Difference between revisions of "Team:AHUT China/New Parts"

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   <div align="center"><img src="https://static.igem.org/mediawiki/2018/e/e9/T--AHUT_China--10121.1.png" width="1015" height="851" alt=""/></div><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: 16px;">&nbsp;&nbsp;&nbsp;Fig. 1-1 Map of CA2-WT recombinant vector</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: 16px;">&nbsp;&nbsp;&nbsp;Fig. 1-1 Map of CA2-WT recombinant vector</p> <br>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/c/c6/T--AHUT_China--10121.2.png" width="496" height="809" alt=""/></div><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: 16px;">&nbsp;&nbsp;&nbsp;Fig. 1-2 Agarose Gel Electrophoresis of CA2-WT recombinant plasmid and its identification by enzyme digestion. Lane M: DL marker; Lane 1: CA2-WT recombinant plasmid; Lane 2: enzyme digestion band of CA2-WT digested by MluⅠ, the length was 1028 bp (the arrow indicated).</p> <br>
 
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                   <p style="text-align: center; font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 36px;"><strong style="font-family: Segoe, 'Segoe UI', 'DejaVu Sans', 'Trebuchet MS', Verdana, sans-serif; font-style: normal; font-weight: 400; color: #000000;">8.30</strong></p>
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                   <p style="text-align: center; font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 36px;"><strong style="font-family: Segoe, 'Segoe UI', 'DejaVu Sans', 'Trebuchet MS', Verdana, sans-serif; font-style: normal; font-weight: 400; color: #000000;">Induced expression of CA2-WT</strong></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: 16px; ">&nbsp;&nbsp;&nbsp;&nbsp;<span style="font-size: 24px">Pilot </span> Expression of Protein<br>&nbsp;&nbsp;&nbsp;“IPTG+ 4h”“IPTG- 4h”</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: 16px; ">&nbsp;&nbsp;&nbsp;&nbsp;<span style="font-size: 24px">The </span>CA2-WT expression plasmid was transformed into E. coli BL21 (DE3), and the cultured liquid was subjected to IPTG-induced CA2 expression, and the bacterial solution was sonicated, followed by SDS-PAGE and Western Blot(Fig. 1-3 and Fig. 1-4) .It shows that the size of CA2 is 30.6 kDa, which is compared with Marker. The position indicated by the arrow in the figure is the CA2 band. It can be seen from lanes 1 and 2 in the figure that the IPTG condition is significantly induced. The expression of CA2, and it can be seen from lanes 3-6, that the induced expression of CA2 is mainly expressed in a soluble form in the supernatant of the bacterial liquid.The above results indicate that we successfully obtained E. coli which expresses CA2.</p>   
 
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  <div align="center"><img src="https://static.igem.org/mediawiki/2018/4/44/T--AHUT_China--830.png" width="420" height="77" alt=""/></div><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: 16px;">&nbsp;&nbsp;&nbsp;After the rocker culture is 4 H, add IPTG(final concentration is 0.5 mm) and then the rocker culture is 4 hours. Each of the two tubes absorbs 200 μL of bacterial fluid, and in the frozen centrifuge, it is 4 °C and 12000 rpm centrifuge. 10 minutes, go to the clear, It is resuspended with 100 μL 1x SDS Loading buffer, then heated in a water bath pot at 100 °C for 10 minutes and stored in a -20 °C refrigerator.</p>  
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  <div align="center"><img src="https://static.igem.org/mediawiki/2018/f/ff/T--AHUT_China--10121.3.png" width="531" height="502" alt=""/></div><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: 16px;">&nbsp;&nbsp;&nbsp;Fig. 1-3 SDS-PAGE analysis for CA2 cloned in pET-30a(+) and expressed in BL21(DE3) strain.</p>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/1/12/T--AHUT_China--10121.4.png" width="843" height="368" alt=""/></div><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: 16px;">&nbsp;&nbsp;&nbsp;Fig. 1-4 Western blot analysis for CA2 cloned in pET-30a(+) and expressed in BL21(DE3) strain.</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-style: normal; font-weight: 400; font-size: 36px; text-align: center;">  &nbsp;&nbsp;&nbsp;<strong style="font-family: Segoe, 'Segoe UI', 'DejaVu Sans', 'Trebuchet MS', Verdana, sans-serif; font-style: normal; font-weight: 400;color: #000000;"> 8.31</strong></h2>
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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-style: normal; font-weight: 400; font-size: 36px; text-align: center;">  &nbsp;&nbsp;&nbsp;<strong style="font-family: Segoe, 'Segoe UI', 'DejaVu Sans', 'Trebuchet MS', Verdana, sans-serif; font-style: normal; font-weight: 400;color: #000000;"> Purification of CA2-WT</strong></h2>
 
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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: 16px;">&nbsp;&nbsp;&nbsp;<span style="font-size: 24px">&nbsp;Protein</span> extraction <br>&nbsp;&nbsp;&nbsp;&nbsp;CA2L203K</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: 16px;">&nbsp;&nbsp;&nbsp;<span style="font-size: 24px">&nbsp;After</span> confirming that CA2 can be induced by E. coli BL21 (DE3), we will further purify the crude protein extract by nickel column purification to obtain purified CA2 protein. The results in the figure (Figures 5 and 6) illustrate that we have obtained a higher purity CA2 protein.
 
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   <div align="center"><img src="https://static.igem.org/mediawiki/2018/2/2d/T--AHUT_China--831.11.png" width="761" height="76" alt=""/></div>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/e/eb/T--AHUT_China--831.12.png" width="657" height="476" 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: 16px;">&nbsp;&nbsp;&nbsp;&nbsp;CA2WT<br>&nbsp;&nbsp;&nbsp;&nbsp;Fig.1-5 CA2 was purified with Ni column; fractions were analyzed by SDS-PAGE. Lane M: Protein marker; Lane 1: Supernatant after cell lysate centrifugation; Lane 2: Flow through; Lane 3: Wash with 50mM Tris, 150mM NaCl, 20 mM Imidazole, pH 8.0; Lane 4: Elute with 50mM Tris, 150mM NaCl, 50 mM Imidazole, pH 8.0; Lane 5: Elute with 50mM Tris, 150mM NaCl, 500 mM Imidazole, pH 8.0.</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: 16px;">&nbsp;&nbsp;&nbsp;&nbsp;CA2WT<br>&nbsp;&nbsp;&nbsp;&nbsp;CA2L203K</p>  
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/e/e4/T--AHUT_China--10121.6.png" width="1178" height="724" alt=""/></div><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: 16px;">&nbsp;&nbsp;&nbsp;Fig. 1-6 SDS-PAGE and Western blot analysis of CA2. Lane 1: Positive control (BSA); Lane 2: purified CA2; Lane 3: Purified CA2.</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-style: normal; font-weight: 400; font-size: 36px; text-align: center;">  &nbsp;&nbsp;&nbsp;<strong style="font-family: Segoe, 'Segoe UI', 'DejaVu Sans', 'Trebuchet MS', Verdana, sans-serif; font-style: normal; font-weight: 400;color: #000000;"> 9.01</strong></h2>
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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-style: normal; font-weight: 400; font-size: 36px; text-align: center;">  &nbsp;&nbsp;&nbsp;<strong style="font-family: Segoe, 'Segoe UI', 'DejaVu Sans', 'Trebuchet MS', Verdana, sans-serif; font-style: normal; font-weight: 400;color: #000000;"> BBa_K2547004 : Construction of CA2（L203K）expression plasmid</strong></h2>
 
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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;"> <span style="font-size: 16px">&nbsp;&nbsp;&nbsp;&nbsp;<span style="font-size: 24px">Purification</span>&nbsp;of Protein <br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Prior to purification</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: 14px;"> <span style="font-size: 16px">&nbsp;&nbsp;&nbsp;&nbsp;<span style="font-size: 24px">We</span>&nbsp;first synthesized the sequence of the mutant CA2, and then cloned it into the expression vector pET-30a(+), and identified the correctness of the obtained recombinant vector by restriction enzyme digestion and sequencing (Fig.2-1 and Fig. 2-2).</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: 14px;"> <span style="font-size: 16px">&nbsp;&nbsp;&nbsp;&nbsp;Fig. 2-1 Map of CA2 (L203K) recombinant vector</p><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: 14px;"> <span style="font-size: 16px">&nbsp;&nbsp;&nbsp;&nbsp;Purified</p><br>
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  <div align="center"><img src="https://static.igem.org/mediawiki/2018/d/d0/T--AHUT_China--10122.2.png" width="657" height="476" alt=""/></div>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/8/8a/T--AHUT_China--91.3.png" width="564" height="75" 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;"> <span style="font-size: 16px">&nbsp;&nbsp;&nbsp;&nbsp;Fig.2-2 Agarose Gel Electrophoresis of CA2(L203K) recombinant plasmid and its identification by enzyme digestion （NdeⅠand Hind Ⅲ）. Lane M: DNA marker; Lane 1: CA2(L203K) recombinant plasmid; Lane 2: enzyme digestion band of CA2(L203K) , the length was 825 bp (the arrow indicated).</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-style: normal; font-weight: 400; font-size: 36px; text-align: center;">  &nbsp;&nbsp;&nbsp;<strong style="font-family: Segoe, 'Segoe UI', 'DejaVu Sans', 'Trebuchet MS', Verdana, sans-serif; font-style: normal; font-weight: 400;color: #000000;">9.03</strong></h2>
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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-style: normal; font-weight: 400; font-size: 36px; text-align: center;">  &nbsp;&nbsp;&nbsp;<strong style="font-family: Segoe, 'Segoe UI', 'DejaVu Sans', 'Trebuchet MS', Verdana, sans-serif; font-style: normal; font-weight: 400;color: #000000;">Induced expression of CA2(L203K)</strong></h2>
 
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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: 16px;">&nbsp;&nbsp;&nbsp;<span style="font-size: 24px">&nbsp;Western</span> blot identification of proteins<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CA2L203K&nbsp;&nbsp;&nbsp;&nbsp;CA2WT</p>  <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: 16px;">&nbsp;&nbsp;&nbsp;<span style="font-size: 24px">&nbsp;The</span> CA2(L203K) expression plasmid was transformed into E. coli BL21 (DE3), and the cultured liquid was subjected to IPTG-induced CA2 (L203K) expression, and the bacterial solution was sonicated, followed by SDS-PAGE(figure 2-3), the size of CA2(L203K) is known to be 30.6 kDa, which is compared with Marker. The position indicated by the arrow in the figure is the CA2(L203K) band. It can be seen from lanes 1 and 2 in the figure that the IPTG condition is significant to the expression of CA2 which was induced, and it can be seen from lanes 3-6 that the induced expression of CA2 was mainly expressed in soluble form in the supernatant of the bacterial liquid. The above results indicated that we successfully obtained E. coli which expresses CA2(L203K). </p>  <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: 14px;"> <span style="font-size: 16px">&nbsp;&nbsp;&nbsp;&nbsp;Fig.2-3 SDS-PAGE analysis for CA2(L203K) cloned in pET-30a(+) and expressed in BL21(DE3) strain.</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-style: normal; font-weight: 400; font-size: 36px; text-align: center;">  &nbsp;&nbsp;&nbsp;<strong style="font-family: Segoe, 'Segoe UI', 'DejaVu Sans', 'Trebuchet MS', Verdana, sans-serif; font-style: normal; font-weight: 400;color: #000000;">9.04</strong></h2>
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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-style: normal; font-weight: 400; font-size: 36px; text-align: center;">  &nbsp;&nbsp;&nbsp;<strong style="font-family: Segoe, 'Segoe UI', 'DejaVu Sans', 'Trebuchet MS', Verdana, sans-serif; font-style: normal; font-weight: 400;color: #000000;">Purification of CA2(L203K) protein</strong></h2>
 
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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: 16px;">&nbsp;&nbsp;&nbsp;<span style="font-size: 24px">&nbsp;Quantification</span> of protein concentrations</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: 16px;">&nbsp;&nbsp;&nbsp;<span style="font-size: 24px">&nbsp;After</span> confirming that CA2(L203K) can be induced by E. coli BL21(DE3), we will further purify the crude protein extract by nickel column purification to obtain purified CA2(L203K) protein. Figure2-4 shows the results. We have obtained a highly purified mutant CA2 protein.</p>   
 
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/6/6e/T--AHUT_China--93.2.png" width="908" height="189" 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;"> <span style="font-size: 16px">&nbsp;&nbsp;&nbsp;&nbsp;Fig.2-4 SDS-PAGE and Western blot analysis of CA2(L203K). Lane 1: Negative control; Lane 2: purified CA2(L203K) protein</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-style: normal; font-weight: 400; font-size: 36px; text-align: center;">  &nbsp;&nbsp;&nbsp;<strong style="font-family: Segoe, 'Segoe UI', 'DejaVu Sans', 'Trebuchet MS', Verdana, sans-serif; font-style: normal; font-weight: 400;color: #000000;">9.05</strong></h2>
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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-style: normal; font-weight: 400; font-size: 36px; text-align: center;">  &nbsp;&nbsp;&nbsp;<strong style="font-family: Segoe, 'Segoe UI', 'DejaVu Sans', 'Trebuchet MS', Verdana, sans-serif; font-style: normal; font-weight: 400;color: #000000;">Determination of protease activity of CA2 and CA2 (L203K)</strong></h2>
 
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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: 16px;">&nbsp;&nbsp;&nbsp;<span style="font-size: 24px">&nbsp;Colorimetric</span> enzyme activity<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Add 250 μl saturated CO2 solution</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: 16px;">&nbsp;&nbsp;&nbsp;<span style="font-size: 24px">&nbsp;We</span> determined the enzymatic activities of wild-type and mutant CA2 by colorimetric and esterase methods. As shown in Figure 2-5 and Figure 2-6, mutant CA2 (L203K) has higher enzymatic activity than wild-type CA2.</p>   
 
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/c/c4/T--AHUT_China--95.1.png" width="789" height="328" alt=""/></div>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/8/8b/T--AHUT_China--10122.5.png" width="618" height="352" alt=""/></div>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/8/82/T--AHUT_China--95.2.png" width="669" height="347" 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;"> <span style="font-size: 16px">&nbsp;&nbsp;&nbsp;&nbsp;Fig.2-5 Colorimetric assay of CA2 activity</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: 16px;">&nbsp;&nbsp;&nbsp;According to the average calculated in the table above, the unit enzyme activity of the mutated carbonic anhydride enzyme is 0.3710(10 5 WAU/μg), and the unit enzyme activity of wild carbonic anhydride enzyme is 0.1755(10 5 WAU/μg).<br>
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Test esterase activity<br>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/5/54/T--AHUT_China--10122.6.png" width="487" height="326" alt=""/></div>
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Determination curve without carbonated anhydrase:</p> <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: 16px;">&nbsp;&nbsp;&nbsp;Fig.2-6 Esterase activity analysis of CA2 protein</p> <br><br><br>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/f/f6/T--AHUT_China--95.3.png" width="903" height="296" alt=""/></div><br>
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<div class="container">
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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: 16px;">&nbsp;&nbsp;&nbsp;Determination curve with carbonated anhydrase（CA2WT）</p> <br>
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              <div class="section_title ">
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/e/e8/T--AHUT_China--95.4.png" width="929" height="330" 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-style: normal; font-weight: 400; font-size: 36px; text-align: center;">  &nbsp;&nbsp;&nbsp;<strong style="font-family: Segoe, 'Segoe UI', 'DejaVu Sans', 'Trebuchet MS', Verdana, sans-serif; font-style: normal; font-weight: 400;color: #000000;">Analysis of Thermal Stability of CA2 and CA2 (L203K)</strong></h2>
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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: 16px;">&nbsp;&nbsp;&nbsp;Determination curve with carbonated anhydrase（CA2L203K）</p> <br>
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                <hr>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/0/0c/T--AHUT_China--95.5.png" width="893" height="306" alt=""/></div>
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                <nbsp><nbsp>
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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: 16px;">&nbsp;&nbsp;&nbsp;Wild carbonic anhydrase activity calculated as 0.5098 mol/g × min<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: 16px;">&nbsp;&nbsp;&nbsp;<span style="font-size: 24px">&nbsp;We</span> examined the activity of carbonic anhydrase in wild-type and mutant CA2 at different times and temperatures by esterase method. The results are shown in Figure 2-7. As the temperature increases, especially at 55 ° C and 65 ° C, The enzymatic activity of wild type CA2 was significantly decreased, while the mutant CA2 still had higher activity, indicating that CA2 (L203K) has better thermal stability.</p>   <br>
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&nbsp;&nbsp;&nbsp;The mutated carbonic anhydrase enzyme activity was calculated to be 1.0392 mol/g * min.
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/f/fb/T--AHUT_China--10122.7.png" width="805" height="867" alt=""/></div>
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</p> <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: 16px;">&nbsp;&nbsp;&nbsp;Fig. 2-7 Activity of purified CA2-WT and CA2 (L203K) under indicated temperatures and time points.</p> <br>
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Revision as of 06:36, 12 October 2018

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Experimental results




    BBa_K2547000: Construction of CA2-WT expression plasmid

     We first synthesized the sequence of human carbonic anhydrase CA2, and then cloned it into the expression vector pET-30a(+), and identified the correctness of the obtained recombinant vector by restriction enzyme digestion and sequencing (Fig. 1-1 and Fig. 1-2).




   Fig. 1-1 Map of CA2-WT recombinant vector



   Fig. 1-2 Agarose Gel Electrophoresis of CA2-WT recombinant plasmid and its identification by enzyme digestion. Lane M: DL marker; Lane 1: CA2-WT recombinant plasmid; Lane 2: enzyme digestion band of CA2-WT digested by MluⅠ, the length was 1028 bp (the arrow indicated).


Induced expression of CA2-WT

    The CA2-WT expression plasmid was transformed into E. coli BL21 (DE3), and the cultured liquid was subjected to IPTG-induced CA2 expression, and the bacterial solution was sonicated, followed by SDS-PAGE and Western Blot(Fig. 1-3 and Fig. 1-4) .It shows that the size of CA2 is 30.6 kDa, which is compared with Marker. The position indicated by the arrow in the figure is the CA2 band. It can be seen from lanes 1 and 2 in the figure that the IPTG condition is significantly induced. The expression of CA2, and it can be seen from lanes 3-6, that the induced expression of CA2 is mainly expressed in a soluble form in the supernatant of the bacterial liquid.The above results indicate that we successfully obtained E. coli which expresses CA2.

 

 


   Fig. 1-3 SDS-PAGE analysis for CA2 cloned in pET-30a(+) and expressed in BL21(DE3) strain.


   Fig. 1-4 Western blot analysis for CA2 cloned in pET-30a(+) and expressed in BL21(DE3) strain.

    Purification of CA2-WT

    After confirming that CA2 can be induced by E. coli BL21 (DE3), we will further purify the crude protein extract by nickel column purification to obtain purified CA2 protein. The results in the figure (Figures 5 and 6) illustrate that we have obtained a higher purity CA2 protein.

 

 


    CA2WT
    Fig.1-5 CA2 was purified with Ni column; fractions were analyzed by SDS-PAGE. Lane M: Protein marker; Lane 1: Supernatant after cell lysate centrifugation; Lane 2: Flow through; Lane 3: Wash with 50mM Tris, 150mM NaCl, 20 mM Imidazole, pH 8.0; Lane 4: Elute with 50mM Tris, 150mM NaCl, 50 mM Imidazole, pH 8.0; Lane 5: Elute with 50mM Tris, 150mM NaCl, 500 mM Imidazole, pH 8.0.


   Fig. 1-6 SDS-PAGE and Western blot analysis of CA2. Lane 1: Positive control (BSA); Lane 2: purified CA2; Lane 3: Purified CA2.

 

 

    BBa_K2547004 : Construction of CA2（L203K）expression plasmid

    We first synthesized the sequence of the mutant CA2, and then cloned it into the expression vector pET-30a(+), and identified the correctness of the obtained recombinant vector by restriction enzyme digestion and sequencing (Fig.2-1 and Fig. 2-2).



    Fig. 2-1 Map of CA2 (L203K) recombinant vector


    Fig.2-2 Agarose Gel Electrophoresis of CA2(L203K) recombinant plasmid and its identification by enzyme digestion （NdeⅠand Hind Ⅲ）. Lane M: DNA marker; Lane 1: CA2(L203K) recombinant plasmid; Lane 2: enzyme digestion band of CA2(L203K) , the length was 825 bp (the arrow indicated).

 

 

   Induced expression of CA2(L203K)

    The CA2(L203K) expression plasmid was transformed into E. coli BL21 (DE3), and the cultured liquid was subjected to IPTG-induced CA2 (L203K) expression, and the bacterial solution was sonicated, followed by SDS-PAGE(figure 2-3), the size of CA2(L203K) is known to be 30.6 kDa, which is compared with Marker. The position indicated by the arrow in the figure is the CA2(L203K) band. It can be seen from lanes 1 and 2 in the figure that the IPTG condition is significant to the expression of CA2 which was induced, and it can be seen from lanes 3-6 that the induced expression of CA2 was mainly expressed in soluble form in the supernatant of the bacterial liquid. The above results indicated that we successfully obtained E. coli which expresses CA2(L203K).



    Fig.2-3 SDS-PAGE analysis for CA2(L203K) cloned in pET-30a(+) and expressed in BL21(DE3) strain.



   Purification of CA2(L203K) protein

    After confirming that CA2(L203K) can be induced by E. coli BL21(DE3), we will further purify the crude protein extract by nickel column purification to obtain purified CA2(L203K) protein. Figure2-4 shows the results. We have obtained a highly purified mutant CA2 protein.


    Fig.2-4 SDS-PAGE and Western blot analysis of CA2(L203K). Lane 1: Negative control; Lane 2: purified CA2(L203K) protein



   Determination of protease activity of CA2 and CA2 (L203K)

    We determined the enzymatic activities of wild-type and mutant CA2 by colorimetric and esterase methods. As shown in Figure 2-5 and Figure 2-6, mutant CA2 (L203K) has higher enzymatic activity than wild-type CA2.


    Fig.2-5 Colorimetric assay of CA2 activity

   Fig.2-6 Esterase activity analysis of CA2 protein




   Analysis of Thermal Stability of CA2 and CA2 (L203K)

    We examined the activity of carbonic anhydrase in wild-type and mutant CA2 at different times and temperatures by esterase method. The results are shown in Figure 2-7. As the temperature increases, especially at 55 ° C and 65 ° C, The enzymatic activity of wild type CA2 was significantly decreased, while the mutant CA2 still had higher activity, indicating that CA2 (L203K) has better thermal stability.


   Fig. 2-7 Activity of purified CA2-WT and CA2 (L203K) under indicated temperatures and time points.