Team:AHUT China/Characterization
Characterization
In 2019, AHUT_China iGEM team has characterized the output of this part in E.coli BL21(DE3) and tested its activity by esterase method which was different from the original method. The result is documented in the experience page and main page of BBa_K2762004.
The sequence of BBa_K2762004 were synthesized and cloned into the expression plasmid pET-30a(+) to obtain the recombinant expression vector. Then the plasmid containing CcaA was introduced into E. coli BL21(DE3) for culture in the medium containing kanamycin, and IPTG was added to induce CcaA expression for 4h. The CcaA Protein was extracted from the bacterial lysate, followed was identified by via SDS-PAGE gel electrophoresis.(Fig.1)
After protein purification, the activity of the enzyme was determined by esterase method different from the original method. As carbonic anhydrase can catalyze the hydrolysis of p-nitrophenyl acetate. Therefore, we intended to use the esterase activity of carbonic anhydrase to catalyze the p-nitrophenyl acetate, and obtain the enzyme activity data by the change of absorbance before and after a certain reaction time. The result indicated that CcaA also showed high enzyme activity. (Fig.2)
