iGEM 2022 — Team shanghai-metropolis

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{% block title %}Notebook{% endblock %}
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# Notebook

## Daily work/Notes

### July 25th

Ice-breaking activity, all members of the team got a chance to go up stage to introduce themselves to the
team. After getting familiar with each other, we started to allocate tasks for the wet team and the dry team
for the next day.

![](https://static.igem.wiki/teams/4278/wiki/notebook/t-shanghai-metropolis-notebook-01.jpg)

### July 26th

Preliminary understanding of PCR and homologous recombination mechanisms. In the afternoon, members started
to obtain target genes from the yeast cell gene pool by the use of primers. Following that, all tubes were
placed in the PCR machine for denaturation, annealing, extension, and final extension processes.

![](https://static.igem.wiki/teams/4278/wiki/notebook/t-shanghai-metropolis-notebook-02.jpg)

### July 27th

Verified the PCR products by making the agarose gel, and ran gel electrophoresis.

![](https://static.igem.wiki/teams/4278/wiki/notebook/t-shanghai-metropolis-notebook-03.jpg)

### July 28th

After screening the agarose gel, we cut the target DNA section and recycled the DNA through centrifuge.

![](https://static.igem.wiki/teams/4278/wiki/notebook/t-shanghai-metropolis-notebook-04.jpg)

### July 29th

Making YEPD and LB nutrients culture agar plates for upcoming experiments. Cultured yeast cells.

![](https://static.igem.wiki/teams/4278/wiki/notebook/t-shanghai-metropolis-notebook-05.jpg)

### July 30th

A day off, the yeast cells were continued to be cultivated.

![](https://static.igem.wiki/teams/4278/wiki/notebook/t-shanghai-metropolis-notebook-06.jpg)

### July 31st

Completed connecting gene fragments. Extracted the yeast cell plasmid through centrifuge, and performed the
double enzyme digestion.

![](https://static.igem.wiki/teams/4278/wiki/notebook/t-shanghai-metropolis-notebook-07.jpg)

### August 1st

After understanding the double enzyme digestion mechanism, the DNA section were inserted into the plasmid.
The plasmid was later put back into the competent state yeast cell.

![](https://static.igem.wiki/teams/4278/wiki/notebook/t-shanghai-metropolis-notebook-08.jpg)

### August 2nd

E. coli, yeast culture coated the plate. The wild type, type A and type B yeast cells were sent to companies
to undergo DNA sequencing tests.

![](https://static.igem.wiki/teams/4278/wiki/notebook/t-shanghai-metropolis-notebook-09.jpg)

### August 3rd

The team went over the whole experiment again to check for any misunderstandings of the contents.

![](https://static.igem.wiki/teams/4278/wiki/notebook/t-shanghai-metropolis-notebook-10.jpg)

### August 4th-8th

Simulated fermentation of alcohol and detection of the content of higher alcohols and ethanol by HPLC.

![](https://static.igem.wiki/teams/4278/wiki/notebook/t-shanghai-metropolis-notebook-11.jpg)

### August 9th-11th

Collate and analyze the data of the fermentation test in lab by stimulating winery.

![](https://static.igem.wiki/teams/4278/wiki/notebook/t-shanghai-metropolis-notebook-12.jpg)

### September 28th

The web pages for all parts of the wiki are completely build.

![](https://static.igem.wiki/teams/4278/wiki/notebook/t-shanghai-metropolis-notebook-13.jpg)

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{% block title %}Engineering{% endblock %}
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# Engineering Success

## 1. Introduction

In the beer brewing process, in addition to the main metabolite ethanol, yeast also produces higher alcohols,
esters, aldehydes, phenols, acids, and other flavor metabolites. Among them, higher alcohol refers to the
general term of a class of alcohols with three or more carbon chain skeletons and is one of the important
chemical substances that form the flavor and taste of wine.

Appropriate higher alcohol content can make the beer taste and aroma full, and the wine body is soft and
harmonious, but if the higher alcohol content is too high, it will lead to the formation of peculiar smells in
beer, which not only affects the drinking taste and flavor quality of beer, but also affects drinkers. The
body produces obvious side effects, which are harmful to the health of the drinker.

The higher alcohols in the finished beer are produced by the growth and reproduction of yeast in the main
fermentation stage of beer. The branched-chain amino acid aminotransferase is decomposed, that is the pathway
of amino acid catabolism; the other is the α-keto acid anabolic pathway. The metabolite pyruvate generated
from glucose through the emp pathway can also generate α-keto acid after a series of reactions that is the
pathway of higher alcohol anabolic.

For this problem, researchers have taken various measures to solve this problem. For example, the higher
alcohol metabolism gene of *Saccharomyces cerevisiae* is modified to achieve the purpose of reducing the
production of higher alcohol.

This project intends to reduce the production of higher alcohols from two types of genetic modification, we
designed to edit the acetate metabolism genes. The alcohol acetyltransferases encoded by ATF1, ATF2, and
Lg-ATF1 genes can catalyze the reaction. At the same time, acetate compounds can also undergo hydrolysis
reactions under the catalysis of the hydrolase encoded by the IAH1 gene to generate corresponding higher
alcohols. Therefore, we replaced the BAT2 gene in the *S. cerevisiae* genome with ATF1 through the
homologous
recombination way.

## 2. Design

The ATF1 gene is inserted into the *S. cerevisiae* expression vector pYES2 and transformed into the S.
cerevisiae strain, which we called SFA-1 (Figure 1). In other words, this plasmid is based on the targeted
modification of the aminotransferase encoding gene, and then targeted modification of the acetate metabolism
gene.

![](https://static.igem.wiki/teams/4278/wiki/engineering/t-shanghai-metropolis-engineering-01.png)

*Figure 1. The schematic map of the pYES2-ATF1 plasmid*

## 3. Build

In order to build our plasmids, The ATF1 was amplified from the genome of *S. cerevisiae*, and the TEF1
promoter
and the CYC1 terminator were amplified from the pHCas9-Nours plasmid. Next, we fused the three fragments by
PCR and extracted the recombinant DNA fragment (Figure 2). Then, we digested the target fragments and the
pYES2 vector with SpeI and SalI, and we used T4 DNA ligase to ligate the fragments and the vector. Then we
transformed the recombinant plasmids into *E. coli* DH5α competent cells and coated on the LB (Amp+)
solid
plates.

![](https://static.igem.wiki/teams/4278/wiki/engineering/t-shanghai-metropolis-engineering-02.png)

*Figure 2. Gel electrophoresis results of the target gene fragments.*

We verified the colonies through colony-PCR (Figure 3), and then we inoculated single colonies (1, 2, 5, and
7) and we send the constructed recombinant plasmid to a sequencing company for sequencing.

![](https://static.igem.wiki/teams/4278/wiki/engineering/t-shanghai-metropolis-engineering-03.jpg)

*Figure 3. verification of the recombinant plasmids by colony-PCR.*

## 3. Functional testing

### a) Construct the engineered *S. cerevisiae* strain

We extracted the correct pYES2-ATF1plasmid and transformed it into the *S. cerevisiae* competent cells
through
the Lithium acetate transform method. Next, we verified the colonies through colony-PCR (Figure 4).

![](https://static.igem.wiki/teams/4278/wiki/engineering/t-shanghai-metropolis-engineering-04.jpg)

*Figure 4. verification of the engineered *S. cerevisiae* by colony-PCR*

### b) Growth Curve measurement

The plasmid was transformed into the *S. cerevisiae* and we named the transformants SFA-1. We
inoculated the
engineered *S. cerevisiae* colonies (SFA-1) in the 5mL YEPD medium and grew at 30℃ and 180 r/min for 12
h. The
above culture 1:100 culture liquid was connected to three bottles of 50mL liquid YEPD medium and incubated
under the same conditions. The absorbance value at 600 nm was measured at 2,4,8,16,24 and 32 h after culture
(Table 1). This result indicates that the genetically modified *S. cerevisiae* SFA-1 showed no harmful
effects
on the growth of the organism itself. Therefore, giving support for applications in the wine fermentation
industry (Table 1).

| Time (h)/Type | SF-1 | WT |
| --- | --- | --- |
| 2 | 0.076 | 0.016 |
| 4 | 0.106666667 | 0.049 |
| 7 | 0.18 | 0.109 |
| 16 | 1.007333333 | 0.639 |
| 24 | 2.558333333 | 1.105 |
| 32 | 2.554666667 | 1.233666667 |

*Table 1. Raw data were obtained at each time period 2, 4, 7, 16, 24, and 32 hours, respectively. Each
strain of WT and SFA-1 was tested with Spectrophotometer three times, to obtain an OD600 absorbance
level.*

## 4. Learn

We have already collected the figures from our experiments. Because of the importance of ATF1 in the aroma
profile of the wine, the yeast with a constitutively overexpressed ATF1 gene produces more isoamyl acetate and
ethyl acetate compared to a wild-type strain. In our project, we overexpressed the ATF1 in the *S.
cerevisiae*
to construct the engineered *S. cerevisiae* strain to control the content of higher alcohols during
fermentation.

In the future, with improved engineering strains, the better quality of the wine would spread all over the
world. ATF1, which we verified its activity in our project, provides a good choice for future research. We
believe that the engineered *S. cerevisiae* which has reduced the production of higher alcohols could be
used to
improve the factories in the future.

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# Experiments

{% include 'experiments/section-1.html' %}
{% include 'experiments/section-2.html' %}
{% include 'experiments/section-3.html' %}
{% include 'experiments/section-4.html' %}
{% include 'experiments/section-5.html' %}

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# Results

## 1. Assemble target gene fragments

As indicated in Figure 1, BAT2 is the DNA strand that is responsible for producing the higher alcohol during
the brewing of wine. A replacement of the BAT2 strand with ATF1 is strongly required in order to reduce the
adverse effects after getting drunk. The BAT2-L-TEF1-ATF1-CYC1-TEFP-NrsR-TEFt-BAT2-R fragment was prepared to
replace the BAT2 fragment in yeast.

![](https://static.igem.wiki/teams/4278/wiki/results/t-shanghai-metropolis-results-01.png)

Figure 1. The map of gene segment integrated into the yeast genome to replace BAT2 with ATF1

### 1.1 Preparation of five fragments of BAT2-L, TEF1, ATF1, CYC1-TEFP-NrsR-TEFt, BAT2-R

According to the experimental design, five fragments of BAT2-L, TEF1, ATF1, CYC1-TEFP-NrsR-TEFt and BAT2-R
should be copied first. In figure 2, the gel electrophoresis results of the above five fragments are shown.

![](https://static.igem.wiki/teams/4278/wiki/results/t-shanghai-metropolis-results-02.jpg)

Figure 2. The gel electrophoresis results of the replicated five fragments.
A: Line: BAT2-R, 501 bp, correct;
Line: CYC1-TEFp-NrsR-TEFt , 1715 bp, correct;
Line: ATF1, 1575 bp, correct.
Line: BAT2-F, 503 bp, correct;
B: Line: TEF1, 400 bp, correct;

According to the comparison between the gel electrophoresis results and the fragment map, we analyzed that
the lengths of the DNA in figure 2A, 2B were consistent with the length of the fragments we needed, so the
replication of the fragments was successful.

### 1.2 fragment assembly

According to the experimental plan, after the five small fragments are cloned, they need to be assembled
together. We combined the DNA strands with the final strands by overlap PCR. The results of gel
electrophoresis of the final fragment BAT2-L-TEF1-ATF1-CYC1-TEFP-NrsR-TEFt-BAT2-R are shown in the figure.

![](https://static.igem.wiki/teams/4278/wiki/results/t-shanghai-metropolis-results-03.jpeg)

Figure 3. The gel electrophoresis results of the final fragments
BAT2-L-TEF1-ATF1-CYC1-TEFP-NrsR-TEFt-BAT2-R.

The red arrow indicates the correct position of the fragment.

After comparing with the marker, it can be found that the marker of the assembled fragment as indicated by
the red arrow in the gel electrophoresis map is between the marker's 4000bp and 5000bp markers. The
assembled fragment is 4,665bp long, which is within the expected range. The result shows the gene strands
are correctly connected and replicated

## 2. Construction of plasmid pYES2-ATF1

### 2.1 Construction of ATF1 gene expression cassette

According to the experimental design, *S. cerevisiae* strain A integrates ATF1 into the genome and replaces
the BAT2 gene. Different from strain A, *S. cerevisiae* strain B uses a plasmid to carry the ATF1 gene, and
the plasmid version highly expresses the ATF1 gene. We called this plasmid pYES2-ATF1.

In order to ensure the successful construction of plasmid pYES2-ATF1, it is essential to construct the ATF1
gene expression cassette. Therefore, we still used PCR and gel electrophoresis to clone and verify whether
the ATF1 gene expression cassette was successfully constructed.

![](https://static.igem.wiki/teams/4278/wiki/results/t-shanghai-metropolis-results-04.png)

Figure 4. The gel electrophoresis results of the ATF1 gene expression cassette.
The red arrow indicates the mark of the gene expression cassette in the figure.

In figure 4, the ATF1 gene expression cassette is 2,256 bp long. The length of DAN as indicated by the red
arrow is between 1,000bp and 2,500bp; it is close to 2,500bp. This suggests that we have successfully
constructed the ATF1 gene expression cassette. At this point, we can safely connect the ATF1 gene expression
cassette to the pYES2 plasmid that has been digested.

### 2.2 Ligation of plasmid vector pYES2 and ATF1 fragment

After confirming that the ATF1 gene expression cassette has been constructed, we need to introduce it into
the pYES2 plasmid that has been cut by Spe I and Sal I restriction enzymes, and ensure that the ATF1 gene
expression cassette is connected to the pYES2 plasmid backbone and form a new pYES2-ATF1 plasmid. We need to
use gel electrophoresis to verify that the *S. cerevisiae* strain SFA-1, that is, *S. cerevisiae* strain B, has
a complete plasmid, so as to ensure the smooth progress of the subsequent strain growth curve and simulated
brewing fermentation test.

![](https://static.igem.wiki/teams/4278/wiki/results/t-shanghai-metropolis-results-05.jpg)

Figure 5. The gel electrophoresis result of the ligated pYES2-ATF1 plasmid.
The red arrow indicates the mark of the plasmid in the figure.

As can be seen on the left side of the figure, there is a clearer mark. After comparing with the marker, we
can prove that the length of plasmid pYES2-ATF1 in the figure is 6000bp to 8000bp. After confirmation with
the map, plasmid pYES2-ATF1 is 7841 bp long, which proves that the marker pointed to by the red arrow is the
pYES2-ATF1 plasmid, that is plasmid pYES2-ATF1. So the construction is successful.

## 3. Growth Curve

We obtained a total of two genetically modified yeast strains, named SF-1 and SFA-1. SF-1 is a strain that
integrates ATF1 into the BAT2 gene of the yeast genome, which results in reduced production of higher
alcohols, thereby reducing drunkenness. SFA-1 is to transfer plasmid pYES2-ATF1 into yeast, and overexpress
ATF1 in the form of plasmid, so as to achieve the purpose of increasing the aroma characteristics of wine. The
third fermenting strain was the wide type (WT) yeast, which served as a negative control without any genetic
modification of yeast.

Subsequently, we performed fermentation experiments based on three yeast strains, SF-1, SFA-1, and WT (wild
type). Therefore, obtaining data between 2, 4, 7, 16, 24, and 32 hours respectively would allow the targeted
organisms to grow and at every time node, we tested the OD600 absorbance for each culture three times using a
spectrophotometer, while each time we blanked the machine using ddH2O, thus, drew the Growth Curve using R
program.

To be more specific, in order to make sure we have grown yeast equally, whenever finish testing the OD600
absorbance value we put them back into the cradle for mixing the nutrients and organisms comprehensively so
that the nutrient level won't be a limiting factor for yeast growths. Considering using the spectrophotometer,
each time, we extracted 1.5ml of the solution from each of the three cultured strains: SF-1, SFA-1, and WT.
Consequently, we transferred the solution into the transparent cuvette one at a time, while blanking the
machine with another previously filled cuvette with double distilled water in it. Through the testing process,
which we tried three times with each strain solution, the machine gave us three OD600 absorbance numbers.

Thus, drew the Growth Curve using the R program. This diagram uses LOG2 to fitting the curve and we calculated
the p-values when comparing with the WT using ANOVA Test as more than two groups were considered.

Table 1, Raw data obtained at each time period 2, 4, 7, 16, 24, 32 hours,
respectively.

| Time (h)/Type | SF-1 | SFA-1 | WT |
| --- | --- | --- | --- |
| 2 0.076 | 0.052 | 0.016 | |
| 4 0.106666667 | 0.108 | 0.049 | |
| 7 0.18 | 0.320333333 | 0.109 | |
| 16 1.007333333 | 1.732333333 | 0.639 | |
| 24 2.558333333 | 2.517666667 | 1.105 | |
| 32 | 2.554666667 | 2.528333333 | 1.233666667 |

![](https://static.igem.wiki/teams/4278/wiki/results/t-shanghai-metropolis-results-07.jpg)

Figure 6. The curve of OD600 absorbance of each strain that R program generated.
Time is on the x-axis and relative absorbance calculated from logarithum is the y-axis, with three keys
SFA-1, SF-1 and Wildtype (WT). p-value is calculated when comparing with WT.

After analysis, we can find that although the growth amount and efficiency of Saccharomyces cerevisiae strains
A and B do not reach or exceed the level of wild-type Saccharomyces cerevisiae strains. However, the slopes of
cerevisiae strain SF-1 and SFA-1 can be relatively equal to those of wild-type cerevisiae strains at different
test time points. This means that the growth efficiency of cerevisiae strains A and B is relatively consistent
with that of the wild-type cerevisiae strain, which improves the competitiveness of our strain products. But
we have at least demonstrated that our Saccharomyces cerevisiae strain can survive, grow and ferment.

## 4. Laboratories fermentation tests

The purpose of the simulated alcohol fermentation test is to verify that the strain can produce ethanol
normally. In this project, this test also proves whether the simple strain we constructed can effectively
reduce the production of higher alcohol.

We use gas chromatography to measure the content of higher alcohols. The following table shows the
experimental data of various alcohols produced by wild-type AQ yeast and SF-1 yeast. We did two sets of
experiments for each yeast. The experimental data showed that the ethanol content of SF-1 yeast was similar to
that of wild-type AQ yeast, indicating that the knockout of BAT2 gene and integration of ATF1 gene had little
effect on the production of ethanol.

Table 2. Gas phase data

| Strain Types/Chemicals | Ethanol (mg/L) | n-Propanol (mg/L) | Isobutanol (mg/L) | Isoamyl alcohol (mg/L) | Phenylethanol (mg/L) | Higher alcohol (mg/L) |
| --- | --- | --- | --- | --- | --- | --- |
| Wild-type AQ yeast① | 9704.7 | 0 | 27.45 | 143.22 | 79.04 | 249.71 |
| Wild-type AQ yeast② | 8836.8 | 0 | 25.07 | 137.56 | 79.96 | 242.59 |
| SF-1① | 7890 | 0 | 0 | 65.95 | 23.86 | 89.81 |
| SF-1② | 8284.5 | 0 | 0 | 68.28 | 29.01 | 97.29 |

Note: tert-amyl alcohol, amyl acetate and 2-ethyl butyric acid are internal standard substances.

We averaged the two sets of experimental data and calculated the percentage of each higher alcohol production,
as shown in the following figures.

![](https://static.igem.wiki/teams/4278/wiki/results/t-shanghai-metropolis-results-08.jpg)

Figure 7. Fitting result of Wild-type AQ yeast and engineered yeast SF-1.

We found that both sets of data of strain SFA-1 were abnormal, and the initial judgment was that the strain
was contaminated by controllable unknown organisms. This is undoubtedly very negative news, we did not do a
good job of protecting the samples during the experiment, which led to controllable contamination.

But the good news is that the higher alcohol production of strain SF-1 decreased by about 152.6 mg per liter
compared to the wild-type strain. is 38.000% of the wild-type strain. This proved that strain A achieved the
experimental assumption and successfully reduced the production of higher alcohols.

However, the ethanol production of strain A was slightly lower than that of the wild-type strain by about
0.15% vol. is about 87.234% of the wild-type strain. This result is estimable and acceptable because the
growth curve data for strain A is the worst of the three strains.

Since strain B has only one more ATF1 gene expression cassette than strain A, it is not difficult to speculate
the test result of strain B. It is speculated that the ethanol production of strain B may be reduced by about
0.1-0.2% vol compared to the wild-type strain, but the production of higher alcohols may be reduced more than
that of strain A, To pessimistic estimate, it is possible to reduce about 155 mg per liter to 170 mg per liter
compared to the wild type strain. If optimistic estimates, strain B can reduce about 180 mg per liter or even
200 mg per liter of higher alcohol compared to the wild-type strain. Although this prediction is radical, it
cannot be ruled out.

In a nutshell, strain A successfully achieved our experimental goal, and although strain B did not prove to be
achieved, it was predicted to be achieved with certainty.

The peak diagram of gas chromatography detection WT AQ yeast-1

The peak diagram of gas chromatography detection WT AQ yeast-2

The peak diagram of gas chromatography detection SF-1 yeast-1

The peak diagram of gas chromatography detection SF-1 yeast-2

The peak diagram of gas chromatography detection SFA-1 yeast-1

The peak diagram of gas chromatography detection SFA-1 yeast-2

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