A.Part contributions

During traditional alcohol fermentation process, exogenous enzymes are required to be added, however, the cost of exogenous enzymes is much too high. It was found that the cost contribution of exogenous enzymes to ethanol produced during the conversion of corn starch was $0.68/gal under the condition that the sugars in the biomass could be converted at maximum theoretical yields. Considerably efforts are in need compulsorily to reduce the cost of exogenous enzymes.

We did overall research on enzymatic hydrolysis method for ethanol fermentation, and it is reported that the condition of enzymatic hydrolysis of SPRs is milder, more efficient and suitable to industrial application, its experiments also indicate that the best method to hydrolyze SPRs is the multienzyme function. Therefore, we identified our two target enzymes; amylase and glucoamylase combination which were proved to work efficiently.

Table 1. Part contributions

Part number	Part name	Contribution type
BBa_K4000000	TEF1 promoter	New Information to an existing Part
BBa_K4000001	GA	New Information to an existing Part
BBa_K4845009	temA	New part
BBa_K4845018	X-2-GA	New part
BBa_K4845019	XII-5-GA	New part
BBa_K4845010	X-2-GA	New part
BBa_K4845011	XII-5-GA	New part
BBa_K4845020	X-2-GA-2	New part
BBa_K4845021	XII-5-GA-2	New part
BBa_K4845012	XII-2-temA	New part
BBa_K4845013	X-3-temA	New part
BBa_K4845014	X-2-GA-2	New part
BBa_K4845015	XII-5-GA-2	New part
BBa_K4845016	XII-2-temA-2	New part
BBa_K4845017	X-3-temA-2	New part

1. Add New Information to an existing Part

A. GA(BBa_K4000001 and BBa_K4000000(TEF1 promoter))

- 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 fragment 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 four gene fragments mentioned above through PCR and observed the outcome through Gel Electrophoresis.

labelled diagram of our 4 constructed plasmids

- DNA sequencing of X-2-GA plasmid

DNA sequencing result of X-2-GA plasmid

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.

- 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.

results of running protein gel electrophoresis to test the function of constructed plasmids

- Improvement: Function Test and Enzyme activity detection

A) Method of Transparent Circle

According to the property of starch that turning 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 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.

Figure 5: 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) Activity detection of recombinase

Figure 6 The results of the detection of enzyme activity.

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. We can observe that the enzyme activity of the recombinant enzyme is highest at pH 5 and 30 ° C.

2. Create New Parts

2.1 temA (BBa_K4845009), XII-2-temA-2(BBa_K4845016) and X-3-temA (BBa_K4845013)

A. Construction and amplification of XI-2-temA plasmid

Figure 7: results of all Over PCR outcomes and labelled diagram of temA gene fragment with promoters and terminators in XI-2-temA plasmid

a) temA is the gene expressing amylase, 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. Both (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.

Construction and amplification of plasmid

Figure 8: 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

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. Both (Band7,8 in the figure) 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.

2.2 XII-5-GA(BBa_K4845019) and X-2-GA (X-2-GA (BBa_K4845018)

B.Construction and amplification of X-2-GA plasmid

: 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

GA is the gene expressing glucoamylase, 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. Both (Figure 9 Band1, 2) are positioned within the expected marker length range observed from the gel electrophoresis, indicating that we successfully connected our target genes together.

C.Construction and amplification of XII-5-GA plasmid

Figure 10: 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

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. Both (Figure 10 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.