Our project tends to enable Saccharomyces cerevisiae to self-secrete alpha-amylase and saccharifying enzyme by inserting four integrated plasmids X-2-GA, XII-5-GA, XI-2-temA and X-3-temA constructed by our group. We picked and extracted two genes expressing to produce alpha-amylase and saccharifying enzyme from bacteria cultured, and we then integrated these genes with plasmid backbones through restriction-ligation method. After obtaining the constructed plasmids, we transformed them into saccharomyces cerevisiae, enabling it to achieve self-secreting enzymes mentioned above. Therefore, we achieve reducing the cost of addition of exogenous enzymes during alcoholic fermentation.
Overview Diagram of our experiment made in Canva.cn
- Construction of plasmids-PCR and Over PCR
We designed 4 recombinant plasmids: X-2-GA, XII-5-GA which are the GA gene fragment expression plasmids, XI-2-temA and X-3-temA which are the temA gene expression plasmids. Also, we extracted promoters and terminators which are required to ligate temA and GA genes into the plasmid backbone from CCTCC M94055 and pHcas9 gene fragments, then we amplified the gene fragments mentioned above through PCR. After obtaining the genes amplified, we observed the outcome through Gel Electrophoresis.
labelled diagram of our 4 target constructed plasmids
A. Construction and amplification of X-2-GA plasmid (Group1)
PCR results of gene fragments of X-2 plasmid and labelled diagram of GA gene fragment with promoters and terminators
a) We previously identified the length of target genes as shown below (from left to right):
X-2-GAP: 690bp
X-2-CYC1t: 278bp
X-2-TEF1: 429bp
X-2-GA: 1579bp (fail)
X-2-GA: 1580bp
X-2-ADH1: 216bp
From our results derived from gel electrophoresis, all gene fragments are correct as well as predicted location according to the indication of marker, but the left X-2-GA gene band didn’t appear which indicate that PCR amplification of it failed (Figure 3). What is more, we had prepared another X-2-GA which is the right one shown. The mere difference between the left X-2-GA and the right one is that the primers used in PCR system are different, GAPp-GA-R1, CYC1t-GA-F1 for the left one, and ADH1t-GA-R1, TEF1p-GA-F1 for the right X-2-GA. Obviously, the second X-2-GA successfully displayed in the gel.
: results of all Over PCR outcomes and labelled diagram of GA gene fragment with promoters and terminators in X-2-GA plasmid：
1:X-2-GAP-GA-CYC1 2:X-2-TEF-GA-ADH1
3:XII-5-GAP-GA-CYC1 4:XII-5-TEF-GA-ADH1
5.XI-2-GAP-temA-CYC1 6.XI-2-TEF-temA-ADH1
7.X-3-GAP-temA-CYC1 8.X-3-TEF-temA-ADH1
b) For the Over PCR of X-2-GA plasmid, we connected X-2-GAP, X-2-GA and X-2-CYC1t together to make X-2-GAP-GA-CYC1 as shown in the labelled figure, and we connected X-2-TEF1, X-2-GA and X-2-ADH1 together to make X-2-TEF-GA-ADH1. The total length of X-2-GAP-GA-CYC1 we expected should be 2485bp, and that of X-2-TEF-GA-ADH1 should be 2160bp, and both (Figure 4 Band1, 2) are positioned within the expected marker length range observed from the gel electrophoresis, indicating that we successfully connected our target genes together.
B. Construction and amplification of XII-5-GA plasmid (Group 2)
PCR results of gene fragments of XII-5 plasmid and labelled diagram of GA gene fragment with promoters and terminators
a) We previously identified the length of target genes as shown below (from left to right):
XII-5-ADH1: 216bp
XII-5-CYC1: 278bp
XII-5-GA: 1580bp
XII-5-GA: 1579bp
XII-5-TEF1: 429bp
XII-5-GA: 1579bp (fail)
XII-5-GAP: 690bp
From our results derived from gel electrophoresis, all gene fragments except the XII-5-GA band in figure 5 are placed on the correct as well as predicted location according to the indication of marker, but the sixth XII-5-GA gene band didn’t show clearly which indicated that PCR amplification of it failed. The mere difference between the third XII-5-GA and the fourth one is that the primers used in PCR system are different, GAPp-GA-R1, CYC1t-GA-F1 for the fourth one, and ADH1t-GA-R1, TEF1p-GA-F1 for the third X-2-GA.
results of all Over PCR outcomes and labelled diagram of GA gene fragment with promoters and terminators in XII-5-GA plasmid
1:X-2-GAP-GA-CYC1 2:X-2-TEF-GA-ADH1
3:XII-5-GAP-GA-CYC1 4:XII-5-TEF-GA-ADH1
5.XI-2-GAP-temA-CYC1 6.XI-2-TEF-temA-ADH1
7.X-3-GAP-temA-CYC1 8.X-3-TEF-temA-ADH1
b) For the Over PCR of XII-5-GA plasmid, we connected XII-5-GAP, XII-5-GA and XII-5-CYC1t together to make XII-5-GAP-GA-CYC1 as shown in the labelled figure, and we connected XII-5-TEF1, XII-5-GA and XII-5-ADH1 together to make XII-5-TEF-GA-ADH1. The total length of XII-5-GAP-GA-CYC1 we expected should be 2485bp, and that of XII-5-TEF-GA-ADH1 should be 2160bp, and both (Figure 6 Band 3,4) are positioned within the expected marker length range according to the results observed from the gel electrophoresis, indicating that we successfully connected our target genes together.
C. Construction and amplification of XI-2-temA plasmid (Group 3)
PCR results of gene fragments of XI-2 plasmid
a ）We previously identified the length of target genes as shown below (from left to right):
1. XI-2-CYC1: 273bp
2. XI-2-ADH1: 214bp
3.4. XI-2-TEF: 423bp
5.6. XI-2-TEF: 423bp (fail)
7.8. XI-2-temA: 1888bp
9.10. XI-2-GAP: 278bp
Our results derived from gel electrophoresis show that except the fifth and the sixth bands in figure 7, all other results correspond to our previous identification stated above, placing within correct length range according to the indication of markers, which means our PCR amplification is successful. XI-2-TEF in Group 3,4 and group 5,6 are basically the samples with the same PCR system, and Band5,6 didn’t appear which implied the PCR amplification of XI-2-TEF failed.
results of all Over PCR outcomes and labelled diagram of temA gene fragment with promoters and terminators in XI-2-temA plasmid
b ) For the Over PCR of XI-2-temA plasmid, we connected XI-2-GAP, XI-2-temA and XI-2-CYC1 together to make XI-2-GAP-temA-CYC1 as shown in the labelled figure, and we connected XI-2-TEF1, XI-2-temA and XI-2-ADH1 together to make XI-2-TEF-temA-ADH1. The total length of XI-2-GAP-temA-CYC1 we expected should be 2804bp, and that of XI-2-TEF-temA-ADH1 should be 2160bp, and both (Figure 8 Band 5,6) are positioned within the expected marker length range according to the results observed from the gel electrophoresis, indicating that we successfully connected our target genes together.
D. Construction and amplification of X-3-temA plasmid (Group 4)
PCR Results of gene fragments within X-3-temA plasmid
a) We previously identified the length of target genes as shown below (from left to right):
1. X-3-CYC1: 273bp
2. X-3-GAP: 278bp
3.4. X-3-TEF: 423bp (4. fail)
5. X-3-temA: 1886bp
6. X-3-ADH1: 214bp (6. fail)
PCR Results of X-3-ADH1 genes specifically
According to the results derived from gel electrophoresis, except Band four and six in (Fail in the stage of PCR gene amplification), other bands of target genes are placed within the correct length range according to the indication of marker, and Band 3 and 4 shared the same sample X-3-TEF to make sure we got at least a successful PCR amplification result. Moreover, since the band 6 X-3-ADH1 didn’t show any DNA stripe, we ran another time of gel electrophoresis (Figure 10), and its position corresponded to our expectation as well, which is very close to the 200bp indicated by the marker.
results of all Over PCR outcomes and labelled diagram of temA gene fragment with promoters and terminators in X-3-temA plasmid
1:X-2-GAP-GA-CYC1 2:X-2-TEF-GA-ADH1
3:XII-5-GAP-GA-CYC1 4:XII-5-TEF-GA-ADH1
5.XI-2-GAP-temA-CYC1 6.XI-2-TEF-temA-ADH1
7.X-3-GAP-temA-CYC1 8.X-3-TEF-temA-ADH1
b) For the Over PCR of X-3-temA plasmid, we connected X-3-GAP, X-3-temA and X-3-CYC1 together to make X-3-GAP-temA-CYC1 as shown in the labelled figure, and we connected X-3-TEF, X-3-temA and X-3-ADH1 together to make X-3-TEF-temA-ADH1. The total length of X-3-GAP-temA-CYC1 we expected should be 2804bp, and that of X-3-TEF-temA-ADH1 should be 2160bp, and both (Band7,8 in the figure 11) are positioned within the expected marker length range according to the results observed from the gel electrophoresis, indicating that we successfully connected our target genes together.
- DNA sequencing
We picked our monoclonal antibodies and sent them to biotechnology company for DNA sequencing, the final results indicated that there were not genetic mutations on our genes, which meant our plasmids were constructed successfully. There are slight differences within a plasmid, for instance, X-2-GA and X-2-GA-2, the difference is that the second one has one more GA gene, and basically the more GA genes, the more stronger ability our constructed yeast cells have to decompose starch, and efficiency is exactly what we what, so we also involve DNA sequencing of X-2plasimd with 2 GA genes.
A. DNA sequencing of X-2-GA plasmid
2: DNA sequencing result of X-2-GA plasmid
3: DNA sequencing result of X-2-GA-2 plasmid
According to the sequencing diagram shown, since there is not much white space appearing in the arrows which are the places where sequencing takes place, it shows that both X-2-GA and X-2-GA-2 plasmids are out of genetic mutations, meaning that our X-2-GA plasmid is constructed successfully.
B. DNA sequencing of XII-5-GA-2 plasmid
4: DNA sequencing result of XII-5-GA-2 plasmid
According to the sequencing diagram shown, almost all the arrows are appearing red except the one located upon the S. fibuligera GA gene in the middle. The DNA sequencing shows that there are no genetic mutation taking place on our constructed XII-5-GA-2 plasmid, meaning that our construction of the plasmid is successful as well.
C. DNA sequencing of XI-2-temA plasmid
5: DNA sequencing result of XI-2-temA plasmid
6: DNA sequencing result of XI-2-temA-2 plasmid
According to the sequencing diagram shown, since there is not much white space appearing in the arrows which are the places where sequencing takes place, it shows that both XI-2-temA and XI-2-temA-2 plasmids are out of genetic mutations, meaning that our XI-2-temA plasmid is constructed successfully.
D. DNA sequencing of X-3-temA plasmid
7: DNA sequencing result of X-3-temA plasmid
18: DNA sequencing result of X-3-temA-2 plasmid
According to the sequencing diagram shown, since there is not much white space appearing in the arrows which are the places where sequencing takes place, it shows that both X-3-temA and X-3-temA-2 plasmids are out of genetic mutations, meaning that our X-3-temA plasmid is constructed successfully.
- Test for plasmid transformation
After we transformed our constructed plasmid into our yeast cell called 1974, we were supposed to check whether the plasmids successfully transformed into the yeast cell. Therefore, we extracted the constructed plasmids from breaking some 1974 yeast cells again to test it through PCR and gel electrophoresis. Eventually our results indicated that we successfully transformed our constructed plasmids into the yeast cell.
A. transformation testing of GA-containing plasmids through PCR and Gel electrophoresis
19: Results of PCR of plasmids extracted from GA-genes-containing 1974 yeast cell
The result gel figure D, F shown in the figure both indicate the length of GA genes, and our expected length of GA genes are around 2200bp just as the length derived from Over PCR of GA genes connecting to its promotor and terminator, which showed that our transformation of plasmids containing GA genes is successful.
B. transformation testing of temA-containing plasmids through PCR and Gel electrophoresis
0: Results of PCR of plasmids extracted from temA-genes-containing 1974 yeast cell
The result shown in figure 20A leads to the length of temA genes fragment of X-3 plasmid, and figure B shows the length of temA genes fragment of XI-2 plasmid. For both of the plasmids, our expected results are around 2000bp since we have successfully constructed the those plasmids before, then we are able to confirm that we have successfully transform our constructed temA-containing plasmid into 1974 yeast cell.
- Protein expression and purification
Our experiment expected proteins expressed by temA genes to be 68.3 kDa, and that expressed by GA genes to be 57.4 kDa as shown in the figure labelled. From the observation of the result, we found that both kinds of proteins satisfied our expectation. This result supports that our experiment and constructed yeast cell successfully functioned from the perspective of molecular level.
1: results of running protein gel electrophoresis to test the function of constructed plasmids
- Function Test and Enzyme activity detection
A) Method of Transparent Circle
According to the property of starch that turns blue as it meets iodine solution, we placed our constructed Saccharomyces cerevisiae in the culture dish with starch solution distributed evenly. If our saccharomyces cerevisiae is successfully constructed, there will be alcohol produced around the strain because of our engineered property of self-secreting amylase and glucoamylase which work to decompose starch into glucose molecules, and those glucose molecules will be fermented by our constructed yeast cells 1974. As shown in the figure 22 A, B, C, our constructed yeast cell did function to turn starch into alcohol, giving the phenomenon that there are transparent circles with respectively diameters of 2.14cm, 2.56cm and 2.23cm around our engineered yeast cell.
2: Transparent circle experiment for the function testing
Diameter of the transparent circle in A: 2.14cm
Diameter of the transparent circle in B: 2.56cm
Diameter of the transparent circle in C: 2.23cm
B) Enzyme Activity Detection in Starch Hydrolyzing Capacity
To verify the GA and temA activity, we measured the enzyme activity of the recombinase. The enzyme activity was measured by the glucose content detection kit. Enzyme activity was expressed as U/mL supernatant, and one unit of enzyme activity was defined as the amount of enzyme required to release 1 μmol glucose per minute. The recombinant was incubated at different pH values (3, 4, 5, 6 and 7) and temperature values (30℃, 40℃, 50℃, 60℃, 70℃ and 80℃) to study the enzymatic properties of the recombinant enzyme.
Figure 23: The enzyme activity of Yeast 1974-GA-temA and Wild Yeast 1974 under different pH value at the same temperature
According to Figure 23 A, we can see that generally, Yeast 1974-GA-temA possesses higher enzyme activity than the wild at 30 oC. In addition, we can tell that when the pH value equals 5, both strains reach their highest enzyme activity of both strains where the temperature is lower than 50oC, and there seem little changes in the enzyme activity responding to the pH values after the temperature equals or is higher than 50oC.
Figure 24: The enzyme activity of Yeast 1974-GA-temA and Wild Yeast 1974 under different temperature at the same pH value
According to Figure 24, when the temperature goes higher, the enzyme activity of both strains decreases basically. Based on the curve trends of Figure 24-A,B,C,E, there is an obvious turning point at 50 oC which we have already pointed out previously. But it is worth noting that when the pH value equals 5, both strains show different trend of enzyme activity against temperature and it will require further research. Compared with the wild, Yeast 1974-GA-temA possesses higher enzyme activity when the pH value is higher than 5.
Figure 25: The comparison of the enzyme activity curves of Yeast 1974-GA-temA under different pH values and different temperature, respectively
After we integrated Figure 23 and Figure 24, we can obtain the comparison graphs in Figure 25. To conclude, the enzyme activity of the recombinant enzyme in Yeast 1974-GA-temA is highest at pH 5 and 30 ° C at the given setting. Also based on our data, the proper condition for our recombinant enzyme will be when the pH value range is 4 to 6 and the temperature is 30 oC around where our recombinant yeast possesses better enzyme activity in starch hydrolyzing capacity than the wild.
C) Direct Test and application of constructed yeast functioning
Figure 26. Application of 1974-GA-temA and alcohol production statistics
We subsequently applied our constructed yeast to produce alcohol in reality, and we measured the amount of alcohol produced, making a chart to showcase the result. We had two sample groups: our constructed yeast 1974-GA-temA and the control group ordinary yeast 1974. Our result indicates that: Firstly, our constructed yeast did function successfully to decompose starch into yeast incrementally over time; secondly, our constructed yeast did bolster the alcohol fermentation efficiency in comparison to the control group ordinary yeast 1974 with apparent difference shown in the chart.