Proof of Concept

Overview

Our project is to design a beer that is antioxidant and with low purine content, which is healthier and has a lower risk to get gout than the beer on the market. We constructed yeast with the genes of our interest, SOD1, PNP1, and chit42. To determine if we have constructed the modified yeast successfully, we performed function assays to confirm it.

Pyrogallol Assay

For the modified yeast with SOD1, what we used to do the assay is pyrogallol, which is a chemical reagent that can autoxidize in the presence of molecular oxygen and can detect the enzyme activity of SOD1 by the inhibition rate of SOD1 to its self-oxidation.

For the modified yeast with SOD1, what we used to do the assay is pyrogallol, which is a chemical reagent that can autoxidize in the presence of molecular oxygen and can detect the enzyme activity of SOD1 by the inhibition rate of SOD1 to its self-oxidation. We used YPD media at the beginning and the result was not as expected. The result of the pyrogallol assay is not greatly different between the medium of the wild-type strain and the SOD1 strain. Then we found that the OD of YPD media is too high at 325 nm. To work it out, we further propose three hypotheses that may influence the experimental results. Firstly, we considered whether the concentration of the protein was too low, so we used the 30kDa tube to collect the protein, but the assay still failed. Then we considered the influence of the YPD media itself. We tested pyrogallol and the absorption peaks at 420 nm wavelength because the oxidation product of Pyrogallol also has a strong absorption peak at 420 nm wavelength, but the absorption peak of YPD is small at this wavelength. According to the result, we found that the concentrated medium still had a certain light absorption value under our experimental wavelength so we changed the media from YPD media to SC media. Unfortunately, the result still has not changed. Thus, we further suspected that the size of the protein was too small for its 3D structure. We used the filtrates of the 30KDa filtration to perform Western Blot to find out if there is the SOD1 in it or if it were filtered in the filtration, if it were filtered, we changed the filtration with 10KDa. The final result of the function assay showed that there was a certain difference between the SOD1 strain and the wild-type strain. Although the difference was not stable, which may be related to the insufficient secretion of SOD1. Due to environmental constraints, we cannot simulate fermentation, and our strain has only been incubated overnight.

DPPH Assay

The DPPH radical scavenging assay is performed by using DPPH assay and then examined the light absorbance is performed by light spectrophotometry at 517 nm. The FY4 strain modified by chit42 containing vectors shows enhanced ability to remove free radicals in solution, as a comparison with the wild-type strain, which demonstrates that the TPS1 pomoter_chit42_alpha factor part works.

PNP1 Activity Assay

PNP1 is an enzyme that could break down the inosine to hypoxanthine, the developer could convert the hypoxanthine into urea acid. Urea acid is measured at a wavelength of OD =293nm. We add the inosine and developer into the PNP1 assay buffer at room temperature to form a reaction mix. Add the supernatant of the wild-type strain and PNP1 strain respectively as the test groups, at the same time, prepare exactly the same reagents except for the developer as the background group. In the assay process, we found the activity is negative instead of positive as we predicted.

Then we do the standard test, which proves all the regents are effective. All the test groups and background groups have a negative slope, and the gap between them is too small. So PNP1 might have no activity during the test.

Reference

Li K, Xing R, Liu S, Li R, Qin Y, Meng X, Li P (2012) Separation of chito-oligomers with several degrees of polymerization and study of their antioxidant activity. Carbohydr Polym 88:896–903

Wang, Z., Bai, X., He, X., & Zhang, B. (2014). Secretion expression of SOD1 and its overlapping function with GSH in brewing yeast strain for better flavor and anti-aging ability. Journal of Industrial Microbiology & Biotechnology, 41(9), 1415-24. doi:http://dx.doi.org.libezproxy.um.edu.mo/10.1007/s10295-014-1481-9