Part:BBa_K4845020
X-2-GA-2
X-2-GA-2
Summary
We constructed this new recombinant plasmid BBa _ K4845020( X-2-GA-2 ) based on the old part BBa _ K4000001 (GA ) which was created by group iGEM21 _ Fujian _ United. And the new part 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.
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. The optimal expression conditions of the protein GA were explored. 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.
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 the old part BBa _ K4000001 (GA ).
Usage and Biology
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 environment[1-3]. 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, 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 to 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 (Figure 1). 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 4-5.
Figure 1: Overview of the methodology of our project design through synthetic biology
Construction Design
To construct GA-containing plasmids, we firstly amplified the GAP, TEF1 promoters, GA key gene and CYC1, ADH1 terminators through PCR. Since we would insert two GA gene fragments, we used two different promoters and two different terminators(Table 1). After the preparation of basic materials, we connected the promoters, GA key gene and terminators through Over PCR to construct GAP-GA-CYC1 and TEF1-GA-ADH1 genes which will be inserted into plasmid skeletons (X-2) later. The design of the two gene fragments is listed in both the form of chart and shown in the visualized diagram (Figure 2).
Figure 2: Visualized blocks-assembly diagram for showing our design of GA DNA template
Upper one: GAP-GA-CYC1 gene of 2485bp
Lower one: TEF1-GA-ADH1 gene of 2160bp
After we obtained our two target GA gene fragments, we inserted them into the X-2 plasmid skeletons through restriction endonuclease digestion and ligation method (Figure 3).
Figure 3: Visualized models of our plasmids designed (X-2-2GA)
Cultivation, Purification, and SDS-PAGE
A. Construction and amplification of X-2-GA plasmid
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 4). 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.
Figure 4: PCR results of gene fragments of X-2 plasmid and labelled diagram of GA gene fragment with promoters and terminators
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 5 Band1, 2) are positioned within the expected marker length range observed from the gel electrophoresis, indicating that we successfully connected our target genes together.
Figure 5: results of all Over PCR outcomes and labelled diagram of GA gene fragment with promoters and terminators in X-2-GA plasmid
We picked our monoclonal antibodies and sent them to the 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 stronger ability our constructed yeast cells have to decompose starch, and efficiency is exactly what we want, so we also involve DNA sequencing of X-2 plasmid with 2 GA genes (Figure 6 and 7).
Figure 6: DNA sequencing result of X-2-GA plasmid
Figure 7: DNA sequencing result of X-2-GA-2 plasmid
B. Test for plasmid transformation
The result gel figure 8 D, F shown in the figure both indicate the length of GA genes, and our expected length of GA genes is around 2200bp just as the length derived from Over PCR of GA genes connecting to its promoter and terminator, which showed that our transformation of plasmids containing GA genes is successful.
Figure 8: Results of PCR of plasmids extracted from GA-genes-containing 1974 yeast cell
C. Protein expression and purification
Our experiment expected proteins expressed by GA genes to be 57.4 kDa as shown in the figure 9 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.
Figure 9: 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 10 A, B, C, our constructed yeast cell did function to turn starch into alcohol, giving the phenomenon that there are transparent circles with respective diameters of 2.14cm, 2.56cm, and 2.23cm around our engineered yeast cell.
Figure 10: 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 11).
Figure 11: The enzyme activity of Yeast 1974-GA-temA and Wild Yeast 1974 under different pH value at the same temperature
According to Figure 11 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 12: The enzyme activity of Yeast 1974-GA-temA and Wild Yeast 1974 under different temperature at the same pH value
According to Figure 12, when the temperature goes higher, the enzyme activity of both strains decreases basically. Based on the curve trends of Figure 12-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 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 13: The comparison of the enzyme activity curves of Yeast 1974-GA-temA under different pH values and different temperature, respectively
After we integrated Figure 11 and Figure 12, we can obtain the comparison graphs in Figure 13. 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 14). 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 14: Alcohol production of yeast 1974 and GA-temA-1974
Reference
[1] Bušić Arijana, Marđetko Nenad, Kundas Semjon, et al. Bioethanol Production from Renewable Raw Materials and Its Separation and Purification: A Review[J]. Food technology and biotechnology, 2018, 56(3): 289-311.
[2] Xu Shuai, Yang Li, Tan Liping, et al. Enzymatic hydrolysis and application of sweet potato residue [ J ]. Journal of Qilu University of Technology. 2021, 35(03): 28-33.
[3] Xin Wang, Bei Liao, Zhijun Li, et al. Reducing glucoamylase usage for commercial-scale ethanol production from starch using glucoamylase expressing Saccharomyces cerevisiae[J]. Bioresources and Bioprocessing, 2021, 8(1): 20.
[4] Rosemary A. Cripwell, Lorenzo Favaro, Marinda Viljoen-Bloom, et al. Consolidated bioprocessing of raw starch to ethanol by Saccharomyces cerevisiae: Achievements and challenges[J]. Biotechnology Advances, 2020, 42: 107579.
[5] LM de Moraes, S Astolfi-Filho, SG Oliver. Development of yeast strains for the efficient utilization of starch: evaluation of constructs that express alpha-amylase and glucoamylase separately or as bifunctional fusion proteins[J]. Applied microbiology and biotechnology, 1995, 43(6): 1067-76.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 3841
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 4858
Illegal BglII site found at 7280
Illegal XhoI site found at 4073 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 4819
Illegal AgeI site found at 6190
Illegal AgeI site found at 6478
Illegal AgeI site found at 7241
Illegal AgeI site found at 8612 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2836
Illegal BsaI.rc site found at 317
Illegal BsaI.rc site found at 4482
Illegal SapI site found at 469
Illegal SapI site found at 4296
Illegal SapI.rc site found at 1814
Illegal SapI.rc site found at 4719
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