iGEM 2021 — Team Fujian_United

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# Team:Fujian United/Notebook

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# Notebook

![](https://static.igem.org/mediawiki/2021/c/c8/T--Fujian_United--img_notebook_1.jpg)

|  |  |
| --- | --- |
| July | |
| 4th | Prepare culture mediums. |
| 5th | Amplify the plasmid fragments by PCR, gel electrophoresis and recycle DNA. ![](https://static.igem.org/mediawiki/2021/d/d6/T--Fujian_United--img_notebook_2.png) |
| 6th | Construction of the 4 plasmids. |
| 7th | Colony PCR, gel electrophoresis, and select single clones. ![](https://static.igem.org/mediawiki/2021/5/57/T--Fujian_United--img_notebook_3.png) |
| 8th | Plasmids extraction from E. Coli, and sent to the DNA sequencing company. |
| 9th | Verify the DNA sequencing resuls and carry out the transformation of plasmids into the Saccharomyces cerevisiae by Heat-Shock approach. |
| 11st | Prepare competent cells of the Saccharomyces cerevisiae, and incubate the yeast single colony in 1ml culture mediums. |
| 12nd | Day off. Due to the limited time of incubation, our Saccharomyces cerevisiae didn't demonstrate efficient growth. Hence, we were not able to carry out the enzyme acitivety tests. |
| 13th | Carry out fermentation performance test by testing the concentrations of glucose and ethanol. |
| August | |
| Repeat the plasmids construction experiments and the yeast culture by AQ yeast. ![](https://static.igem.org/mediawiki/2021/e/e0/T--Fujian_United--img_notebook_4.jpg) | |
| September | |
| Carry out enzyme activity tests and fermentation performance tests. | |

---

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# Team:Fujian United/Engineering

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# Engineering

# Background

Alcohol (ethanol) is an important chemical in the fields of food, medicine, and biofuels. The
traditional fermentation method uses corn or tapioca starch as raw material to produce alcohol,
which is obtained by rectification after fermentation by *Saccharomyces cerevisia*e. The raw
materials of corn or tapioca starch must be pretreated by crushing first, and then α-amylase
(liquefaction enzyme) is added to hydrolyze the α-1,4-glucosidic bond in the starch under high
temperature conditions to cut the starch into short-chain pastes of varying lengths. The
liquefaction enzyme needs to be added exogenously. If *Saccharomyces cerevisiae* can express
the liquefaction enzyme and/or saccharification enzyme genes heterologously, so that the yeast can
hydrolyze starch and produce sugar by itself, the enzymes can be reduced. Add the amount to reduce
costs and improve efficiency.

#

We considered to express the glucoamylase gene GA derived from *Saccharomycopsis fibuligera* in
*Saccharomyces cerevisiae*. In order to test the effect of gene expression time-specificity on
the alcohol production of strains fermented starch liquefaction mash, codon optimized GA were
controlled by 3 different promoters. The three promotors are the constitutive promoter TEF1, the
glucose-inducible promoter HXT7, and the glucose-repressive promoter ICL1. The transcription of GA
gene also controlled by strong terminator CYC1.

# Build

The Golden gate modular one-step non-marking cloning construction method was used to construct
plasmids pYES2-TGC, pYES2-HGC and pYES2-IGC (Figure 1). The recombinant plasmids were transformed
into *Saccharomyces cerevisiae*.

![](https://static.igem.org/mediawiki/2021/4/40/T--Fujian_United--img_engineering_1.jpg)
Figure 1. Schematic map of Glucoamylase expression plasmids. pYES2-ctl is control plasmid.

# Test

In the test, the glucoamylase activity of the culture supernatant of the obtained strains and the
ability of fermented corn starch to reduce enzymes to produce alcohol are tested, and the income is
calculated. The GA activity of *S. cerevisiae* strains harboring various plasmids was
determined using the glucoamylase activity assay kit. As shown in Figure 2, the control stain (left
column) exhibited no GA activity. The strains harboring pYES2-TGC and pYES2-HGC plasmids, which
means the GA’s expression was driven by the constitutive promoter TEF1 and glucose inducible
promoter HXT7, showed almost the same and high GA activity. However, the strain in which the
expression of GA was under the glucose repressive promoter ICL1, exhibited a very low GA activity,
this is because the substrate of the glucoamylase assay kit is glucose, the expression of the GA
cassette was inhibited.

![](https://static.igem.org/mediawiki/2021/4/40/T--Fujian_United--img_engineering_2.jpg)
Figure 2. S. cerevisiae strains harboring various plasmids glucoamylase activity determination.

\*\*Statistical significance between indicated strains, p < 0.01. ns, not
significant. Data represent the means of two independent colonies.

To verify the GA secretion capacity of the GA-expressing *S. cerevisiae* strains, we measured
the
glucose concentration inside the cell during the corn starch fermentation process. As shown in
Figure 3A, at the initial stage (0 h), when the GA was added during the “starch-to-glucose” process,
higher contents of glucose were detected than the process without GA addition. This is because GA
can degrade starch to make more glucose. Along with the fermentation process, the glucose
concentration in the pYES2-ctl containing strain decreased obviously (Figure 3B), due to the without
more glucose production, glucose was utilized by the strain. However, the pYES2-TGC and pYES2-HGC
containing stains showed higher glucose concentration without GA addition, this is because the GA
produced by the strains could hydrolysis starch to prepare glucose, in other words, the
GA-expressing strains could reduce the enzyme usage. In contrast to this, when the GA was added in
the medium preparation process, all the strains showed almost the same glucose concentration at 24
h.

![](https://static.igem.org/mediawiki/2021/6/61/T--Fujian_United--img_engineering_3.jpg)
Figure 3. Glucose concentration inside the cell during the GA-expressing S. cerevisiae strains corn starch fermentation process.

# Learn

Genetically engineered yeast can produce glucoamylase enzymes. The GA-expressing strains showed a
comparable glucose production capacity with the GA addition in the medium.

---

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# Team:Fujian United/Experiments

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# Experiments

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# Team:Fujian United/Results

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# Results

# 1. Glucoamylase expression plasmids construction

![](https://static.igem.org/mediawiki/2021/8/8e/T--Fujian_United--img_results_1.jpg)
Fig.1 Glucoamylase expression plasmids in this project

Glucoamylase (GA) is one of the key enzymes during the starch to glucose process. A genetically
engineered GA expressing *Saccharomyces cerevisiae* strain is strongly required to save the
enzyme cost in starch based bioethanol industry.

To express the glucoamylase gene under different strength promoters, we designed three plasmids
together with a control plasmid pYES2-ctl (Fig. 1A), that is, GA is expressed under constitutive
promoter TEF1 (Fig. 1B), GA is expressed under glucose inducible promoter HXT7 (Fig. 1C), and GA is
expressed under glucose repression promoter ICL1 (Fig. 1D).

![](https://static.igem.org/mediawiki/2021/2/25/T--Fujian_United--img_results_2.jpg)
Fig. 2 Construction of Saccharomycopsis fibuligera derived glucoamylase expressing plasmids

The Golden gate cloning method was used to prepare the GA-expressing plasmids. The basic parts of the
plasmids such as the pYES2 backbone, GA coding sequence, promoters, and terminators were all
amplified successfully firstly (Fig. 2 A and B), then the multiplex GA expression cassettes were
assembled using the overlap PCR method, the resulting parts were cut with *Sap*I restriction
enzyme and ligated with the backbone, which was cut with the same enzyme. The primer pairs
YZ-IGEM-1/CYC1t-R were set to verify the positive colonies (Fig. 2C). We chose 24 colonies to verify
whether the plasmids were correct or not using the colony PCR, the positive rate for the plasmids
pYES2-TGC, pYES2-HGC, and pYES2-IGC were 11/24, 17/24, and 17/24, respectively. Two or three
positive colonies were sequenced to verify further.

# 2. GA plasmids transformation

![](https://static.igem.org/mediawiki/2021/3/35/T--Fujian_United--img_results_3.jpg)
Fig. 3 GA-expressing plasmids transformation and positive *S. cerevisiae* transformants selection using 50 μg/mL (A) and 350 μg/mL (B) hygromycin. Top left, pYES2-ctl. Top right, pYES2-TGC. Bottom left, pYES2-HGC. Bottom right, pYES2-IGC.

The constructed GA-expressing plasmids were chemically transformed into the *S. cerevisiae*
CEN.PK2-1C strain, the positive transformants were selected against a relatively low concentration
of hygromycin (50 μg/mL) firstly to ensure colony growth. As shown in Fig. 1A, plasmids pYES2-ctl,
pYES2-TGC, pYES2-HGC, and pYES2-IGC seemed all transformed into the CEN.PK2-1C strain. Then, we
streaked colonies from the low concentration hygromycin plates onto the high concentration
hygromycin (350 μg/mL) plates again to verify the positive transformants.

![](https://static.igem.org/mediawiki/2021/b/b3/T--Fujian_United--img_results_4.jpg)
Fig. 4 Verification of the plasmids pYES2-ctl (A), pYES2-TGC (B), pYES2-HGC and pYES2-IGC (C) transformation via colony PCR in CEN.PK2-1C strain.

The colonies which grew on the high concentration hygromycin plates were subjected to colony PCR to
verify the plasmids transformation again. From Fig. 4 we can see that positive bands implied the
plasmids transformation successfully.

# Glucoamylase activity assay

![](https://static.igem.org/mediawiki/2021/7/7f/T--Fujian_United--img_results_5.jpg)
Fig. 5 *S. cerevisiae* strains harboring various plasmids glucoamylase activity determination. \*\*Statistical significance between indicated strains, p < 0.01. ns, not significant. Data represent the means of two independent colonies.

The GA activity of *S. cerevisiae* strains harboring various plasmids was determined using the
glucoamylase activity assay kit. As shown in Fig. 5, the control stain (left column) exhibited no GA
activity. The strains harboring pYES2-TGC and pYES2-HGC plasmids, which means the GA’s expression
was driven by the constitutive promoter TEF1 and glucose inducible promoter HXT7, showed almost the
same and high GA activity. However, the strain in which the expression of GA was under the glucose
repressive promoter ICL1, exhibited a very low GA activity, this is because the substrate of the
glucoamylase assay kit is glucose, the expression of the GA cassette was inhibited.

# Fermentation test

![](https://static.igem.org/mediawiki/2021/6/61/T--Fujian_United--img_results_6.jpg)
Fig. 6 Glucose concentration inside the cell during the GA-expressing *S. cerevisiae* strains corn starch fermentation process.

To verify the GA secretion capacity of the GA-expressing *S. cerevisiae* strains, we measured the glucose concentration inside the cell during the corn starch fermentation process. As shown in Fig. 6A, at the initial stage (0 h), when the GA was added during the “starch-to-glucose” process, higher contents of glucose were detected than the process without GA addition. This is because GA can degrade starch to make more glucose. Along with the fermentation process, the glucose concentration in the pYES2-ctl containing strain decreased obviously (Fig. 6B), due to the without more glucose production, glucose was utilized by the strain. However, the pYES2-TGC and pYES2-HGC containing stains showed higher glucose concentration without GA addition, this is because the GA produced by the strains could hydrolysis starch to prepare glucose, in other words, the GA-expressing strains could reduce the enzyme usage. In contrast to this, when the GA was added in the medium preparation process, all the strains showed almost the same glucose concentration at 24 h. Overall, when the GA was removed from the medium preparation, the GA-expressing strains showed a comparable glucose production capacity with the GA addition situation.

---

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