Method: Colorimetric Uricase and Uric Acid Assay Kit
Since we are measuring the uricase activity and the concentration of uric acid, a commercial uricase assay kit from Abcam (ab65344)is used. In the assay, uricase activity can convert uric acid to allantonin and H2O2. Under the catalyzation of horseradish peroxidase (HRP), the H2O2 produced will react with a fluorescence probe to give colorimetric signal. A red color would be observed if there is H2O2. The absorbance can then be read by plate reader at 570 nm.
Two standard curves were plotted to covert the absorbance reading to the concentration of uricase and uric acid later. The equations of the curves were obtained, with R square nearly equals to 1.
Result:BBa_K3058000 expressed Functional Uricase
Uricase assay was used to determine the uricase activity of our biobrick BBa_K3058000. Briefly, BBa_K3058000 DH5a colonies were picked to grow in 5ml LB solution overnight. The starter cultures were inoculated into 200ml LB and allowed to grow for 6 hours. When the OD600 reached to 0.9, cells were harvested by centrifugation. The total protein was extracted by freeze-thaw method and finally diluted into 1XPBS . Uricase assay was then performed according to the manufacturer's protocol. The absorbance of uricase and controls were being monitored at regular time intervals. Compare with the control, our BBa_K3058000-expressing E.coli could give a bright red color in the assay, indicating a strong uricase activity. The absorbance was qualified. According to student t’s test, the difference between the control and our sample is significant, with p-value smaller than 0.05. According to the standard curve plotted, the concentration of uricase is about 25uM/mL on average. This assay proved that this biobrick worked as expected.
Result: BBa_K3058001 expressed HIU
Uricase assay was also used to determine the effect of HIU hydrolase expressed by BBa_K3058001. Using the same steps above, BBa_K3058001 colonies were cultured and harvested by centrifugation. The total protein was extracted and Uricase assay was then performed. The absorbance of uricase and controls were being monitored at regular time intervals. The result revealed that HIU hydrolase does not have significant result on the uricase activity.