, the enzyme has the fastest reaction rate at 37 °C and is inactivated at 50 °C, which is not suitable for industrial applications of large-scale CO2 capture. Therefore, we plan to obtain engineered CA2 mutants with high thermal stability by using genetic engineering technology, laying the foundation for subsequent industrial applications. The overall design for our project is as follows (Fig. 2).</p>
 
                   <p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 18px;" >As we have described in the background, traditional CO2 capture technology is still in its early stages and is often characterized by high energy consumption and low efficiency. The goal of the project is to develop a new kind of low energy, high efficiency and environmentally friendly CO2 capture method. Based on this goal, we intend to use human carbonic anhydrase 2 (CA2) as the research object, because CA2 can efficiently catalyze CO2 hydration to produce HCO3- (Fig. 1), which can achieve efficient capture of CO2, however, the enzyme has the fastest reaction rate at 37 °C and is inactivated at 50 °C, which is not suitable for industrial applications of large-scale CO2 capture. Therefore, we plan to obtain engineered CA2 mutants with high thermal stability by using genetic engineering technology, laying the foundation for subsequent industrial applications. The overall design for our project is as follows (Fig. 2).</p>
<br>
+
<br><br><br>
 
<div align="center"><img src="
 
<div align="center"><img src="
 
https://static.igem.org/mediawiki/2018/e/ed/T--AHUT_China--_design333.jpg" width="650"  alt=""/></div>
 
https://static.igem.org/mediawiki/2018/e/ed/T--AHUT_China--_design333.jpg" width="650"  alt=""/></div>
  <p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆';  font-size: 14px;text-align: center;">Fig. 1 The catalytic mechanism of CA2 </p>  <br>
+
  <p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆';  font-size: 14px;text-align: center;">Fig. 1 The catalytic mechanism of CA2 </p>  <br><br><br>
 
<div align="center"><img src="
 
<div align="center"><img src="
 
https://static.igem.org/mediawiki/2018/8/8b/T--AHUT_China--_liucheng.jpg" width="650"  alt=""/></div>
 
https://static.igem.org/mediawiki/2018/8/8b/T--AHUT_China--_liucheng.jpg" width="650"  alt=""/></div>
  <p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆';  font-size: 14px;text-align: center;">Fig. 2 The overall design model for our project </p>   
+
  <p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆';  font-size: 14px;text-align: center;">Fig. 2 The overall design model for our project </p>  <br><br><br><br>
 
<h3>The detailed design procedure is described as follows:</h3><br>
 
<h3>The detailed design procedure is described as follows:</h3><br>
 
<p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 18px;" >1. Established the design principles of carbonic anhydrase 2 (CA2)
 
<p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 18px;" >1. Established the design principles of carbonic anhydrase 2 (CA2)

Revision as of 01:15, 18 October 2018

Royal Hotel Royal Hotel







Design


As we have described in the background, traditional CO2 capture technology is still in its early stages and is often characterized by high energy consumption and low efficiency. The goal of the project is to develop a new kind of low energy, high efficiency and environmentally friendly CO2 capture method. Based on this goal, we intend to use human carbonic anhydrase 2 (CA2) as the research object, because CA2 can efficiently catalyze CO2 hydration to produce HCO3- (Fig. 1), which can achieve efficient capture of CO2, however, the enzyme has the fastest reaction rate at 37 °C and is inactivated at 50 °C, which is not suitable for industrial applications of large-scale CO2 capture. Therefore, we plan to obtain engineered CA2 mutants with high thermal stability by using genetic engineering technology, laying the foundation for subsequent industrial applications. The overall design for our project is as follows (Fig. 2).




Fig. 1 The catalytic mechanism of CA2




Fig. 2 The overall design model for our project





The detailed design procedure is described as follows:


1. Established the design principles of carbonic anhydrase 2 (CA2) With the help of computer-aided analysis software Discovery Visual Studio, we established the design principles of CA2 to predict the ideal mutation sites for this protein:


1) Maintain the 3D structure of enzyme;

2) Modify the interactions between residues around active sites;

3) Improve the rigidity of active sites;

4) Shorten the distance of proton transfer.

2. Molecular docking of enzyme-substrate.

Molecular docking with Autodock was performed to investigate the docking conformation of the substrate at the catalytic site and to analyze the interaction between the residues at the catalytic site and the substrate. Effects of the secondary and tertiary structure of the catalytic sites on the catalytic process were further investigated by using Autodock and Discovery Visual Studio. The mutation sites and substitution residues were set, and then the molecular docking of the recombinase was carried out to compare the enzyme-substrate docking conformation before and after recombination. Suitable mutation sites and replacement residues were selected to improve their catalytic properties.



3. Enzyme-solvent kinetics simulation.

Kinetic simulation was conducted by Gromacs software to investigate the conformation of the enzyme under aqueous solvent conditions at normal/high temperature conditions and to analyze the root mean square fluctuation of its individual residues. According to the results above, unstable residues were chosen to mutate, and the advanced structure of the enzyme and its rheology before and after recombination were further compared by Gromacs and Discovery Visual Studio software, then suitable mutation sites and replacement residues were selected to improve their stability.



4. Construction of vectors

Next, we are preparing to construct wild-type and mutant CA2 prokaryotic expression vectors by using genetic engineering technology. The coding sequences of CA2-WT and mutant CA2 were both optimized and synthesized, then cloned into the expression vector pET-30a(+), respectively. The correctness of the obtained recombinant vectors were identified by restriction enzyme digestion and sequencing.



5. Expression and purification of proteins

Then, we induce the expression of wild-type and mutant CA2 protein in E.coli BL21 (DE3) with isopropyl-1-thio-β-Dgalactopyrasonide (IPTG) induction. Briefly, recombinant plasmids of the wild-type and mutant CA2 were transformed into E. coli BL21 (DE3) and positive clones were screened by kanamycin resistance. Then, the recombinant E. coli BL21 (DE3) were propagated and its expression was induced with IPTG. Cells were lysed by sonication on ice, and the obtained crude extracts were centrifuged to separate supernatant and debris, and both fractions were subjected to SDS-PAGE and Western Blot.

After confirming that wild-type and mutant CA2 could be expressed in our chassis E. coli BL21 (DE3), protein of wild-type and mutant CA2 were further purified with nickel column for the following CO2 capture.



6. Identification of the function

In order to verify the function of the mutant protein, we prepared the colorimetric or esterase method to measure the Km and Vmax value of the wild-type and mutant CA2, and compare the thermostability of the two proteins by esterase method. Finally, we determine whether the activity of the mutant protein changes and whether the thermal stability is significantly improved.