Part:BBa_K2762004:Experience

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Contribution from iGEM2018 AHUT_China

yu Zhang

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 form the original method. The result was documented in the experience page and the main page of BBa_K2762004.

The sequence of BBa_K2762004 was synthesized and cloned it 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 culturing in the medium containing kanamycin, and IPTG was added to induce CcaA expression for 4h. The CcaA Protein was extracted from the bacterial lysates followed by identification via SDS-PAGE gel electrophoresis.(Fig. 1)

Fig. 1 SDS-PAGE analysis of CcaA protein extracted from lysates of expressed in E.coli BL21(DE3) strain

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 showed that CcaA also showed high enzyme activity. (Fig. 2)

Fig 2 The enzyme activity of CcaA analyzed by esterase method