iGEM 2022 — Team suzhou-union

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

## 8/14

1. We know each other and get familiar with each other quickly through ice-breaking games.
2. In order to let us to understand our project in depth, the wet team coach led the students to do a project
   review.
3. The probationary group captains were elected. we also created an official account on WeChat, designed the
   team name and Logo, and conceive a series of teams’ souvenir.

![](https://static.igem.wiki/teams/4124/wiki/notebook/t-suzhou-union-notebook-01.jpg)

## 8/15

1. We learned the safety knowledge related to the laboratory and the matters needing attention in the
   experimental operation;
2. LB and YPD media were made.

![](https://static.igem.wiki/teams/4124/wiki/notebook/t-suzhou-union-notebook-02.jpg)

## 8/16

We learned about the principle and application of PCR, after which each one performed the process of
configuring the PCR system for ACC1, FAS1, and FAS2, followed by PCR reactions for these three enzymes and the
process of running gel, and finally observed the experimental results.

![](https://static.igem.wiki/teams/4124/wiki/notebook/t-suzhou-union-notebook-03.jpg)

Table 1: PCR Process
![](https://static.igem.wiki/teams/4124/wiki/notebook/t-suzhou-union-notebook-04.png)

## 8/17

First, we use Gel DNA Extraction Kit to extract ACC1, FAS 1 and FAS2 genes that were got yesterday. Then we
divided into three groups to transform ACC1, FAS1 and FAS2 respectively. After that the connected target genes
were transformed into Escherichia coli, thus completing the plasmid construction.

Table 2: Gel DNA Extraction Kit component
![](https://static.igem.wiki/teams/4124/wiki/notebook/t-suzhou-union-notebook-05.png)

## 8/18

We studied the coating plate using the plasmid that had been transformed yesterday and did the yeast
inoculation experiment.

![](https://static.igem.wiki/teams/4124/wiki/notebook/t-suzhou-union-notebook-06.jpg)

## 8/19

We checked if the PCR transformation products from yesterday grew on the solid culture plates, after which
they performed colony PCR, use Gel DNA Extraction Kit to extract genes, and placed into the media overnight
for incubation. At the same time, the preliminary preparation of yeast competent cell was carried out.

![](https://static.igem.wiki/teams/4124/wiki/notebook/t-suzhou-union-notebook-07.jpg)

## 8/20

Preparation for the yeast competent cell.

Preparation for the yeast competent cell.

At last, we transform the plasmid that we extracted to the yeast competent cell.

![](https://static.igem.wiki/teams/4124/wiki/notebook/t-suzhou-union-notebook-08.jpg)

## 8/22-26

Selection of monoclonal strains, add then into YPD media to do the fermentation cultivation. After that, using
microplate reader to get the growing curve of fermentation strain. The fermentation samples were sent to test
the content of ethyl hexanoate

![](https://static.igem.wiki/teams/4124/wiki/notebook/t-suzhou-union-notebook-09.jpg)

Table 3: the value on the microplate reader
![](https://static.igem.wiki/teams/4124/wiki/notebook/t-suzhou-union-notebook-10.jpg)

## 8/27

Individual and team photo shooting

Assigning the task of writing wikis and parts

![](https://static.igem.wiki/teams/4124/wiki/notebook/t-suzhou-union-notebook-11.jpg)

## 8/28-9.12

Write wikis and parts; Prepare the Powerpoint for pre-presentation.

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{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
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# Engineering

## Introduction

Because the fragrant flavor of Baijiu is produced by FAEEs, the concentration of this substance is also the
standard for the quality of Baijiu, thus the output of FAEEs from S. cerevisiae is important. However, due to
the single strain fermentation process of Baijiu, the production of FAEEs is not enough causing the flavor to
become insufficient. Therefore, if the FAEEs produced by yeast is increased, the flavor of Baijiu can be more
abundant and fragrant.

The content of this project is to improve the FAEEs production ability of *S.cerevisiae* cells by
overexpressing FAS1, FAS2 or ACC1. Under the function of Malonyl-CoA, ACC1, FAS1, and FAS2 synthesize fatty
acids, fatty Acyl-CoAs produce FAEEs (fig.1). Therefore, as long as we successfully construct one of the three
overexpression plasmids, and transferred it into yeast, the production of FAEEs can be significantly
increased.

![](https://static.igem.wiki/teams/4124/wiki/engineering/t-suzhou-union-engineering-01.jpg)

*Figure 1. The metabolic pathway of producing FAEEs.*

## Design

In this experiment, we designed two plasmids YPF1K-FAS1 and YPF2K-FAS2. Because the PGK promoter have strong
ability in expression in yeast, and the URA3 usually be used as an auxotrophic screening tag in
uracil-deficient yeast, we choose Yep352-pPGK1 and BY4741 as plasmid-vector and strain host respectively. The
DNA sequences of FAS1 and FAS2 were inserted into the XhoI site of the Yep352-pPGK1 vector, so that produce
two recombinant plasmids YPF1K-FAS1 (fig.2A) and YPF2K-FAS2 (fig.2B).

![](https://static.igem.wiki/teams/4124/wiki/engineering/t-suzhou-union-engineering-08.jpg)

*Figure 2. The map of plasmid YPF1K-FAS1 and YPF2K-FAS2.*

## Build

Firstly, the exogenous gene FAS1 and FAS2 were amplified by PCR, and the Yep352-pPGK1 was linearized by XhoI
restriction enzyme digestion(fig.3). Then, the DNA fragments and plasmid-vector were ligated by homologous
recombination, and then transferred into E.coli DH10b.

![](https://static.igem.wiki/teams/4124/wiki/engineering/t-suzhou-union-engineering-02.jpg)

*Figure 3. Results of PCR amplification of FAS1 and FAS2 fragments.*

DH10B(YPF1K-FAS1 and YPF2K-FAS2) colonies were obtained using single-fragment recombination. The sequencing
results showed that the constructed YPF1K-FAS1 and YPF2K-FAS2 contained multiple synonymous mutations, but all
of them also contained individual mutation sites, which would lead to changes in the corresponding amino acids
of the protein. Pick clones with fewer mutations in them. The mutation site was then back-mutated using
site-directed mutagenesis

![](https://static.igem.wiki/teams/4124/wiki/engineering/t-suzhou-union-engineering-03.jpg)

*Figure 4. Sequencing results for corrected mutations of YPF1K-FAS1 and YPF2K-FAS2.*

After back-mutating, it is transferred into competent cells of *E. coli* DH10b. The correct recombination
plasmids YPF1K-FAS1 and YPF2K-FAS2 were extracted from *E. coli* DH10b with concentrations up to 2μg/μL.
Then, the plasmid was transferred into competent yeast cells BY4741 to improve the FAEEs production capacity
of yeast cells by using LiAc/SS carrier DNA/PEG method[1]. Finally, yeast clones were identified by direct
PCR. The result showed that there were 6147bp (FAS1) and 5685bp (FAS2) bands indicated that transformation
success (fig.5).

![](https://static.igem.wiki/teams/4124/wiki/engineering/t-suzhou-union-engineering-04.jpg)

*Figure 5. Results of PCR identification of yeast clones.*

## Test

In order to test the ability of our engineered yeast strain we constructed to produce Luzhou-flavor baijiu,
the engineered yeast strain YPF1K-FAS1/BY4741(YEP-FAS1) and YPF2K-FAS2/BY4741(YEP-FAS2) was placed in 110 mL
of YP20D liquid medium and fermented at 30 °C and 220 rpm for 60 h. Two parallels were set for each strain,
and 1 ml of fermentation broth was taken out every 10 hours to measure the growth of the strain. The growth
curve of the strain during the fermentation process is shown in figure 6. It can be seen that the growth of
our engineered yeast strain after overexpressing the FAS1 or FAS2 gene is comparable to that of the negative
control yeast strain (YEP), indicating that the genetic engineering modification will not affect the growth of
the strain, which is in line with expectations.

*Table 1. Fermentation time statistics table*
![](https://static.igem.wiki/teams/4124/wiki/engineering/t-suzhou-union-engineering-05.jpg)

![](https://static.igem.wiki/teams/4124/wiki/engineering/t-suzhou-union-engineering-06.jpg)

*Figure 6. Fermentation effect comparison*

According to the results of detection of ethyl caproate content(fig.9), compared with the control group,
YEP-FAS1 and YEP-FAS2 engineering bacteria can produce more ethyl caproate, and the fermentation content of
YEP-FAS1 engineering bacteria is significantly higher. From the peak graph of ethyl caproate, our engineering
bacteria really ferment to produce ethyl caproate. This is also a supplement to the wiki results. Our
experiment can provide reference for other iGEM teams, and provide some guidance for subsequent industrial
production, which verifies the engineering success.

![](https://static.igem.wiki/teams/4124/wiki/results/t-suzhou-union-results-08.jpg)

Figure 7. The ethyl caproate peak of YEP-FAS1 detectedby GC-MS.

![](https://static.igem.wiki/teams/4124/wiki/results/t-suzhou-union-results-09.jpg)

Figure 8. Partial enlarged view of ethyl caproate peak of YEP-FAS1 detected by GC-MS.

Table 2. The peak area value of ethyl caproate fermented of three strains by GC-MS detection.

|  | Retention time(min) | Peak area |
| --- | --- | --- |
| YEP-1 | 7.886 | 49152276 |
| YEP-2 | 7.885 | 59295100 |
| YEP-1 | 7.884 | 64329291 |
| YEP-2 | 7.888 | 80959256 |
| YEP-FAS1-1 | 7.889 | 536339098 |
| YEP-FAS1-2 | 7.885 | 426329012 |
| YEP-FAS1-3 | 7.889 | 417371253 |
| YEP-FAS1-4 | 7.884 | 407642903 |
| YEP-FAS2-1 | 7.893 | 490448534 |
| YEP-FAS2-2 | 7.896 | 312597902 |
| YEP-FAS2-3 | 7.886 | 147231169 |
| YEP-FAS2-4 | 7.885 | 128083095 |

![](https://static.igem.wiki/teams/4124/wiki/results/t-suzhou-union-results-10.jpg)

Figure 9. Relative value of ethyl caproate fermented of three strains by GC-MS detection.

## Learn

After the fermentation time of each group continues data processing and drawing, it can be clearly seen from
the figure 6 that after the optimization and improvement of our experiment, through the characterization of OD600,
the brewing yield is significantly higher than that of the control group(Fig.6), indicating that our
experiment has been successful and also It is hoped that our experiments can provide reference for other iGEM
teams and provide certain guidance for subsequent industrial production.

## Reference

[1] R Daniel Gietz & Robert H Schiestl Nat. Protoc. 2, 1–4 (2007); doi:10.1038/nprot.2007.17; published
online: 31 January 2007; corrected online 4 December 2008.

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{% block title %}Experiments{% endblock %}
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# Experiments

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

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{% extends "layout.html" %}
{% block title %}Results{% endblock %}
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# Results

## 1. Plasmids construction

As indicated in Figure 1, the synthesis principle of ethyl caproate is that acetyl coenzyme A is generated
from acetic acid, and malonyl coenzyme A is generated under the action of ACC1, and then converted into fatty
acid, fatty alcohol and ethyl caproate under the action of FAS1 and FAS2. Therefore, the resultant quantity of
ethyl caproate can be increased by ACC1, FAS1 and FAS2.

![](https://static.igem.wiki/teams/4124/wiki/results/t-suzhou-union-results-01.jpg)

Fig.1 Synthesis principle of ethyl caproate

Firstly, we construct three plasmids, which are YPA1k-ACC1, YPA1k-FAS1 and YPA2k-FAS2. PCR is the abbreviation
of polymerase chain reaction, which is a method of enzymatic synthesis of specific DNA fragments in vitro. PCR
is an examination of DNA or RNA of various pathogens such as bacteria, viruses and fungi by using in vitro
amplification technology. Therefore, the gene fragments of ACC1 (cut into ACC1-1 ACC1-2 because ACC1 is too
long) and FAS1 and FAS2 can be replicated and amplified by PCR and extended until it can be observed. The
results (Figure 2) indicated that FAS1, FAS2, ACC1-a and ACC1-b DNA strands are correctly replicated.

![](https://static.igem.wiki/teams/4124/wiki/results/t-suzhou-union-results-02.jpg)

Figure 2 FAS1, FAS2, ACC1-a and ACC1-b DNA strands are correctly replicated
Identification of FAS1, FAS2, ACC1-a and ACC1-b DNA strands by agarose gel electrophoresis.

Secondly, we used double enzyme digestion to construct YPA1k-ACC1, YPA1k-FAS1 and YPA2k-FAS2 plasmids. Then,
the constructed YPA1K-ACC1, YPA1K-FAS1 and YPA2k-FAS2 plasmids were added to the competent cells and mixed
well. After 30min ice bath, the plasmid was subjected to 42℃ heat shock for 45s. 1 mL of antibiotic free LB
medium was added to the EP tube of the mixture of plasmid and competent cells and mixed well. After shaking
for 1 h at 600 rpm at 37°C, 100 mL of bacterial solution was coated on the solid medium containing ampicillin
or kanamycin, and inverted in the 37°C bacterial incubator for 12 h, Finally, the monoclonal colonies shown in
the figure were successfully obtained.

![](https://static.igem.wiki/teams/4124/wiki/results/t-suzhou-union-results-03.jpg)

Figure 3 FAS1, FAS2, ACC1-a and ACC1-b DNA strands are correctly replicated

The constructed plasmids are added to the transformation reagent and prepared yeast competent cells. The mix
is then applied onto the YBP culture plates and left under 37℃ overnight to ensure colony growth.

Thirdly, PCR was used to verify the monoclonal colony of the strain. As indicated in Figure 4, the plasmids
were successfully constructed.

![](https://static.igem.wiki/teams/4124/wiki/results/t-suzhou-union-results-04.jpg)

Figure 4 YPA1k-FAS1, YPA2k-FAS2 and YPA1k-ACC1 plasmids are correctly replicated
Identification of YPA1k-FAS1, YPA2k-FAS2 and YPA1k-ACC1 plasmids by agarose gel electrophoresis.

Fourthly, gene sequencing is used to double verification. As indicated in Figure 5, YPA1k-FAS1 and YPA2k-FAS2
sequences were correct.

![](https://static.igem.wiki/teams/4124/wiki/results/t-suzhou-union-results-05.jpg)

Figure 5 Sequencing of YPA1k-FAS1 and YPA2k-FAS2 plasmids
Identification of YPA1k-FAS1 and YPA2k-FAS2 by gene sequencing.

## 2. Strain functional test

To ensure the growth of the cells in the culture medium, we determined the optical density at 600nm (OD600)
(Table 1).

Table.1 OD600 of the culture medium

|  |  |  |  |
| --- | --- | --- | --- |
|  | ACC1 | FAS1 | FAS2 |
| OD600 | 0.237 | 0.075 | 0.064 |

After guaranteeing the growth in cells, the culture medium is sent to a professional company to detect the
concentration of FAEEs. Results show that the concentration also significantly increased compared with the
control group.

![](https://static.igem.wiki/teams/4124/wiki/results/t-suzhou-union-results-06.jpg)

Figure 6 Fermentation growth curve
Concentration values at different time points.

We collected samples that have been fermented for 60 hours and send them to the company to measure the content
of ethyl hexanoate. We first used conventional gas chromatography (GC) methods to detect ethyl hexanoate in
the sample, and found that the peaks of the ester and alcohol crossed and could not be completely separated or
the content of ethyl hexanoate could not be detected. Subsequently, through interviews with experts and
research literature, we learned that alcohols and esters can be separated using the gas chromatography-mass
spectrometry (GC-MS) method. So we re-fermented, collected 60 hours of fermentation samples, and sent them to
the company to measure GC-MS.

![](https://static.igem.wiki/teams/4124/wiki/results/t-suzhou-union-results-08.jpg)

Figure 7. The ethyl caproate peak of YEP-FAS1 detectedby GC-MS.

![](https://static.igem.wiki/teams/4124/wiki/results/t-suzhou-union-results-09.jpg)

Figure 8. Partial enlarged view of ethyl caproate peak of YEP-FAS1 detected by GC-MS.

According to the results of detection of ethyl caproate content(fig.9), compared with the control group,
YEP-FAS1 and YEP-FAS2 engineering bacteria can produce more ethyl caproate, and the fermentation content of
YEP-FAS1 engineering bacteria is significantly higher. From the peak graph of ethyl caproate, our engineering
bacteria really ferment to produce ethyl caproate. This is also a supplement to the wiki results. Our
experiment can provide reference for other iGEM teams, and provide some guidance for subsequent industrial
production, which verifies the engineering success.

Table1. The peak area value of ethyl caproate fermented of three strains by GC-MS detection.

|  | Retention time(min) | Peak area |
| --- | --- | --- |
| YEP-1 | 7.886 | 49152276 |
| YEP-2 | 7.885 | 59295100 |
| YEP-1 | 7.884 | 64329291 |
| YEP-2 | 7.888 | 80959256 |
| YEP-FAS1-1 | 7.889 | 536339098 |
| YEP-FAS1-2 | 7.885 | 426329012 |
| YEP-FAS1-3 | 7.889 | 417371253 |
| YEP-FAS1-4 | 7.884 | 407642903 |
| YEP-FAS2-1 | 7.893 | 490448534 |
| YEP-FAS2-2 | 7.896 | 312597902 |
| YEP-FAS2-3 | 7.886 | 147231169 |
| YEP-FAS2-4 | 7.885 | 128083095 |

![](https://static.igem.wiki/teams/4124/wiki/results/t-suzhou-union-results-10.jpg)

Figure 9. Relative value of ethyl caproate fermented of three strains by GC-MS detection.

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