iGEM 2023 — Team hangzhou-biox

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

---

* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

{% endblock %}

{% extends "layout.html" %} {% block title %}Project Description{% endblock %}
{% block lead %}{% endblock %} {% block page\_content %}

## Engineering success

#### Categories

#### **Abstract**

Our project aimed to increase vitamin B2 production in
the baker’s yeast *Saccharomyces cerevisiae* to produce vitamin B2-enriched food. We successfully overexpressed ADE4, RIB1, and RIB7 (all
combinations) in *S. cerevisiae* S288C. The engineered strains can grow normally in
YPD media and dough. The test results showed that the more
genes got overexpressed, the more vitamin B2 was produced.
Our best strain, co-overexpressing all three genes, increased the vitamin B2 content by 193%
in liquid YPD media, and by 91% in steamed buns.

#### 1. **Design**

**1.1 Yeast selection:** ***Saccharomyces cerevisiae*** **S288C**

*S. cerevisiae*, commonly known as baker's yeast, stands as one of the
most frequently utilized food microorganisms worldwide. It
has been used in baking and winemaking for thousands of
years, therefore being our perfect choice. Another
advantage of using *S. cerevisiae* for vitamin B2 production is that it has a
well-studied natural riboflavin synthesis pathway
(Gudipati et al., 2014). The *S. cerevisiae* S288C is a prototrophic strain capable of
dough-leavening. S288C is fully sequenced and widely used
in labs, which enables us to anticipate reliable outcomes
during molecular manipulations.

**1.2 Plasmid selection**

*S. cerevisiae* S288C is susceptible to G418, which makes the G418
resistance gene (KanMX) a good selection marker (Vickers
et al., 2013). And since we aimed to overexpress genes in *S. cerevisia*e, a plasmid containing a strong promoter was needed. pCEV-G4-Km is a classic *S. cerevisiae*-*Escherichia coli* shuttle cloning and expression vector containing
KanMX and the TEF1 promoter. TEF1 promoter from the yeast *Yarrowia lipolytica* has been proven one of the strongest promoters of *S. cerevisiae* (Partow et al., 2010).

![](https://static.igem.wiki/teams/4883/wiki/yuqi-eng1.png)

Figure 1. Plasmid map of pCEV-G4-Km.

**1.3 Overexpression of ADE4, RIB1, and RIB7**

According to the discovered riboflavin synthesis pathway,
GTP is the precursor of riboflavin (Kowalski et al., 2008; Gudipati et al., 2014). So, our first try was to overexpress GTP. With all the ADE enzymes, we chose to overexpress the
first enzyme, phosphoribosylpyrophosphate
amidotransferase, encoded by ADE4. This was a tentative
choice since no research has been done to determine the
rate-limiting step in the pathway. Overexpression of ADE4
should lead to overexpression of IMP, which is the
precursor of both GTP and ATP. Excess of GTP should lead
to a higher amount of vitamin B2, and excess of ATP should
facilitate cell growth as the energy carrier.

RIB genes are directly responsible for riboflavin
synthesis. With RIB1, 7, 2, 3, 4, and 5 being discovered,
we had a broad range of choices. Research in *Ashbya gossypii* and *Pichia pastoris* has shown that overexpression of any RIB gene leads to
the overexpression of riboflavin, and the more RIB genes
get overexpressed, the more vitamin B2 gets produced
(Ledesma‐Amaro et al., 2015; Marx et al., 2008). This
year, due to the time limit, we planned to only
overexpress the first two enzymes, RIB1 (GTP
cyclohydrolase II) and RIB7
(diaminohydroxyphoshoribosylaminopyrimidine deaminase). We
plan to include more genes in the future.

![](https://static.igem.wiki/teams/4883/wiki/eng2.png)

Figure 2. GTP and riboflavin synthesis pathways of *S. cerevisiae* (Kowalski et al., 2008; Gudipati et al., 2014). Green
circles indicate genes overexpressed in this project.

Then, we designed the overexpression vectors for ADE4, RIB1, and RIB7. BamHI and NheI would be used to linearize the pCEV-G4-Km. Each of the genes would be inserted between
the two restriction sites, forming pCEV-G4-ADE4-Km,
pCEV-G4-RIB1-Km, and pCEV-G4-RIB7-Km

![](https://static.igem.wiki/teams/4883/wiki/eng3.png)

Figure 3. A. Plasmid map of pCEV-G4-ADE4-Km; B. Plasmid map of pCEV-G4-RIB1-Km; C. Plasmid map of
pCEV-G4-RIB7-Km.

**1.4 Multi-gene co-overexpression**

To determine which gene influences the production of
vitamin B2 most and to determine how multiple genes work
together, we planned to co-overexpress ADE4, RIB1, and
RIB7 in all possible combinations. The combinations
included RIB1+ADE4, RIB7+ADE4, RIB1+RIB7, and
RIB1+RIB7+ADE4 .

The 2A self-cleaving peptides cleave a longer peptide
into two shorter peptides. For the polycistronic gene
expression system to work in our yeast, we planned to put
multiple genes downstream of one PTEF1 promoter and insert the 2A peptide sequences
between genes. ERBV-1 and PTV have been proven to have the
highest cleavage efficiency in *S. cerevisia*e (Souza-Moreira et al., 2018). Therefore, RIB1-PTV-ADE4,
RIB7-PTV-ADE4, RIB1-ERBV-1-RIB7, and
RIB1-ERBV-1-RIB7-PTV-ADE4 would be assembled, with the
stop codon of the prior genes deleted. Two different 2A
peptides should be used in RIB1-ERBV-1-RIB7-PTV-ADE4 to
avoid unexpected recombination.

Then, we designed the overexpression vectors for combinations of ADE4, RIB1,
and RIB7. BamHI and NheI would be used to linearize the pCEV-G4-Km. Each of the gene combinations would be
inserted between the two restriction sites, forming pCEV-G4-RIB1-PTV-ADE4-Km, pCEV-G4-RIB1-ERBV-1-RIB7-Km,
and pCEV-G4-RIB7-PTV-ADE4-Km. Specifically, we planned to
build the longest plasmid
pCEV-G4-RIB1-ERBV-1-RIB7-PTV-ADE4-Km based on constructed
CEV-G4-RIB7-PTV-ADE4-Km.

![](https://static.igem.wiki/teams/4883/wiki/eng4.png)

Figure 4. A. Map of RIB1-PTV-ADE4; B. Plasmid map of pCEV-G4-RIB1-PTV-ADE4-Km; C. Map of RIB1-PTV-RIB7; D. Plasmid map of pCEV-G4-RIB1-PTV-RIB7-Km; E.  Map of
RIB7-ERBV-1-RIB4; F. Plasmid map of pCEV-G4-RIB7-ERBV-1-RIB4-Km; G. Map of
RIB1-ERBV-1-RIB7-PTV-ADE4; H. Plasmid map of pCEV-G4-RIB1-ERBV-1-RIB7-PTV-ADE4-Km.

#### 2. **Build**

**2.1 Vector construction: single ADE4, RIB1, and
RIB7**

ADE4 (1533 bp), RIB1 (1035 bp), and RIB7 (732 bp) were
acquired by PCR (Phusion High-Fidelity PCR Master Mix, Thermo Fisher,
Waltham, MA, USA) using the genomic DNA of *S. cerevisia*e S288C as the template. BamHI and NheI (FastDigest,
Thermo Fisher, Waltham, MA, USA) were used to linearize
pCEV-G4-Km. Gibson Assembly (ClonExpress Ultra One Step Cloning Kit, Vazyme, Nanjing,
Jiangsu, China) was performed to insert each gene between the restriction
sites. The recombinant vectors pCEV-G4-ADE4-Km, pCEV-G4-RIB1-Km, and pCEV-G4-RIB7-Km were then transformed into *S. cerevisiae* S288C using the LiAc/SS Carrier DNA/PEG method
(Gietz et al., 2006).

![](https://static.igem.wiki/teams/4883/wiki/eng5.png)

Figure 5. A. Gel electrophoresis of colony PCR products
for verification of correct transformation of plasmid
pCEV-G4-ADE4-Km into *E. coli* DH5α; B. Gel electrophoresis of colony PCR products for
verification of correct transformation of plasmid
pCEV-G4-RIB1-Km into *E. coli* DH5α; C. Gel electrophoresis of colony PCR products for
verification of correct transformation of plasmid
pCEV-G4-RIB7-Km into *E. coli* DH5α.

**2.2 Multi-gene co-overexpression**

ADE4, RIB1, and RIB7 with different homologous overlaps
were acquired by PCR (Phusion High-Fidelity PCR Master Mix, Thermo Fisher,
Waltham, MA, USA) using the genomic DNA of *S. cerevisia*e S288C as the template. BamHI and NheI (FastDigest,
Thermo Fisher, Waltham, MA, USA) were used to linearize
pCEV-G4-Km. Gibson Assembly (ClonExpress Ultra One Step Cloning Kit, Vazyme, Nanjing,
Jiangsu, China) was performed to insert each gene combination between the
restriction sites.

*Problem*

We managed to construct pCEV-G4-RIB1-PTV-ADE4-Km, pCEV-G4-RIB7-PTV-ADE4-Km, and
pCEV-G4-RIB1-ERBV-1-RIB7-Km. However, when we tried to use
pCEV-G4-RIB7-PTV-ADE4-Km as the template to amplify
RIB7-PTV-ADE4 by PCR, gel electrophoresis showed no bands. We had to adjust the
PCR program to get the fragment we needed.

*Problem solved*

After several failed PCRs, we tried touchdown PCR from
68°C to 52°C. Although the
gel electrophoresis results were still smeared, bands at
around 2400 bp were identifiable. The fragments were
successfully collected through proper gel extraction,
enabling the construction of pCEV-G4-RIB1-ERBV-1-RIB7-PTV-ADE4-Km.

![](https://static.igem.wiki/teams/4883/wiki/eng6.png)

Figure 6. A. Gel electrophoresis of colony PCR products for
verification of correct transformation of plasmid pCEV-G4-RIB1-PTV-ADE4-Km (1-4), pCEV-G4-RIB7-PTV-ADE4-Km
(5-8), and pCEV-G4-RIB1-ERBV-1-RIB7-Km (9-12) into *E. coli* DH5α; B. Gel electrophoresis of colony PCR products for
verification of correct transformation of plasmid pCEV-G4-RIB1-ERBV-1-RIB7-PTV-ADE4-Km into *E. coli* DH5α.

The recombinant vectors were then transformed into *S. cerevisiae* S288C.

#### 3. **Test**

**3.1 Growth rate tests**

First of all, we wanted to make sure that our engineered
yeasts can still grow normally. Each of the strains was
inoculated into flasks containing liquid YPD media and
cultured at 30 ℃, 180 rpm. On hours 0, 8, 24, 32, 48, 56,
and 72, the OD600s were measured using a NanoDrop One
spectrophotometer (Thermo Fisher, Waltham, MA, USA).

![](https://static.igem.wiki/teams/4883/wiki/success8544.png)

Figure 7. Growth curves of engineered *S. cerevisiae* strains in liquid YPD media for 72h.

All the strains had similar growth rates, which meant
that the overexpression of ADE4, RIB1, and RIB7 did not
dramatically influence cell metabolites. In general,
strains overexpressing more than one gene showed a lower
OD600 when reached the stationary stage. This might be due
to the matter and energy used to reproduce being moved to
the overproduction of vitamin B2.

**3.2 Vitamin B2 production tests #1: in liquid YPD
media**

Then, we started to test the vitamin B2 production by
each strain. The WT *S. cerevisiae* S288C and seven engineered strains were inoculated
into YPD media, and cultured at 30 ℃, 180 rpm. On hours 0,
4, 8, 24, 28, 32, 48, 52, 56, and 72, the riboflavin
concentrations in the supernatants were measured using
high-performance liquid chromatography (HPLC) according to
GB 5009.85—2016, National Standard of the People’s
Republic of China.

![](https://static.igem.wiki/teams/4883/wiki/success9503.png)

Figure 8. Vitamin B2 contents in liquid YPD media
containing engineered *S. cerevisiae* strains for 72h.

No obvious differences between the strains can be seen in
the first 10 hours. This might be due to the low yeast
density in the media before 10 hours, which was confirmed
by Figure 7. After 10 hours, results confirmed that the WT *S. cerevisiae* S288C is capable of producing a small amount of
vitamin B2. The overexpression of any single ADE4, RIB1,
or RIB7 gene increased vitamin B2 production by about 40%.
Compared to RIB7 (33% increase), ADE4 (44% increase) and
RIB1(46% increase) were the better genes. *S. cerevisiae* showed massive vitamin B2 overproduction when two
genes were co-overexpressed. RIB1-RIB7, RIB1-ADE4, and
RIB7-ADE4 increased the vitamin B2 contents by 96%, 131%,
and 111% respectively, all higher than that of the single
gene overexpression strains. Amazingly, *S. cerevisiae* S288C overexpressing all three genes produced the
most vitamin B2, resulting in a nearly three-fold increase
(193% higher).

**3.3 Vitamin B2 production tests #2: in self-made
steamed buns**

To determine whether our engineered yeasts can help to
make vitamin-B2-enriched food, we made steamed buns using
our yeasts in the lab. For each strain, 50 g of bread
flour was mixed with 0.5 g of yeast and 50 mL of water.
The dough was placed in plastic bags and incubated at 30
°C for until it rose.

In our lab, it took the dough about 4 hours to rise.
However, most recipes say it should rise in less than 1.5
hours. This difference might be due to our original strain
choice. We used a laboratory strain, S288C, as our
chassis, but the recipes use commercial yeast strains. It turned out that S288C was not as efficient as the
commercial strains in bread-making, which indicated that
we should change our chassis to the commercial strains in
the future.

Then, we put the dough in a food steamer for 15 minutes.
The riboflavin concentrations in the buns were measured
using high-performance liquid chromatography (HPLC).

![](https://static.igem.wiki/teams/4883/wiki/success11523.png)

Figure 9. A. Self-made teamed bun using *S. cerevisiae* S288C carrying pCEV-G4-RIB7-Km; B. Vitamin B2 contents in steamed buns leavened by
engineered *S. cerevisiae* strains. \*: p < 0.01; \*\*: p < 0.001.

All the strains helped to make the vitamin-B2-enriched
buns. Overexpression of RIB1, RIB7, ADE4, and RIB1-RIB7
contributed to a small amount of vitamin B2 increase (all
< 25% increase, p > 0.01). RIB1-ADE4 and RIB7-ADE4
overexpression strains significantly (p < 0.01)
increased the vitamin B2 amounts in the steamed buns by
67% and 64% respectively. The highest overproduction of
vitamin B2 came from the strain stacking RIB1, RIB7, and
ADE4, resulting in a 91% increase (p < 0.0001).

#### 4. **Learn**

We proved that overexpression of ADE4, RIB1, and RIB7
in *S. cerevisiae* could lead to vitamin B2 overproduction without
influencing cell growth rate. We successfully found the
strain that produced the most vitamin B2 in bun-making,
which is the strain co-overexpressing all three genes. In
the future, we plan to introduce more ADE and RIB genes
into *S. cerevisiae* to reach higher vitamin B2 production. We also plan to switch our chassis from the lab strain *S. cerevisiae* S288C to some commercial strains.

#### **References**

Gietz RD, Woods RA. Yeast transformation by the LiAc/SS
Carrier DNA/PEG method. Methods Mol Biol. 2006;313:107-20.
doi: 10.1385/1-59259-958-3:107. PMID: 16118429.

Gudipati, V.; Koch, K.; Lienhart, W.-D.; Macheroux, P.
The Flavoproteome of the Yeast Saccharomyces Cerevisiae. *Biochimica et Biophysica Acta (BBA) - Proteins and
Proteomics* **2014**, *1844* (3), 535–544. DOI:10.1016/j.bbapap.2013.12.015.

Kowalski, D.; Pendyala, L.; Daignan-Fornier, B.; Howell,
S. B.; Huang, R.-Y. Dysregulation of Purine Nucleotide
Biosynthesis Pathways Modulates Cisplatin Cytotoxicity in *Saccharomyces Cerevisiae*. *Molecular Pharmacology* **2008**, *74* (4), 1092–1100. DOI:10.1124/mol.108.048256.

Ledesma-Amaro, R.; Serrano-Amatriain, C.; Jiménez, A.;
Revuelta, J. L. Metabolic Engineering of Riboflavin
Production in Ashbya Gossypii through Pathway
Optimization. *Microbial Cell Factories* **2015**, *14* (1). DOI:10.1186/s12934-015-0354-x.

Marx, H.; Mattanovich, D.; Sauer, M. Overexpression of
the Riboflavin Biosynthetic Pathway in Pichia Pastoris. *Microbial Cell Factories* **2008**, *7* (1). DOI:10.1186/1475-2859-7-23.

Partow, S.; Siewers, V.; Bjørn, S.; Nielsen, J.; Maury,
J. Characterization of Different Promoters for Designing a
New Expression Vector in Saccharomyces Cerevisiae. *Yeast* **2010**, *27* (11), 955–964. DOI:10.1002/yea.1806.

Souza-Moreira TM, Navarrete C, Chen X, Zanelli CF,
Valentini SR, Furlan M, Nielsen J, Krivoruchko A.
Screening of 2A peptides for polycistronic gene expression
in yeast. FEMS Yeast Res. 2018 Aug 1;18(5). doi:
10.1093/femsyr/foy036. PMID: 29617770.

Vickers CE, Bydder SF, Zhou Y, Nielsen LK. Dual gene
expression cassette vectors with antibiotic selection
markers for engineering in Saccharomyces cerevisiae.
Microb Cell Fact. 2013 Oct 25;12:96. doi:
10.1186/1475-2859-12-96. PMID: 24161108; PMCID:
PMC4231455.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

---

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

{% endblock %}