Overview

We successfully constructed all four plasmids required to meet our aim eventually. To introduce, these four plasmids(X-2-2GA, XII-5-2GA, X-3-2temA，XI-2-2temA) are grouped into two separate rounds of engineering according to their key genes (GA, temA) expressed. To be more specific, GA is the gene expressing glucoamylase, and temA is the gene expressing amylase.X-2-2GA and XII-5-2GA share the same gene sequence with length of 8802bp, but different plasmid backbones (X-2 and XII-5).X-3-2temA and XI-2-2temA share the same gene sequence with length of 9433bp (total plasmid length), but different plasmid backbones (X-3 and XI-2). What is more, 2GA or 2temA indicate the number of the key genes inserted in the plasmid, we inserted two key genes into each plasmid to promote starch decomposing efficiency by expressing more amylases or glucoamylases. Furthermore, the reason why we constructed four plasmids that two of them share the same gene sequence but difference plasmid backbones is that two same plasmid backbones are not able to be transformed into a single cell together since they share the same replicon(ori) (Novick; Austin and Nordstrom; Bouet, Nordstrom and Lane; Diaz, Rech and Bouet; Helinski; Johnson and Nolan; Meacock and Cohen).

Table 1: clear information expressed in the form of chart to ensure comprehension

Plasmid Name	Key genes	Length of plasmid	Enzyme expressed	Round
X-2-(2)GA	GA*2	8802bp	Glucoamylase	1
XII-5-(2)GA	GA*2	8802bp	Glucoamylase	1
X-3-(2)temA	temA*2	9433bp	Amylase	2
XI-2-(2)temA	temA*2	9433bp	Amylase	2
Success?	√	√	√	√

The whole project design does follow the methodology of synthetic biology (Figure 1), and our experiment involved the engineering design cycle（DBTL） comprehensively.

Figure1: A general diagram to showcase the build and test parts of our experiment process

Engineering Design (DBTL) Cycles

1. Engineering Round1 (plasmids with GA key genes)

1) Design: X-2-2GA and XII-5-2GA plasmids

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. 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, XII-5) later. The design of the two gene fragments is listed in both the form of chart and shown in the visualized diagram.

Table 2: clear information in the form of chart to show our target GA gene design

Order of GA template	Promoter gene	Key gene	Terminator gene
GAP-GA-CYC1	GAP	GA	CYC1
TEF1-GA-ADH1	TEF1	GA	ADH1

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 and XII-5 plasmid skeletons through restriction endonuclease digestion and ligation method.

Figure3: Visualized models of our plasmids designed (X-2-2GA, XII-5-2GA)

2) Build: X-2-2GA and XII-5-2GA plasmids

A1. Construction and amplification of X-2-GA plasmid

Figure 4: 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):

From our results derived from gel electrophoresis, all gene fragments are placed on the correct as well as predicted location according to the indication of marker, but the left X-2-GA gene band didn’t appear. What is more, we had prepared another X-2-GA which is the right one shown.

Figure 5: results of all Over PCR outcomes and labelled diagram of GA gene fragment with promoters and terminators in X-2-GA plasmid

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 (Band1, 2) are positioned within the expected marker length range observed from the gel electrophoresis, indicating that we successfully connected our target genes together.

B1. Construction and amplification of XII-5-GA plasmid

Figure 6: PCR results of gene fragments of XII-5 plasmid and labelled diagram of GA gene fragment with promoters and terminators

From our results derived from gel electrophoresis, all gene fragments except the XII-5-GA band 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.

Figure 7: results of all Over PCR outcomes and labelled diagram of GA gene fragment with promoters and terminators in XII-5-GA plasmid

a) 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 (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.

A2. DNA sequencing of X-2-GA plasmid

Figure 8: DNA sequencing result of X-2-GA plasmid

Figure 9: 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.

B2. DNA sequencing of XII-5-GA-2 plasmid

Figure 10: 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.

A. transformation testing of GA-containing plasmids through PCR and Gel electrophoresis

Figure11: Results of PCR of plasmids extracted from GA-genes-containing 1974 yeast cell

The result gel figure 11 D, F showed that our transformation of plasmids containing GA genes is successful.

3) Test: X-2-2GA and XII-5-2GA plasmids

B. SDS-PAGE Protein testing and purification

Figure 12: results of running protein gel electrophoresis to test the function of constructed plasmids

Our experiment expected proteins expressed by GA genes to be 57.4 kDa as shown in the figure labelled. From the observation of the result, we found that GA-expressing proteins did satisfy our expectation. This result supports that our experiment and constructed GA-containing plasmids successfully functioned from the perspective of molecular level.

A. Method of Transparent Circle—Functionally Testing the whole 1974 yeast cell with 4 constructed plasmids

Figure 13: 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

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.

B. Direct Test and application of constructed yeast functioning

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.

4) Learn from Round1

From the experiment, we do believe that we achieved engineering success and our GA plasmids constructed are successful and functioning as expected as well. What is more, we failed to amplify the GA genes twice for both X-2-GA and XII-5-GA in PCR, we thought it might be due to incomplete extraction of template which leads to low concentration of target genes processed by PCR. Also, we should practice more on adding agents into the gel. Still, we can increase the sample size of test of the function of our constructed yeast

2. Engineering Round2 (plasmids with temA key genes)

1)Design: X-3-2temA and XI-2-2temA plasmids

In order to construct temA-containing plasmid, we firstly amplified the GAP, TEF1 promoters, temA key genes and CYC1, ADH1 terminators through PCR. Since we would insert two temA genes into the same plasmid, we used two different promoters and two different terminators. With 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 (X-3, XI-2) later. What is more, the design of our target genes is displayed both in the form of table and the form of visualized diagram.

Table 4: clear information in the form of chart to show our target temA gene design

Order of temA template	Promoter gene	Key gene	Terminator gene
GAP-temA-CYC1	GAP	temA	CYC1
TEF1-temA-ADH1	TEF1	temA	ADH1

Figure14: 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 X-3 and XI-2 plasmid skeletons through restriction endonuclease digestion and ligation method.

Figure15: Visualized models of our plasmids designed (X-3-2temA, XI-2-2temA)

2)Build: XI-2-2temA and X-3-2temA plasmids

A1. Construction and amplification of XI-2-temA plasmid

Figure 16: 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, 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.

Figure 17: 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 (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.

B1. Construction and amplification of X-3-temA plasmid

Figure 18: 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)

According to the results derived from gel electrophoresis, except Band four and six, 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, and its position corresponded to our expectation as well, which is very close to the 200bp indicated by the marker.

Figure 19: results of all Over PCR outcomes and labelled diagram of temA gene fragment with promoters and terminators in X-3-temA plasmid

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) 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.

A2. DNA sequencing of XI-2-temA plasmid

Figure 20: DNA sequencing result of XI-2-temA plasmid

Figure 21: DNA sequencing result of XI-2-temA-2 plasmid

According to the sequencing diagram shown, our XI-2-temA plasmid is constructed successfully.

B2. DNA sequencing of X-3-temA plasmid

Figure 22: DNA sequencing result of X-3-temA plasmid

Figure 23: DNA sequencing result of X-3-temA-2 plasmid

According to the sequencing diagram shown, our X-3-temA plasmid is constructed successfully.

C. transformation testing of temA-containing plasmids through PCR and Gel electrophoresis

Figure24: Results of PCR of plasmids extracted from temA-genes-containing 1974 yeast cell

The result shown in figure A 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. We have successfully constructed those plasmids and we are able to confirm that we have successfully transform our constructed temA-containing plasmid into 1974 yeast cell.

3 ) Test: X-3-2temA and XI-2-2temA plasmids

D. SDS-PAGE Protein testing and purification

Figure 25: results of running protein gel electrophoresis to test the function of constructed plasmids

Our experiment expected proteins expressed by temA genes to be 68.3 kDa as shown in the figure labelled. From the observation of the result, we found that temA-expressing proteins satisfied our expectation. This result supports that our experiment and constructed temA-containing plasmids successfully functioned from the perspective of molecular level.

E. Method of Transparent Circle—Functionally Testing the whole 1974 yeast cell with 4 constructed plasmids

Figure 26: 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

F. Direct Test and application of constructed yeast functioning

Application of 1974-GA-temA and alcohol production statistics

4 ) Learn from Round2

After the experiment, we had a discussion with our instructor and we learnt that for transparent circle experiment, we should add a control group to show the difference between starch-distributing-culture dish with our constructed yeast and that without our constructed yeast.

Reference

Austin, S., and K. Nordstrom. "Partition-Mediated Incompatibility of Bacterial Plasmids." Cell 60.3 (1990): 351-4. Print.

Bouet, J. Y., K. Nordstrom, and D. Lane. "Plasmid Partition and Incompatibility--the Focus Shifts." Mol Microbiol 65.6 (2007): 1405-14. Print.

Diaz, R., J. Rech, and J. Y. Bouet. "Imaging Centromere-Based Incompatibilities: Insights into the Mechanism of Incompatibility Mediated by Low-Copy Number Plasmids." Plasmid 80 (2015): 54-62. Print.

Helinski, D. R. "A Brief History of Plasmids." EcoSal Plus 10.1 (2022): eESP00282021. Print.

Johnson, T. J., and L. K. Nolan. "Plasmid Replicon Typing." Methods Mol Biol 551 (2009): 27-35. Print.

Meacock, P. A., and S. N. Cohen. "Genetic Analysis of the Inter-Relationship between Plasmid Replication and Incompatibility." Mol Gen Genet 174.2 (1979): 135-47. Print.

Novick, R. P. "Plasmid Incompatibility." Microbiol Rev 51.4 (1987): 381-95. Print.