Latest revision as of 09:47, 10 October 2023

XI-2-temA-2

XI-2-temA-2

Composite Part BBa_K4845016 (XI-2-temA-2)

Construction Design

Based on BBa_K4845002 (XI-2-backbone), we added the following parts:

• BBa_K4845004 (GAP promoter)
• BBa_K4000002 (CYC1 terminator)
• BBa_K4845007 (ADH1 terminator)
• BBa_K4845009 (temA)
• BBa_K4000000 (TEF1 promoter)

These parts were added to BBa_K4845016 (XI-2-temA-2). It was constructed to increase two promoters and terminators, two temA genes on the XI-2-backbone plasmid, improve the enzyme activity of glucoamylase, and further improve the ability of yeast to decompose starch.

In order to construct BBa_K4845016 (XI-2-temA-2), we firstly amplified the GAP, TEF1 promoters, temA key genes, and CYC1, ADH1 terminators through PCR (Table 1). Since we would insert two temA genes into the same plasmid, we used two different promoters and two different terminators. With the preparation of basic materials, we linked the promoters, key genes, and terminators together through Over PCR to construct GAP-temA-CYC1 and TEF1-temA-ADH1 genes which will be inserted into the two plasmid skeletons (XI-2) later (Figure 1). What is more, the design of our target genes is displayed both in the form of a table and the form of visualized diagram.

Figure 1: Visualized blocks-assembly diagram for showing our design of temA DNA template

Upper one: GAP-temA-CYC1 gene of 2804 bp

Lower one: TEF1-temA-CYC1 gene of 2482 bp

After we obtained our target temA gene fragments, we inserted them into XI-2 plasmid skeletons through restriction endonuclease digestion and ligation method (Figure 2).

Figure 2: Visualized models of our plasmids designed (XI-2-2temA)

Engineering Principle

In alcoholic fermentation, α-amylase cannot hydrolyze α-1,6 glycosidic bonds. The complete hydrolysis of starch requires the synergistic effect of α-amylase and glucoamylase, but α-amylase is considered to be more important than glucoamylase, because the hydrolysis of starch into oligosaccharides by α-amylase may be the rate-limiting step. Therefore, the glucoamylase was integrated into the plasmid, and the starch α-1,4 glycosidic bond was rapidly hydrolyzed, and the α-1,6 glycosidic bond and α-1,3 glycosidic bond were slowly hydrolyzed, and the final product was all glucose.

Cultivation, Purification and SDS-PAGE

A. DNA sequencing of XI-2-temA 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 (Figure 3 and 4).

Figure 3: DNA sequencing result of XI-2-temA plasmid

Figure 4: DNA sequencing result of XI-2-temA-2 plasmid

B. Transformation testing of temA-containing plasmids through PCR and Gel electrophoresis

The figure 5 shown in figure A leads to the length of temA genes fragment of XI-2 plasmid. We have successfully constructed those plasmids and we are able to confirm that we have successfully transformed our constructed temA-containing plasmid into 1974 yeast cell.

Figure 5: Results of PCR of plasmids extracted from temA-genes-containing 1974 yeast cell

C. Protein expression and purification

Our experiment expected proteins expressed by temA genes to be 68.3 kDa as shown in the figure labeled (Figure 6). From the observation of the result, we found that the proteins satisfied our expectation. This result supports that our experiment and constructed yeast cell successfully functioned from the perspective of the molecular level.

Figure 6: Results of running protein gel electrophoresis to test the function of constructed plasmids

Characterization/Measurement

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 7A, 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 2.14 cm, 2.56 cm, and 2.23 cm around our engineered yeast cell.

Figure 7: Transparent circle experiment for the function testing

Diameter of the transparent circle in A: 2.14 cm

Diameter of the transparent circle in B: 2.56 cm

Diameter of the transparent circle in C: 2.23 cm

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°C, 40°C, 50°C, 60°C, 70°C, and 80°C) to study the enzymatic properties of the recombinant enzyme.

Figure 8: The enzyme activity of Yeast 1974-GA-temA and Wild Yeast 1974 under different pH value at the same temperature

According to Figure 8, we can see that generally, Yeast 1974-GA-temA possesses higher enzyme activity than the wild at 30°C. 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 50°C, and there seem little changes in the enzyme activity responding to the pH values after the temperature equals or is higher than 50°C.

Figure 9: The enzyme activity of Yeast 1974-GA-temA and Wild Yeast 1974 under different temperature at the same pH value

According to Figure 9, when the temperature goes higher, the enzyme activity of both strains decreases basically. Based on the curve trends of Figure 9-A, B, C, E, there is an obvious turning point at 50°C which we have already pointed out previously. But it is worth noting that when the pH value equals 5, both strains show a 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 10: The comparison of the enzyme activity curves of Yeast 1974-GA-temA under different pH values and different temperature, respectively

After we integrated Figure 8 and Figure 9, we can obtain the comparison graphs in Figure 10. 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°C 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 11). 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 11: Alcohol production of yeast 1974 and GA-temA-1974

Reference

1. Innis MA, Holland MJ, McCabe PC, Cole GE, Wittman VP, Tal R, Watt KW, Gelfand DH, Holland JP, Meade JH (1985) Expression, glycosylation, and secretion of an Aspergillus Glucoamylase by Saccharomyces cerevisiae. Science 228(4695):21–26.
2. Ashkari T, Nakamura N, Tanaka Y, et al. Rhizopus Raw-Starch-Degrading Glucoamylase: Its Cloning and Expression in Yeast[J]. Agricultural and Biological Chemistry, 2014, 50(4).
3. Wu Xiaoping, Li Wenqing, Luo Jinxian. Expression of α-amylase and glucoamylase and construction of engineered Saccharomyces cerevisiae. Journal of Sun Yat-sen University (Natural Science Edition), 1999(02): 81-85.

Sequence and Features