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Revision as of 08:44, 14 October 2018
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Notebook
8.29
Pick Clon
![](https://static.igem.org/mediawiki/2018/0/02/T--AHUT_China--829.png)
8.30
Pilot Expression of Protein
“IPTG+ 4h”“IPTG- 4h”
![](https://static.igem.org/mediawiki/2018/4/44/T--AHUT_China--830.png)
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
![](https://static.igem.org/mediawiki/2018/2/2d/T--AHUT_China--831.11.png)
![](https://static.igem.org/mediawiki/2018/e/eb/T--AHUT_China--831.12.png)
CA2WT
CA2L203K
![](https://static.igem.org/mediawiki/2018/e/ee/T--AHUT_China--831.2.png)
![](https://static.igem.org/mediawiki/2018/3/3f/T--AHUT_China--831.22.png)
9.01
Purification of Protein
Prior to purification
![](https://static.igem.org/mediawiki/2018/c/cc/T--AHUT_China--91.1.png)
![](https://static.igem.org/mediawiki/2018/7/7b/T--AHUT_China--91.2.png)
Purified
![](https://static.igem.org/mediawiki/2018/8/8a/T--AHUT_China--91.3.png)
![](https://static.igem.org/mediawiki/2018/6/65/T--AHUT_China--91.4.png)
9.03
Western blot identification of proteins
CA2L203K CA2WT
![](https://static.igem.org/mediawiki/2018/6/60/T--AHUT_China--93.1.png)
9.04
Quantification of protein concentrations
![](https://static.igem.org/mediawiki/2018/6/6e/T--AHUT_China--93.2.png)
![](https://static.igem.org/mediawiki/2018/8/8b/T--AHUT_China--93.3.png)
9.05
Colorimetric enzyme activity
Add 250 μl saturated CO2 solution
![](https://static.igem.org/mediawiki/2018/c/c4/T--AHUT_China--95.1.png)
![](https://static.igem.org/mediawiki/2018/8/82/T--AHUT_China--95.2.png)
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:
![](https://static.igem.org/mediawiki/2018/f/f6/T--AHUT_China--95.3.png)
Determination curve with carbonated anhydrase(CA2WT)
![](https://static.igem.org/mediawiki/2018/e/e8/T--AHUT_China--95.4.png)
Determination curve with carbonated anhydrase(CA2L203K)
![](https://static.igem.org/mediawiki/2018/0/0c/T--AHUT_China--95.5.png)
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.