Goal: To construct three types of genetically modified Saccharomyces cerevisiae "BADY", which had the function of antioxidants as well as purine-reduce.
Results: The genetically modified Saccharomyces cerevisiae "BADY" was constructed successfully and worked to secret target protein as we expected. chit42 GM yeast showed a certain antioxidant effect, but the free radical removal effect of SOD1 GM yeast and the purine removal effect of PNP1 GM yeast was not obvious. We analyzed the reasons and proposed solutions, which will be continuously improved in subsequent experiments
We aimed to connect and amplify the purchased fragment(50bp ADE2 insertion site + Promoter + MF alpha + 6 x glycine + Coding Gene ) and vector to construct our plasmid.
1. Amplify of the Interest Fragment
Firstly, amplification and purification of the DNA fragments we want to insert into the backbone (pFA6a vector) and the backbone preparing for the Gibson assembly were successfully done by PCR. The results of these can be seen in the gel (Figure 1 & Figure 2).
Figure 1. Agarose electrophoresis of purchase fragment SOD1, PNP1, and chit42.
Figure 2. Agarose electrophoresis of the vector pFA6a-3HA-kanMX6
2. Plasmid Construction
After the plasmids were constructed by Gibson assembly, they were successfully introduced into TOP10 cells by calcium transformation. Single colonies of TOP10 cells were picked up from the plate containing 50 μg/ mL KanMX media. The colony PCR was done twice to make sure our genes are imported correctly.
Figure 3. Agarose electrophoresis of colony PCR in order to select successfully transformed strain (SOD1 strain)
Figure 4. Agarose electrophoresis of colony PCR in order to select successfully transformed strain (PNP1 strain)
Figure 5. Agarose electrophoresis of colony PCR in order to select successfully transformed strain (chit42 strain)
Then, No. 6 SOD1 colony, No. 8 PNP1 colony, and No. 8 chit42 colony were selected for the second purification.
Figure 6. Agarose electrophoresis of colony PCR in order to select successfully transformed strain (second round)
After that, plasmids from TOP10 cells were extracted and sent to the company to sequence so that every bp of our fragment is correct. It is worth mentioning that by comparing the sequencing results and the structure designed by us, we found that there is a silence mutation in the HA tag of SOD1 plasmids. There was no variation in the rest of the SOD1 plasmids. The plasmids carrying the other two target genes were not mutated.
3. Construction of homologous fragments in yeast
Before the construction of homologous fragments in yeast, the plasmids we built before were firstly extracted from the Top 10 cells. Then, we constructed the insertion fragment using two primers. Among them, the reverse primer was attached with 50bp ADE2 homologous fragment, which enabled it to insert ADE2 site accurately together with the 50bp homologous fragment of the plasmid:
Forward primer: gctgcaggtcgacggatccccATGGATTCTAGAACAGTTGG
Figure 7. Agarose electrophoresis of Homologous fragments in order to detect and purify (SOD1 strain, PNP1 strain)
Figure 8. Agarose electrophoresis of Homologous fragments in order to detect and purify (chit42 strain)
In addition, all the length we labeled in the figures above means the expected length of the PCR product.
We aimed to transform the insertion fragment to the ADE2 site of the Saccharomyces cerevisiae FY4 strain genome.
To transform the insertion fragment to FY4 strain, we used the Lithium Acetate and Heat shock method
After two days of cultivation, the red colonies were picked up from the plate with G418（250ug/ml）and then we did colony PCR (figure 4 and 5) to confirm whether our genes were inserted into the yeast genome or not.
We first successfully constructed a strain containing exocrine SOD1, which will be referred to as "SOD1 Strain" later. After more attempts, we also successfully constructed the strain containing exocrine PNP1 and chit42. The following are the GM Strain and the wild-type strain of GM growing on the YPD plate.
Figure 1. The wild-type strain (left) and SOD1 strain (right).
Figure 2. The wild-type strain (left) and PNP1 strain (right).
Figure 3. The wild-type strain (left) and chit42 strain (right).
It's obvious that the GM yeast showed a red color on the plates.
Figure 4. Agarose electrophoresis of Yeast Colony PCR in order to detect (chit42 strain and PNP1 strain)
Figure 5. Agarose electrophoresis of Yeast Colony PCR in order to detect (SOD1 strain)
To ensure that our target protein is both expressed and successfully secreted out of the cell, we extracted protein from GM yeast cells and their corresponding yeast-free medium using the TCA method for Western Blot to verify their existence. The following are the results of Western blot analysis of extracted proteins.
Figure 1. SDS-PAGE electrophoresis of the Intracellular protein extract and growth medium protein extract of genomic modified SOD1 strain and wild type strain. 1. Intracellular protein extract of SOD1 strain. 2. Intracellular protein extract of the wild-type strain. 3. growth medium protein extract. 3. medium protein extract of SOD1 strain. 4. growth medium protein extract of the wild-type strain.
Figure 2. SDS-PAGE electrophoresis of the Intracellular protein extract and growth medium protein extract of genomic modified PNP1, chit42 strain (with 8% ethanol to inducing during growth), and the wild-type strain. 1. Intracellular protein extract of PNP1 strain. 2. Intracellular protein extract of chit42 strain. 3. Intracellular protein extract of the wild-type strain. L. protein ladder 4. growth medium protein extract of PNP1. 5. growth medium protein extract of chit42 strain. 6. growth medium protein extract wild type. 7. growth medium protein extract of chit42 strain (the strain without ethanol inducing during growth). 8. positive control
Since we did not detect the protein in the growth medium of chit42 we tried more. Different concentrations of ethanol were added to induce endochitinase 42 secretion during the growth of the chit42 strain.
Figure 3. SDS-PAGE electrophoresis of the growth medium protein extract of genomic modified chit42 strain and wild type strain (with different concentrations of ethanol using to express induce chit42 during growth). 1. protein extract of chit42 strain without ethanol induce 2. protein extract of chit42 strain with 2% ethanol induce 3. protein extract of chit42 strain with 4% ethanol induce 4. protein extract of chit42 strain with 8% ethanol induce. 5. protein extract of the wild-type strain. 6. positive control.
Auto-oxidation Test of Pyrogallol
Overview: Pyrogallol is a 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.
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
Figure 2. pyrogallol function assay of SOD1 strain and the wild-type strain. And we add H2O in the self oxidation group as control.
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.
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.
Figure 8. Pyrogallol assay after changing the medium of culturing yeast from YPD medium to SC medium that added a group of the SC media without yeast was added as control
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.
The DPPH radical scavenging assay is performed by using DPPH assay, (Chen et al. 2014). Prior to the real test, the chit42 yeast and the wild-type strain are incubated in SC medium overnight until the OD reaches 0.5-0.7. Then, 8% ethanol was added to induce the production, and the yeast was incubated under the same circumstances for another 18 hours: 25 degrees and rotating speed at 220 rpm. Before the examination, yeast was centrifuged, the supernatant was filtered to do the DPPH test. Briefly, 50ul samples with the addition of 200 ul 51.54 mg/L DPPH solution in 95% ethanol are labeled as As. Besides, Ao is by mixing 50 ul ethanol with 200ul DPPH solution of the same concentration. Additionally, Ar is made by adding 50 ul samples to 200 ul 95% ethanol. During the test, the three groups are triple-examined by using 96 microwell plates. Each group is incubated at 25 degrees in darkness for 30 minutes. After that, all three groups in the same well plate examined the light absorbance are performed by light spectrophotometry at 517 nm. And the DPPH radical scavenging rate of each sample can be deduced by the equation below:
DPPH radical scavenging rate= (1-(As-Ar)/A0)*100%
Figure 1. DPPH function assay of the chit42 strain with ethanol concentration: 8%
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. The result demonstrates that the TPS1 pomoter_chit42_alpha factor part works, the 5% difference of DPPH scavenging rate indicates chit42 strain produces higher levels of chitooligosaccharide than wild type.
Figure 2. DPPH function assay of the wild-type and chit42 strain induced by 4% ethanol
We also examine the DPPH quenching activity under 4 % ethanol as western blot detects protein deposits when chit42 is induced by 4% ethanol. After induce wild type and chit42 strain with 4% ethanol, the result from figure 2 indicates a higher potency to eliminate radicals compared with chit42 under induction of 8% ethanol, given that DPPH scavenging rate rise by 10% under 4% induction and the rate rises by 5% under 8% ethanol induction. Combining two experiments above, there is a high possibility that the best optimal concentration of ethanol needs to be identified, even the operational concentration of 8% of ethanol according to literature still functions quite well.
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 supernatant of fermentation broth into the reaction mix, then measure the OD at 293nm every two minutes for 30 minutes, the slope of the graph will show the activity.
We add the inosine and developer into the PNP1 assay buffer at room temperature to form a reaction mix. Add the supernatant of 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. Dividing into U.V. transparent plate(96-well). Then measure the absorbance at OD=293nm every two minutes for 30 minutes.
In the assay process, we found the slope is negative instead of positive as we predicted (Figure 1).
Figure 1. PNP activity assay curve
Then we did the standard test, it proves all the regent are effective (Figure 2).
Figure 2. Standard test curve
All the test groups and background groups have a negative slope, and the gap between them is too small. We can’t detect the activity of PNP1, probably because the protein is degraded, the expression in the SC medium is insufficient.