Part:BBa_K3789011
SOD enzyme secretion system
This composite part consists of pGK1 promoter, Mating factor alpha prepro-leader (MF alpha), 6 x glycine, SOD1 coding sequence, and ADH1 terminator. The product of this system is a secretable superoxide dismutase 1 enzyme(SOD) together with a HA tag. MF alpha enables the enzyme to be secreted. The 6 glycines between MF alpha and SOD1 coding sequence avoid the interruption between them, enabling them to form correct spatial structure.
Auto-oxidation Test of Pyrogallol:
Overview: Pyrogallol is the chemical reagent that can autoxidize in the presence of molecular oxygen as it serves both as the source and as a scavenger of oxygen-free radicals. It is often used to detect the enzyme activity of SOD1 by the inhibition rate of SOD1 to its self-oxidation. In our project, we also used pyrogallol to evaluate the activity of SOD1 that our GM yeast produced.
At first, we cultured both the SOD1 strain and the wild-type strain using the YPD medium overnight. Then, the YPD medium was centrifuged, and the supernatant was extracted and filtered to obtain the yeast-free medium. To detect SOD1 activity in this medium, we used the pyrogallol assay. Under the condition of the alkaline solution containing EDTA, we mixed pyrogallol and water, YPD medium, YPD Media grown by SOD1, and YPD media grown by the wild-type yeast, respectively. At the same time, we also made a mixture without pyrogallol as blank. After mixing, they were quickly divided into 96-well plates. Each tube of the sample is added to three Wells. During the following half-hour, we measured the absorbance value of the solution at the wavelength of 325nm every minute until 30 minutes.
However, We found that the results of the Pyrogallol assay showed that the medium of the wild-type strain and SOD1 strain does not differ greatly in inhibiting Pyrogallol's autoxidation. According to this result, we did mainly three improvements:
1. Using a size of 30kDa centrifugal filter devices to concentrate our medium.
2. Change the medium of culturing yeast from YPD medium to SC medium.
3. Using a size of 10kDa centrifugal filter devices to concentrate our medium.
Preliminary Experiment
It is worth mentioning that before we did the Pyrogallol function assay of the medium of strain, we first used a known antioxidant vitamin C as a positive control to test the effectiveness of pyrogallol.
According to this result, ascorbic acid concentration above 0.1mg/ mL can effectively inhibit pyrogallol autoxidation. Therefore, our Pyrogallol function assay can be proved to be meaningful.
Pyrogallol Function Assay Result:
According to this result, we found that the medium of SOD1 and the wild-type strain both had a strong effect on pyrogallol self oxidation, but there was not much difference between SOD1 and the wild-type. In order to further verify this result, we decide to change H2O into YPD medium in the self oxidation group to determine whether YPD medium itself has an impact on Pyrogallol auto-oxidation.
Figure 3 shows the result of the pyrogallol assay at wavelength 325 nm after changing H2O into YPD medium in the self oxidation group. We found that the slope difference between the Wild Type group, the SOD1 group, and the self oxidation group was very small, which in fact could not effectively prove that the SOD1 group had a better inhibitory effect on Pyrogallol autoxidation.
In order to improve the Function assay, we used the 30kDa centrifugal filter devices to filter our medium (the size of SOD1 is 29.8kda) in the following experiment. The benefits of doing this are mainly in two aspects: 1. The concentration medium increases the concentration of our target protein. 2. Filter out some small miscellaneous proteins to reduce the influence of background.
Figures 4 and 5 are the results of the Pyrogallol assay after the YPD medium was filtered by the 30 kDa centrifuge. In particular, we also tested pyrogallol at 420 nm wavelength, because the oxidation product pyrogallol-quinone of Pyrogallol also has a strong absorption peak at 420 nm wavelength. At this wavelength, the absorption peak of the YPD medium is smaller, which can better exclude the influence of the YPD medium on the experiment itself. However, the slopes of the SOD1 strain and the wild-type strain were still almost the same.
Considering the high absorption peak of Yeast Extract-Peptone-Dextrose (YPD) medium in solution and the inclusion of yeast extract in YPD media. We decided to replace the YPD medium with the synthetic complete (SC) medium to culture our strain. The reason we choose to use the SC medium is that, firstly, the absorption peaks of the SC medium were much smaller in the 325 nm wavelength used in our pyrogallol assay. Secondly, the components of SC Media are all known. Compared with YPD containing yeast extract, it may have less background influence on the experiment itself. The below figures show the composition difference between SC medium and YPD medium.
Preliminary Experiment
Before we officially started the second round of function assay, we did two things to ensure the necessity and validity of replacing with the SC medium.
Firstly, the absorption peaks of YPD and SC medium at 325 nm and 420 nm were compared and measured. According to this result, we can know that the problem of background absorption peaks is basically solved well.
Secondly, we detected and compared the difference of growth rate between Wild Type Strain and Genetic Modified Strain in the original YPD media and the newly replaced SC Media.
By comparison, we found that the YPD medium and SC medium had little influence on the growth speed of the wild-type strain. However, for GM Strain, their growth rate in SC media is much less than that in the YPD medium.
This pre-experiment provided convenience for our subsequent Function assay because according to this growth curve, we roughly inferred in the following Function assay how long we should culture our Strain in advance to make it reach a better state.
According to the result above, we found that the concentrated medium still had a certain absorption value under our experimental wavelength. And considering that the 30 kDa filter we used before may not be able to concentrate our protein effectively because the size of our protein is 29.8 kDa and the 3D structure formed by it, we speculated that it might leak out from the gap of the filter. So we used the new 10kDa centrifugal filters to concentrate the strain medium.
Figure 9 and Figure 10: Pyrogallol assay after changing the medium of culturing yeast from YPD medium to SC medium and use a 10kDa filter to concentrate the medium. overall result: Combined with the results of our six or seven function assays, we did not successfully prove the efficacy of SOD1. Although there was a certain difference between the SOD1 strain and the wild-type strain in the results of the function assay, we found that the difference was not stable after repeated experiments, and it was considered as an error of the experiment itself. As for the reason for this result, we speculate that it may be related to the insufficient secretion of SOD1. Due to the limitation of time and experimental environment, we have no way to expand the cultivation of yeast and the scale and time. For future experiments, we plan to enhance the simulation of yeast fermentation conditions to some extent and hope that it will increase the antioxidant capacity of the SOD1 strain.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1023
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1023
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1802
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1023
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1023
Illegal NgoMIV site found at 1646 - 1000COMPATIBLE WITH RFC[1000]
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