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

 
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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 carbonic anhydrase gene into E. coli.</p>
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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;" >&nbsp;&nbsp;To ensure that E. coli-producing carbonic anhydrase 2(CA2) normally absorbs carbon dioxide in an industrialized environment, we first simulate the protein molecule expressed by a computer to obtain a thermally stable carbonic anhydrase 2(CA2), which produces a thermally stable carbonic anhydrase 2 (CA2), 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>
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<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>
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<h3>The detailed design procedure is described as follows:</h3><br>
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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;" >1. Established the design principles of carbonic anhydrase 2 (CA2)
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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 anhydrase 2(CA2) 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>
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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;" >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>
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2. Molecular docking of enzyme-substrate.<br><br>
&nbsp;&nbsp;Our team acquired the sequence of carbonic anhydrase from the human body and contacted Biotech to help us synthesize the carbonic anhydrase gene in its entire sequence. The mutant carbonic anhydrase 2(CA2) used by our team absorbs carbon dioxide more strongly than other mammals, plants, algae, and carbonic anhydrase 2(CA2) produced by bacteria. Carbonic anhydrase 2(CA2) in human body has the fastest reaction rate at 37 °C, and is inactivated at 50 °C, but its maximum reaction rate can reach 106 s-1, which is the fastest catalytic carbonic anhydrase 2(CA2).
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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 anhydrase structure(Fig. 1):</h3> <br>
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<br><br>
                <div align="center"><img src="
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https://static.igem.org/mediawiki/2018/5/5b/T--AHUT_China--_design111.jpg" width="501" height="340" 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 CO2 release in human carbonic anhydrase II crystals </p>               
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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;" >
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&nbsp;&nbsp;</p><br>
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<h3>&nbsp;&nbsp;&nbsp;&nbsp;
 
Increased thermal stability of carbonic anhydrase 2(CA2):</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;" >&nbsp;&nbsp;&nbsp;&nbsp;In order to make the carbonic anhydrase 2(CA2) suitable for the industrial environment to absorb carbon dioxide, later we use molecular simulation technology, the amino acid as the basic unit, the mutations of residues on the secondary structure of the carbonic anhydrase, 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.</p>
 
<h3>&nbsp;&nbsp;&nbsp;&nbsp;Thermal stability studies of CA2-WT and CA2 (L203K) protein:</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;We then investigated the effect of temperature on CA2 activity by esterase activity assay. As shown in (Fig. 2), as the temperature increases, especially at 55 °C and 65 °C, the enzymatic activity of CA2-WT was significantly decreased, while the mutant CA2 still retain relatively high activity, indicating that CA2 (L203K) was more stable at high temperature and retained its activity.
+
3. Enzyme-solvent kinetics simulation.<br><br>
 +
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>
<div align="center"><img src="
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<br><br>
https://static.igem.org/mediawiki/2018/0/0d/T--AHUT_China--_design222.jpg" width="501" height="640" 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 Activity of purified CA2-WT and CA2 (L203K) protein under indicated temperatures and time points</p>
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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;" >
<h3>&nbsp;&nbsp;&nbsp;&nbsp;Enzymatic properties of carbonic anhydrase:</h3>
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4. Construction of vectors<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;" >&nbsp;&nbsp;&nbsp;&nbsp;Carbonic anhydrase is currently the most efficient enzyme for catalyzing the hydration of CO2. Carbonic anhydrase can catalyze CO2 andH2O produces HCO3-( Fig. 3). Under natural conditions, the reaction rate is extremely low, but the reaction rate is greatly accelerated under the catalysis of carbonic anhydrase, and the reaction rate can reach 104-106 reactions per second. The main rate limiting step is the diffusion rate of the substrate . In animals, carbonic anhydrase is mainly involved in the acid-base balance and CO2 transport in the blood; in plants, carbonic anhydrase mainly helps the chloroplast to absorb CO2.</p><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.
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</p>
<div align="center"><img src="https://static.igem.org/mediawiki/2018/e/ed/T--AHUT_China--_design333.jpg" width="724" height="400" alt=""/></div><br><div align="center">
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<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: 14px;text-align: center;">Fig. 3 The mechanism of hydration of carbon dioxide catalyzed by carbonic anhydrase 2(CA2)</p> <br> </p></div>
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<h3>Application of carbonic anhydrase 2 (CA2) in carbon dioxide concentration:</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;There are two kinds of enzymatic trapping techniques: non-immobilized carbonic anhydrase trapping technology and immobilized carbonic anhydrase trapping technology. Non-immobilized Carbonic anhydrase trapping technology is the first carbonic anhydrase capture technology, This method directly using free carbonic anhydrase CO2 capture, at this time the activity of carbonic anhydrase is low, about 30% of the activity, and this method is not conducive to the re-use of enzymes(Fig. 4); in order to compensate for the deficiency of non-immobilized carbonic anhydrase technology, Some people will silica and other inorganic compounds as a carrier of the carbonic anhydrase, thereby curing the carbonic anhydrase, it is found that the activity of carbonic anhydrase at this time to maintain about 60%, and the easy recovery of carbonic anhydrase , so our team is using immobilized carbonic anhydrase capture technology. </p><br>
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5. Expression and purification of proteins<br><br>
<div align="center"><img src="https://static.igem.org/mediawiki/2018/b/bf/T--AHUT_China--_design4444.jpg" width="724" height="600" alt=""/></div><br><div align="center">
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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>
<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. 4 Schematic diagram of immobilized carbonic anhydrase capture CO2</p> <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></div>
+
</p>
<h3>references:</h3>
+
<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;" >[1]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>
+
[2]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<br>
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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;" >
[3]S L. Structure and mechanism of carbonic anhydrase [J]. Pharmacology & Therapeutics, 1997, 74(1): 1.<br>
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6. Identification of the function<br><br>
[4]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>          
+
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.
 +
</p>
 
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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.