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                       <div align="center"><h2>Design</h2></div>
 
                       <div align="center"><h2>Design</h2></div>
 
<hr>
 
<hr>
                   <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;" >&nbsp;&nbsp;&nbsp;&nbsp;As we have described in the background, traditional carbon dioxide collection techniques are still in its early stages, characterized by high consumption and low efficiency. We want low-energy, large-scale, efficient collection of carbon dioxide, in order to achieve this goal, we by the carbon anhydride enzyme gene into E. coli.</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: 18px;" >&nbsp;&nbsp;To ensure that E. coli-producing carbonic anhydride (CA-II) normally absorbs carbon dioxide in an industrialized environment, we first simulate the protein molecule expressed by a computer to obtain a thermally stable carbonic anhydride (CA-II), which produces a thermally stable carbonic anhydride (CA-II), so that it can normally absorb carbon dioxide in a factory-like environment.
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 +
                   <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 CO<span style="font-size: 13px">2</span> 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 CO<span style="font-size: 13px">2</span> capture method. Based on this goal, we intend to use human carbonic anhydrase 2 (CA2) as the research object, because CA2 can efficiently catalyze CO<span style="font-size: 13px">2</span> hydration to produce HCO<span style="font-size: 13px">3</span><sup>-</sup> (Fig. 1), which can achieve efficient capture of CO<span style="font-size: 13px">2</span>, 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 CO<span style="font-size: 13px">2</span> 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>
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<br><br><br>
 +
<div align="center"><img src="
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https://static.igem.org/mediawiki/2018/e/ed/T--AHUT_China--_design333.jpg" width="750"  alt=""/></div>
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<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>
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<div align="center"><img src="
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https://static.igem.org/mediawiki/2018/8/8b/T--AHUT_China--_liucheng.jpg" width="650"  alt=""/></div>
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<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>
 +
<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)
 +
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:
 
</p>
 
</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;" >&nbsp;&nbsp;&nbsp;&nbsp;E. Coli's carbonic anhydride enzyme (CA-II) promotes CO2 hydration to produce co32-, which binds to the free ca2+ in the environment to form calcium carbonate deposits, thereby achieving the purpose of absorbing carbon dioxide, and producing inorganic products that can be used.</p>
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<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) Maintain the 3D structure of enzyme; <br><br>
 +
2) Modify the interactions between residues around active sites; <br><br>
 +
3) Improve the rigidity of active sites; <br><br>
 +
4) Shorten the distance of proton transfer.<br><br>
 +
</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;" >
 
  <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;" >
&nbsp;&nbsp;Selection of carbonic anhydride enzymes:<br>
+
2. Molecular docking of enzyme-substrate.<br><br>
Our team acquired a sequence of the human Body's carbonic anhydride, and contacted biotech companies to help us synthesize the carbonic anhydride gene in full sequence. The Carbon-anhydride (CA-II) activity of the human being used by our team is more active than that of other mammals, plants, algae, and bacteria-producing carbonic anhydride (CA-II). The carbon anhydride (CA-II) extracted in the human body at 37 ℃ the fastest reaction rate, 50 ℃ conditions, But its maximum reaction rate can reach 106 s-1, the fastest catalytic rate of carbonic anhydride (CA-II).
+
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.
 
  </p>
 
  </p>
  <h3>Carbonic anhydride Enzyme structure:</h3> <br>
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<br><br>
                <div align="center"><img src="
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https://static.igem.org/mediawiki/2018/3/31/T--AHUT_China--_design1.jpg" width="501" height="340" 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: 18px;" >
+
&nbsp;&nbsp;&nbsp;&nbsp;Christonpherks,JamesJL.Biotechnology for the acceleration of carbon dioxide capture and sequestration[J].Science Direct,2011,22:818-823.
+
</p><br>
+
+
 
 
<h3>&nbsp;&nbsp;&nbsp;&nbsp;Increased thermal stability of carbonic anhydride (CA-II):</h3>
+
  <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;" >
  <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;" >&nbsp;&nbsp;&nbsp;&nbsp;In order to make the carbonic anhydride enzyme (CA-II) suitable for the industrial environment to absorb carbon dioxide, later we use molecular simulation technology, the amino acid as the basic unit, the residual radical mutation on the two-stage structure of carbon anhydride, and the influence of molecular conformation, to obtain the best amino acid mutation sites, The thermal stability of enzymes was improved without affecting the enzyme Activity.<br>
+
3. Enzyme-solvent kinetics simulation.<br><br>
&nbsp;&nbsp;Note: This part also the experimental data of the thermal stability of the carbon anhydride enzyme and the three-dimensional map of the fixed-point mutant base of the carbonic anhydride gene have not been added and referenced
+
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.
 
  </p>
 
  </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;" >&nbsp;&nbsp;&nbsp;&nbsp;The mechanism of hydration of carbon dioxide catalyzed by carbonic anhydride enzyme (CA-II:</p><br>
+
<br><br>
+
<div align="center"><img src="https://static.igem.org/mediawiki/2018/c/c9/T--AHUT_China--_design2.jpg" width="724" height="400" alt=""/></div><br><div align="center"><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;" >
+
4. Construction of vectors<br><br>
+
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.
                <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;" >&nbsp;&nbsp;&nbsp;&nbsp;Duda D, Tu C , Qian M, et al , 2001.Structural and kinetic analysis of the chemical rescue of the proton transfer function of carbonic an- hydrase Ⅱ [ J] .Biochemistry , 40 (6):1741—1748 <br>
+
Elder I , Han S , Tu C , et al , 2004.Activation of carbonic anhydrase Ⅱ by active-site incorporation of histidine analogs [ J] .Arc Bioch Biophys, 421:283—289
+
</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;" >&nbsp;&nbsp;&nbsp;&nbsp;Application of carbonic anhydride enzyme (ca-ii) in carbon dioxide concentration
+
 
  </p>
 
  </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;" >&nbsp;&nbsp;&nbsp;&nbsp;There are two kinds of enzymatic trapping techniques: non-immobilized carbon anhydride trapping technology and immobilized carbon anhydride trapping technology. Non-immobilized Carbon anhydride trapping technology is the first carbon anhydride capture technology, This method directly using free carbon anhydride enzyme CO2 capture, at this time the activity of carbonic anhydride enzyme is low, about 30% of the activity, and this method is not conducive to the re-use of enzymes; in order to compensate for the deficiency of non-immobilized carbonic anhydride enzyme technology, Some people will silica and other inorganic compounds as a carrier of the carbon anhydride enzyme, thereby curing the carbon anhydride enzyme, it is found that the activity of carbon anhydride at this time to maintain about 60%, and the easy recovery of carbonic anhydride enzyme, so our team is using immobilized carbon anhydride enzyme capture technology. </p><br>        
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<br><br>
<div align="center"><img src="https://static.igem.org/mediawiki/2018/d/df/T--AHUT_China--_design3.jpg" width="724" height="484" alt=""/></div><br><div align="center"><br>
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          <h6>&nbsp;&nbsp;&nbsp;&nbsp;Reference documentation</h6> <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;" >
+
<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;" >
&nbsp;&nbsp;&nbsp;&nbsp;S L. Structure and mechanism of carbonic anhydrase [J]. Pharmacology & Therapeutics, 1997, 74(1): 1.
+
5. Expression and purification of proteins<br><br>
Supuran C T, Conroy C W, Maren T H. Is cyanate a carbonic anhydrase substrate? [J]. Proteins-structure Function & Bioinformatics, 2015, 27(2): 272-8.</p>                
+
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.<br><br>
 +
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.  
 +
</p>
 +
<br><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;" >
 +
6. Identification of the function<br><br>
 +
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.
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Latest revision as of 01:39, 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.