New Improved Part: BBa K4845020 (X-2-GA-2)
Existing Part: BBa _ K4000001 (GA, Group: iGEM21_Fujian_United)
We constructed a new recombinant plasmid BBa _ K4845020( X-2-GA-2 ) based on the old part BBa _ K4000001 (GA ). And X-2-GA-2 was transferred into yeast 1974 for protein expression, enzyme activity detection, quantitative and qualitative detection of its ability to decompose starch. It was proved by iodine staining experiment that X-2-GA-2 could decompose starch, and the ability of recombinant Saccharomyces cerevisiae to decompose starch was further characterized by measuring alcohol production.
Compared with the old part BBa _ K4000001 (GA ), the new recombinant plasmid BBa _ K4845020 ( X-2-GA-2 ) has the two main improvements. First, we added two GAs to the backbone X-2 plasmid to improve the ability of decompose starch. At the same time, the optimal expression conditions of the protein GA were explored, and added the test experiments. the starch degradation ability of recombinant X-2-GA-2 in Saccharomyces cerevisiae was characterized by quantitative and qualitative detection methods. Secondly, on the basis of adding α-amylase, the glucoamylase was further integrated into the chromosome of S.cerevisiae.Based on the hydrolysis circle, the starch degradation ability of the above strains was tested. The hydrolase activity of the fermentation supernatant of the obtained strains was tested, and the ability of the strains to ferment raw starch of sweet potato residue to produce alcohol was tested. Finally, S.cerevisiae strains with autocrine α-amylase and glucoamylase were obtained to achieve the goal of saving enzyme dosage.The results showed that the alcohol production capacity of the S.cerevisiae strains with autocrine α-amylase and glucoamylase was indeed higher than that of iGEM21 _ Fujian _ United.
The problems we are going to solve are that the cost of exogenous enzymes during alcohol fermentation process is much too high, and wasted sweet potato residue is harmful to the environment1-3. Therefore, as long as we enable the saccharomyces cerevisiae to self-secrete alpha-amylase and glucoamylase which function to completely hydrolyze starch into glucose molecules through synthetic biology, then we can largely reduce the cost of exogenous enzymes, and we can also put sweet potato residue into use as a raw material of alcoholic fermentation to turn the pollution problems into profits and efficiency smartly. To be more specific, we will transform plasmids BBa _ K4845020 ( X-2-GA-2 ) containing genes that express alpha-amylase and glucoamylase into our yeast 1974(Figure 1)4-5.Therefore, we achieve reducing the cost of addition of exogenous enzymes during alcoholic fermentation.
Figure1: Overview of the methodology of our project design through synthetic biology (made in Canva.cn)
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 2A, 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.
Figure 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 3).
Figure 3: The enzyme activity of Yeast 1974-GA-temA and Wild Yeast 1974 under different pH value at the same temperature
According to Figure 3A, 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 4: The enzyme activity of Yeast 1974-GA-temA and Wild Yeast 1974 under different temperature at the same pH value
According to Figure 4, when the temperature goes higher, the enzyme activity of both strains decreases basically. Based on the curve trends of Figure 4-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 5: The comparison of the enzyme activity curves of Yeast 1974-GA-temA under different pH values and different temperature, respectively
After we integrated Figure 3 and Figure 4, we can obtain the comparison graphs in Figure 5. 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) Determination of alcohol production
To further and directly verify the normal functioning of our constructed yeast, we applied them to produce alcohol in reality, thus obtained this bar chart. Figure shows that the 1974-GA-temA did produce incremental alcohol over time, and it does boost the alcohol production a lot in comparison to the control group yeast 1974(Figure 6). The alcohol production capacity of 2022 iGEM21 _ Fujian _ United was 0.18g / L. The alcohol production of our combined yeast 1974-GA-temA was 0.23 g / L, which proved that the alcohol production capacity of our combined yeast 1974-GA-temA was improved.
Figure 6.Alcohol production of yeast 1974 and GA-temA-1974
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