Difference between revisions of "Team:AHUT China/Notebook"
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<hr> | <hr> | ||
<nbsp><nbsp> | <nbsp><nbsp> | ||
| − | <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: | + | <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;"> <span style="font-size: 24px"> Protein</span> extraction <br> CA2L203K</p> |
<p> </p> <p> </p> | <p> </p> <p> </p> | ||
<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> | <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> | ||
| Line 87: | Line 87: | ||
<hr> | <hr> | ||
<nbsp><nbsp> | <nbsp><nbsp> | ||
| − | <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: | + | <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;"> <span style="font-size: 24px"> Western</span> blot identification of proteins<br> CA2L203K CA2WT</p> <br> |
<div align="center"><img src="https://static.igem.org/mediawiki/2018/6/60/T--AHUT_China--93.1.png" width="971" height="167" alt=""/></div> <br> | <div align="center"><img src="https://static.igem.org/mediawiki/2018/6/60/T--AHUT_China--93.1.png" width="971" height="167" alt=""/></div> <br> | ||
<br><br> | <br><br> | ||
| Line 95: | Line 95: | ||
<hr> | <hr> | ||
<nbsp><nbsp> | <nbsp><nbsp> | ||
| − | <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: | + | <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;"> <span style="font-size: 24px"> Quantification</span> of protein concentrations</p> |
<br> | <br> | ||
<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> | <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> | ||
| Line 105: | Line 105: | ||
<hr> | <hr> | ||
<nbsp><nbsp> | <nbsp><nbsp> | ||
| − | <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: | + | <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;"> <span style="font-size: 24px"> Colorimetric</span> enzyme activity<br> Add 250 μl saturated CO2 solution</p> |
<br> | <br> | ||
<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> | <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> | ||
<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> | <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> | ||
| − | <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: | + | <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;"> 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> |
Test esterase activity<br> | Test esterase activity<br> | ||
Determination curve without carbonated anhydrase:</p> <br> | Determination curve without carbonated anhydrase:</p> <br> | ||
<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> | <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> | ||
| − | <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: | + | <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;"> Determination curve with carbonated anhydrase（CA2WT）</p> <br> |
<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> | <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> | ||
| − | <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: | + | <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;"> Determination curve with carbonated anhydrase（CA2L203K）</p> <br> |
<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> | <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> | ||
| − | <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: | + | <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;"> Wild carbonic anhydrase activity calculated as 0.5098 mol/g × min<br> |
The mutated carbonic anhydrase enzyme activity was calculated to be 1.0392 mol/g * min. | The mutated carbonic anhydrase enzyme activity was calculated to be 1.0392 mol/g * min. | ||
</p> <br> | </p> <br> | ||
Revision as of 05:17, 13 October 2018
Notebook
8.29
Pick Clon
8.30
Pilot Expression of Protein
“IPTG+ 4h”“IPTG- 4h”
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.
8.31
Protein extraction
CA2L203K
CA2WT
CA2L203K
9.01
Purification of Protein
Prior to purification
Purified
9.03
Western blot identification of proteins
CA2L203K CA2WT
9.04
Quantification of protein concentrations
9.05
Colorimetric enzyme activity
Add 250 μl saturated CO2 solution
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).
Test esterase activity
Determination curve without carbonated anhydrase:
Determination curve with carbonated anhydrase（CA2WT）
Determination curve with carbonated anhydrase（CA2L203K）
Wild carbonic anhydrase activity calculated as 0.5098 mol/g × min
The mutated carbonic anhydrase enzyme activity was calculated to be 1.0392 mol/g * min.
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Anhui University of Technology is located in the national civilized city, Nanjing metropolitan core cities - Ma'anshan City, Anhui Province. Anhui University of Technology is an engineering, metallurgical features, engineering, economics, management, arts, science, law and other disciplines coordinated development of key construction in Anhui multidisciplinary university, with "The Master, Bo" complete the multi-level education training system.
