iGEM 2025 — Team jlu-nbbms

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}This serves as a chronological record of your team's progress throughout the season. It documents your
daily activities, experiments, discussions, and decisions.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5564/banner/notebook.webp)

* Brainstorm
* Project promotion
* MVA biosynthesis
* MVA to oleanolic acid
* Oleanolic acid to Ro
* UDPG biosynthesis

# Brainstorm

2025.01.06

We have formally established the 2025 JLU-NBBMS
team.

2025.01.14

First Brainstorming Session

This marks our inaugural brainstorming session,
requiring team members to share their conceptual
ideas.

Min Yingxuan proposed employing the MBR method
to isolate carbon sources from oily industrial
wastewater, then utilising synthetic biology to
enable chassis organisms to utilise these carbon
sources, ultimately synthesising ginsenosides.

You Mingyu proposed utilising engineered
Bacillus subtilis to degrade harmful substances
such as phenolic acids in soil, thereby
improving ginseng growth conditions and
enhancing saponin yield. He further suggested
immobilising the engineered strain using biochar
to ensure experimental safety.

Zhang Jiayi and Li Ruoxi proposed constructing
anti-diabetic ginsenoside nanocarriers based on
DNA origami. They utilised Y-shaped scaffold DNA
tiling technology to prepare rigid DNA
tetrahedral ROTs. A multifunctional nanoparticle
system featuring folic acid and tripeptide
dual-modified PLGA was developed to
progressively overcome continuous absorption
barriers.

Ou Liyang proposed constructing a multi-enzyme
assembly system within Saccharomyces cerevisiae
to synthesise ginsenosides, alongside designing
sensors and adhesion proteins to facilitate
yeast colonisation at intestinal tumour sites.

Chang Xuanhe proposed preparing ginsenosides as
swellable nanoparticles to load trace amounts of
ginsenoside Rg3, with lipid carrier NLC
enhancing ginsenoside targeting and delivery
efficiency.

Sun Jiayin proposed constructing a mesenchymal
stem cell-ginseng exosome fusion vesicle as a
carrier, containing specific molecular tools.
The carrier could be engineered to target
diseased cells, thereby treating psoriasis.

Song Jie proposed utilising compartmentalised
expression technology to engineer ginsenoside
production, followed by the self-assembly of
traditional Chinese medicine components into
natural small-molecule gels exhibiting
pharmacological effects such as anti-ageing
properties.

![](https://static.igem.wiki/teams/5564/notebook/nobs001.webp)

2025.02.08

Second Brainstorming Session

This marks our second brainstorming session.
Following the initial session, we conducted
preliminary feasibility analyses of our ideas,
exchanged questions and feedback, and selected
the most viable projects for group work. This is
our first presentation since forming the groups.

Group One: Synthesis of Ginsenosides from Edible
Oil Industry Wastewater Building upon the
initial presentation, the team gained deeper
insights into the Membrane Bioreactor (MBR)
process. They proposed a stepwise synthesis
pathway from edible oils to ginsenosides,
identified key genes and synthesis strategies
through literature review, and outlined a design
concept for a gene oscillator. However, the
approach was questioned due to the large number
of genes involved, which would prevent sustained
product output post-oscillation, necessitating
revision. The team proposed organelle
compartmentalisation for production, though this
requires integration with the synthesis process.

The second group focused on improving soil for
ginseng continuous cropping using
biochar-immobilised Bacillus subtilis. Following
concerns raised during the initial presentation
about the safety of the engineered strain, the
team optimised the genetic circuit based on the
principle of population effects. They designed a
suicide switch to enhance project safety and
refined the biochar preparation and modification
protocols.

The third group developed a ginsenoside DNA
origami sponge for moisture retention and
anti-photoaging. Building upon their initial
presentation, they designed a three-dimensional
spatial structure for the origami sponge,
enabling oxidative stress gating during
ginsenoside experiments.

The fourth group focused on Saccharomyces
cerevisiae expressing ginsenosides, colonised at
colon cancer sites to provide adjuvant therapy.
Building upon their initial presentation, they
refined the customisation and therapeutic
systems, constructing a relatively complete
sensor.

![](https://static.igem.wiki/teams/5564/notebook/nobs009.webp)
![](https://static.igem.wiki/teams/5564/notebook/nobs010.webp)
![](https://static.igem.wiki/teams/5564/notebook/nobs011.webp)
![](https://static.igem.wiki/teams/5564/notebook/nobs012.webp)

# Project promotion

2025.02.10

First Principal Investigator (PI)
Presentation:
This marked our inaugural formal engagement with
the PI. During this session,
each group presented their design
proposals, with the PI providing specific feedback to
refine project selection.

(1) The first group focused on
synthesising ginsenosides from industrial
waste oils. Members presented in four sections, providing
an in-depth explanation of the MBR process. They
outlined a stepwise synthesis pathway
from oils to ginsenosides (from oils to
MVA, and from MVA to ginsenosides), identified key genes and synthetic
strategies, proposed a design concept for a
genetic oscillator, and divided the synthesis
into two systems (production and control
systems).
The Principal Investigator (PI) expressed
initial approval, noting the project's strong embodiment of
synthetic biology principles alongside
environmental sustainability. The system design
was deemed relatively robust with high
experimental feasibility.
The PI offered suggestions for
refinement, highlighting that the extensive target gene
selection and numerous synthesis modules risked
exceeding yeast's plasmid capacity limits. The
final recommendation was to prioritise key
elements during design.

(2) The second group proposed
using biochar-immobilised Bacillus subtilis
to remediate ginseng-contaminated
soils. Team members suggested employing synthetic
biology to degrade phenolic acids, thereby
reducing phytohormone toxicity, and utilising
biochar to immobilise Bacillus subtilis for
synergistic soil remediation. The PI noted this
project well aligns with iGEM's founding
principles but highlighted the difficulty in
modifying biochar and the relatively low
experimental feasibility.

(3) The third group proposed
a ginsenoside DNA origami suction cup for
moisture retention and anti-photoaging. Team members suggested employing DNA origami
principles to enable DNA self-assembly into
suction cups, thereby addressing skin photoaging
caused by UV radiation. The PI acknowledged the
project's strong innovation but highlighted
challenges in achieving the research objectives
and forming the multi-enzyme complex.

(4) The fourth group proposed
colonising colon cancer sites with
Saccharomyces cerevisiae expressing
ginsenosides for adjuvant therapy. Team members suggested constructing
therapeutic and colonisation systems. The PI
noted that building multi-enzyme complexes
requires not only multiple genes but also
connecting necessary components, involving
numerous plasmids with potentially limited
efficacy.

Summary:
Based on project feasibility, synthetic
biology principles, and the original intent
of environmental sustainability and
efficiency, the Principal Investigator ultimately
selected the project 'Synthesis of Ginsenosides
from Waste Oils and Fats'. This was deemed
highly feasible, consistent with synthetic
biology characteristics, and capable of
achieving favourable outcomes leveraging the
laboratory platform.

![](https://static.igem.wiki/teams/5564/notebook/nopp001.webp)
![](https://static.igem.wiki/teams/5564/notebook/nopp002.webp)
![](https://static.igem.wiki/teams/5564/notebook/nopp003.webp)
![](https://static.igem.wiki/teams/5564/notebook/nopp004.webp)
![](https://static.igem.wiki/teams/5564/notebook/nopp005.webp)
![](https://static.igem.wiki/teams/5564/notebook/nopp006.webp)

2025.03.09

This marks the inaugural group discussion
to determine the project's thematic
focus.

Improvements over
previous designs include:

(1)
Selection of a specific ginsenoside
type—Oleanolic acid-type ginsenoside
Ro.
This compound exhibits well-defined
pharmacological effects yet remains
underutilised in biosynthetic approaches. Given
its anti-ageing and antioxidant properties,
subsequent applications may extend to cosmetic
production.

(2)
Establishing Saccharomyces cerevisiae as
the biological chassis, owing to its native MVA pathway in the
cytoplasm and essential saponin synthesis
systems including cytochrome monooxygenases and
glycosyltransferases.

(3)
Determining the method for generating
acetyl-CoA from fatty acids—by targeting key MVA pathway enzymes to the
peroxisomes.

(4) Establishing the specific Ro synthesis
strategy—including glycosyltransferase and key
genes for saponin aglycone synthesis.

Proposing multiple optimisation
approaches—reducing bypass metabolism,
optimising key enzymes, enhancing promoter
efficiency, and improving fermentation
conditions.

2025.03.15

We visited Professor Gao Renjun at the
School of Life Sciences to interview him
regarding enzyme modification.
We provided Professor Gao with a detailed
explanation of the project and raised questions
concerning the enzyme modification engineering.

The improvements made
were:

(1) For the glycerol to 2,3-OSQ metabolism
pathway,
it is preferable to utilise the organism's
own metabolic pathway. This approach reduces the number of plasmids
required and prevents metabolic disruption in
the yeast.

(2) Project focus should
prioritise the subsequent conversion of OA
to Ro; thus, the ergosterol pathway need not be
inhibited to avoid compromising yeast viability.

(3)
A website for glycosyltransferases was
recommended. Screening enzymes from databases via
molecular dynamics simulations is advised to
reduce time expenditure, or AI methods could
accelerate progress.

(4) Given the numerous glycosylation steps in
ginsenoside synthesis requiring
substantial UDPG consumption, enhancing endogenous UDPG pathways is
advised.

Summary: Professor Gao emphasised holistic
project management, advocating targeted
focus on the latter stages. Regarding enzyme
engineering, he proposed two more feasible
approaches: AI integration and
database-driven screening.

![](https://static.igem.wiki/teams/5564/notebook/nopp007.webp)
![](https://static.igem.wiki/teams/5564/notebook/nopp008.webp)

2025.3.19

Second PI Report

This time, incorporating expert feedback and
group discussion outcomes, we present specific
optimisation proposals, including:

(1)
Reducing the additional glycerol metabolic
pathway
by utilising acetyl-CoA generated through its
natural metabolic pathway.

Redesigning the Ro pathway to generate
glycosylation pathways, allocating distinct metabolic fluxes to R1
and 4a. Through in vitro optimisation of the
glycosyltransferase, we
have engineered differing metabolic flux levels
between the two pathways. This ensures that the
enzyme UGT73F3 exhibits significantly greater
affinity for 4a than for CE.

2025.04.03

Visited the School of Pharmacy to interview
Professor Zhang Xinmin, an expert in synthetic biology. We provided
Professor Zhang with a detailed explanation of
the project and conducted a preliminary
assessment of its experimental feasibility.

Improvements include:

(1) When loading two to three genes onto a
single plasmid vector,
employing a promoter-terminator fusion
approach
would be more conducive to protein expression.
Additionally, note that different selection
conditions should be chosen for different
plasmids; consideration should be given to using
nutritional selection conditions to reduce
experimental costs.

(2)
The final ginsenoside Ro product should be
validated using high-performance liquid
chromatography (HPLC).
As unexpected outcomes may arise during
experimentation, separate experimental groups
should be established with distinct experimental
conditions.

(3)
This project embodies ecological
conservation and waste recycling
principles.
Glycerol is inexpensive, being the primary
by-product of biodiesel production, and numerous
studies currently explore its use as a feedstock
for value-added products. The approach of
introducing key enzymes into peroxisomes for
fatty acid metabolism via the mevalonate pathway
demonstrates innovation. However, prior
formulation of the culture medium composition is
essential.

![](https://static.igem.wiki/teams/5564/notebook/nopp009.webp)
![](https://static.igem.wiki/teams/5564/notebook/nopp010.webp)

2025.04.10

This was a group discussion during which we
assigned roles to members of the
experimental group
and, incorporating Professor Zhang's insights,
highlighted common pitfalls in plasmid design.
We determined that the plasmids we require must
synthesise the following functions:

(1) Utilising fatty acids to generate
acetyl-CoA for MVA synthesis

(2) Producing the key enzyme necessary for
oleanolic acid (OA) generation

(3) The key enzyme required for converting
oleanolic acid (OA) into the rare
ginsenoside Ro

(4) The key enzyme required for the UDPG
pathway

2025.04.10-04.25

Team members design plasmid maps, determine
vector types, screen for labels, and select
promoter-terminator combinations and tag types.

2025.04.29

A further visit to Professor Zhang Xinmin's
laboratory was made to finalise specific
experimental details, during which the application of carbon
sources was discussed in depth.
Given that fatty acids are categorised as
either saturated or unsaturated, with
unsaturated fatty acids existing in liquid
form and saturated fatty acids in solid
form, the formulation of the culture medium must
consider how yeast can utilise fatty acids. This
may involve various approaches, such as the
addition of emulsifiers.

2025.05.01-06.01

Group members shall procure materials relevant
to the experiment as required by the
experimental needs and under the guidance of the
Principal Investigator, and shall finalise the
experimental protocols and schedule.

2025.08.20

Visited Professor Zhang Zuoming, an expert in
synthetic biology at the School of Life
Sciences, for an interview.

Upon obtaining the HPLC yield results for
the rare ginsenoside Ro, we observed that
alongside the target product peak, the HPLC
results also revealed a deglycosylation
product of Ro—a disaccharide
ginsenoside.
Based on the Ro synthesis process, we
hypothesised this was caused by accumulation of
intermediate products.
We consulted Professor Zhang regarding this
issue, proposing the following
suggestions:

(1) Determine whether the issue lies with
plasmid expression or a defect in the
metabolic pathway encoded by the key
gene?

(2) Validate successful plasmid transfection and
expression via
PCR and Western Blot analysis.

(3)
Assess enzyme activity through in vitro
enzymatic assays.
Professor Zhang suggested that the heterologous
gene, derived from the plant Panax notoginseng,
may undergo excessive glycosylation upon
transfection into yeast, leading to enzyme
inactivation.

(4)
Conduct in vitro experiments to assess
conversion efficiency across both
pathways, and implement a 'source-cutting and
flow-interception approach through dry
experiments.

# Convert glycerol and fatty acids into MVA

Cultivation and Cryopreservation of Yeast

Experimental time: 2025.06.25

Experimental personnel: Zhao Xiaoqing, Sun
Jiayin

Objectives of the Experiment

1. Cultivate and freeze-store Saccharomyces
cerevisiae to prepare for subsequent plasmid
transfection in the production of competent
cells.

2. Prepare the plasmid as a solution for
subsequent transfection.

Experimental Equipment and Reagents

Equipment

Shaker, refrigerator, laminar flow hood,
spectrophotometer

Reagents

YPD medium, plasmid powder, sterile water,
glycerol

Reagent Preparation

Table 1 YPD Medium(500mL)

| Reagent | Amount |
| --- | --- |
| Peptone | 10g |
| Yeast extract | 5g |
| H2O | 500mL |
| glucose | 10g |

Experimental Procedure

Cultivation of Saccharomyces
cerevisiae

(1) Irradiate the ultra-clean workbench with
ultraviolet light for 30 minutes beforehand.
Prepare YPD medium and microwave-treat it in
advance for later use;

(2) Take a 50 mL centrifuge tube and pour 5 mL
of YPD liquid medium into it;

(3) Pick a single colony of Saccharomyces
cerevisiae W303 from the culture plate and
inoculate into the tube. Incubate on a shaking
incubator (30°C, 200 rpm);

(4) After approximately 7 hours, measure the
OD600. If OD600 = 0.3, add 2 mL of the culture
to 100 mL of YPD medium.

Prepare the plasmid

(1) Retrieve the plasmid powder tube and
centrifuge (4μg);

(2) Add 30μL sterile water to the tube;

(3) Transfer 10μL to an EP tube, then add 32μL
sterile water for later use;

(4) Store the remaining 20μL plasmid at -20°C.

Freeze-preserve experimental strains

Take an EP tube, add 750μL bacterial suspension
and 750μL glycerol (50%) in a 1:1 ratio, mix,
and store at -80°C. Replenish the remaining
bacterial suspension in the centrifuge tube with
appropriate medium.

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/nomv02.webp)
![](https://static.igem.wiki/teams/5564/notebook/nomv03.webp)

Analysis and Discussion

Yeast exhibited robust growth and is suitable
for preparing competent cells for subsequent
experiments.

Precautions

(1) Maintain aseptic technique when isolating
single-colony yeast to prevent contamination by
extraneous microorganisms.

(2) When preparing YPD medium, avoid excessive
temperatures during autoclaving. Typically,
autoclave at 115°C for 15 minutes; higher
temperatures may cause glucose to darken.

(3) Ensure culture duration does not exceed
recommended limits. If optical density (OD)
exceeds 1.2, the strain is unsuitable for
subsequent procedures.

Preparation of Sc-Trp Medium and Competent Cells

Experiment Date: 2025.06.29

Experiment operator: Zhao Xiaoqing, Sun Jiayin

Objectives

1. Prepare Sc-Trp medium and plate it to
facilitate subsequent cultivation of
Saccharomyces cerevisiae following plasmid
transfection.

2. Prepare competent cells for plasmid
transfection.

Experimental Equipment and Reagents

Equipment

Laminar flow hood, spectrophotometer, autoclave,
shaking incubator

Reagents

SC-Trp, arginine, adenine, glucose, YNB,
ammonium sulphate, agar, sterile water, LiAC/TE
buffer

Reagent Preparation

Table 1 Sc-Trp Medium (200 mL)

| Reagent | Amount |
| --- | --- |
| Sc-Trp | 0.4g |
| L-Arginine | 0.04g |
| adenine | 0.2g |
| glucose | 4g |
| agar | 4g |
| YNB | 1.6g |
| (NH4)2SO4 | 5g |
| H2O | 200mL |

Experimental Procedure

Preparation of SC-Trp Medium

(1) Take 0.4 g SC-Trp, 0.04 g arginine, 0.2 g
adenine, and 4 g glucose; add to 60 mL water and
place in an empty bottle;

(2) Take 4 g agar and add to 120 mL water; place
in another empty bottle and add a rotor;

(3) Place both bottles alongside an empty 100 mL
bottle into an autoclave for sterilisation
(115°C, 15 minutes);

(4) Weigh 1.6 g of YNB yeast and 5 g of ammonium
sulphate into a beaker. Add 100 mL of sterilised
water and stir with a stirrer rod until
completely dissolved;

(5) Sterilise the solution by filtration through
a 0.22μm filter, then transfer to a 100mL
sterile autoclave bottle for later use;

(6) Take 20mL of the sterilised YNB solution and
add to the sterilised agar bottles. While hot,
pour the SC-Trp solution into the agar bottles
containing YNB and mix thoroughly using a
magnetic stirrer;

(7) Swiftly pour the mixture into sterile Petri
dishes to form plates. Allow to stand at room
temperature for 4 hours before refrigerating for
later use.

Preparation of Competent Cells

(1) Place sterile water in a 4°C refrigerator
for later use. Dilute LiAC/TE to 0.1 mol/L for
later use;

(2) Measure the OD600 of the Saccharomyces
cerevisiae culture from the previous day.
Proceed to the next step if OD600 = 0.8;

(3) Take 50 mL of the culture, centrifuge (3500
rpm, 2 min), and discard the supernatant;

(4) Resuspend in 50 mL of cold sterile water,
centrifuge (3500 rpm, 2 min), and discard the
supernatant;

(5) Resuspend in 1 mL 0.1 mol/L LiAC/TE buffer,
centrifuge briefly (4000 rpm, 30 s), and discard
supernatant;

(6) Resuspend in 400 μL 0.1 mol/L LiAC/TE
buffer, mix thoroughly, and refrigerate
overnight on ice.

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/nomv05.webp)
![](https://static.igem.wiki/teams/5564/notebook/nomv06.webp)

Precautions

(1) After filtration and sterilisation, the YNB
solution must be rapidly poured into agar while
hot to prevent uneven distribution causing
abnormal solidification of the medium.

(2) Once prepared, the medium must be promptly
poured into plates to prevent premature
solidification due to prolonged exposure.

(3) Monitor the OD value of Saccharomyces
cerevisiae to prevent excessive incubation time,
which may render the sample unsuitable for
subsequent experiments.

Plasmid ① transfection; Saccharomyces
cerevisiae
culture post-transfection

Experiment date: 2025.06.30

Experiment operator: Wang Quanhao

Experimental Objectives

(1) Utilise prepared competent cells for plasmid
transfection;

(2) Transfect Saccharomyces cerevisiae competent
cells with Plasmid ① to utilise fatty acids;

(3) Prepare YPD medium and Trp-deficient solid
medium for future use;

Experimental Equipment and Reagents

Equipment

Laminar flow hood, shaker, 30°C incubator, water
bath, rocking incubator, oven, electronic
balance, magnetic stirrer, autoclave

Reagents

PEG, LiAc (1 mol/L), salmon sperm, plasmid,
yeast competent cells, YPD medium, PEG, DMSO
Glucose, peptone, arginine, adenine, SC-Trp;

Reagent Preparation

3.1 YPD medium (500 mL)

| Reagent | Amount |
| --- | --- |
| Peptone | 10g |
| Yeast extract | 5g |
| H2O | 500mL |
| glucose | 10g |

3.2 Trp-deficient solid medium (180 mL)

| Reagent | Amount |
| --- | --- |
| SC-Trp | 0.4 g |
| Arginine | 0.04 g |
| Adenine | 0.2 g |
| 20% Glucose | 4 g |
| H2O | 60 mL |
| Take another empty bottle and add the rotor. | |
| Agar | 120 g |
| H2O | 120 mL |

3.3 Preparation of YNB Medium

| Component | Consumption (100mL) |
| --- | --- |
| YNB for yeast culture | 1.6g |
| (NH4)2SO4 | 5g |
| H2O | 100mL |

Experimental Procedure

Plasmid Transfection

1.1 Boil salmon sperm in water for 5 minutes,
then promptly place into an ice-water mixture
(on ice).

1.2 Add approximately 50 μL of the yeast
competent cells prepared the previous day to
each Eppendorf tube. Centrifuge (4000 rpm, 30
seconds) and discard the supernatant.

1.3 Add sequentially to the EP tube: 42 μL
plasmid, 10 μL salmon sperm (10 mg/mL), 240 μL
PEG, and 36 μL LiAc (1 mol/L). Mix thoroughly by
repeated pipetting with a 1 mL pipette, then
incubate at 30°C for 30 minutes.

1.4 Add 36 μL DMSO to each EP tube. Mix
thoroughly by pipetting and heat-shock at 42°C
for 20 minutes.

Post-transfection yeast culture

2.1 Remove EP tubes from water bath. Centrifuge
(3000 rpm, 20 seconds) and discard supernatant.
Resuspend in 1 mL YPD medium and incubate on
shaking incubator for 3 hours.

2.2 Centrifuge (3000 rpm, 20 s), discard
supernatant, resuspend in 100 μL water. Spread
culture onto plates (Sc-Trp) using glass beads
and incubate at 30°C for approximately 3 days.

Preparation of Trp-deficient solid medium

3.1 Prepare Trp-deficient solution as per
Section 3.2 under 'II', then prepare YNB
solution as per Section 3.3.

3.2 Place glass bottles containing the solutions
into an autoclave for sterilisation (115°C, 15
min).

3.3 Sterilise the YNB solution by filtration
through a 0.22 μm filter, then transfer to a 100
mL sterile autoclave bottle for storage.

3.4 Take 20 mL of the sterilised YNB solution
and add to the Trp double-deficient solution,
mixing thoroughly.

3.5 Pour the medium into sterile Petri dishes
(approximately 15-20 mL per dish). Allow to
solidify at room temperature before inverting
and storing at 4°C (prevent condensation
accumulation).

Preparation of YPD Medium

4.1 Prepare YPD medium as per Section 3.1 of
Part II. Follow the remaining steps as per
Section 4 of Part III. Sterilise by UV
irradiation as a liquid medium.

Precautions

1. Salmon sperm must be pre-boiled for 5 minutes
in boiling water, then rapidly transferred to an
ice-water mixture to preserve exogenous
plasmids.

2. All reagents required for plasmid
transfection must be thoroughly mixed to
maximise cell membrane permeability and enhance
transfection efficiency.

3. Incubation times in the cell culture
incubator and heat shock in the water bath must
be strictly controlled to prevent reduced
transfection efficiency due to prolonged
exposure.

![](https://static.igem.wiki/teams/5564/notebook/nomv07.webp)
![](https://static.igem.wiki/teams/5564/notebook/nomv08.webp)

Growth curve measurement of single yeast clones
containing plasmid ① in different glycerol-fatty
acid media

Experiment date: 2025.07.02

Experiment operator: Sun Jiayin, Min Yingxuan,
Wang Jingning

Objective

Yeast strains transformed with plasmid ① and the
wild-type w303 strain were used as controls,
while various glycerol-fatty acid media were
prepared to screen for the yeast strain
exhibiting the highest selective utilization of
a specific fatty acid.

Equipment and Reagents

Equipment

Laminar flow hood, autoclave, shaker, microplate
reader, 96-well plates

Reagents

Sc-Trp、Arg、Adenine
sulfate、ddH2O、Glycerol、Lauric acid、Oleic
acid、Palmitic acid

Reagent Preparation

(1) Place the following ingredients into a clean
bottle;

| Component | Amount Consumed (180 mL) |
| --- | --- |
| SC-Trp | 0.4g |
| Arginine | 0.04g |
| Adenine sulfate | 0.2g |
| Water | 180 milliliters |

(2) Add X g of solid stearic acid and Y g of
lauric acid separately into two 1.5 mL
centrifuge tubes. Add 150 μL of anhydrous
ethanol to each centrifuge tube and dissolve in
a 60 °C water bath. Immediately transfer to
sterile caps of clean 50 mL centrifuge tubes.
Subsequently, dry the dissolved solids in a fume
hood until no residual liquid remains on the
surfaces of the centrifuge tubes and all
anhydrous ethanol has completely evaporated.

(3) Transfer dried palmitic acid and lauric acid
separately into EP tubes, add 1.8 mL of water
and 0.18 mL of Tween-80, then dissolve
completely by heating in a water bath with
continuous stirring.

(4) Take another EP tube, add Z mL of oleic
acid, M mL of glycerol, and 0.18 mL of Tween-80,
then heat in a water bath and mix thoroughly.

(5) Preheat the solution obtained in step (1)
while stirring continuously. Add palmitic acid,
lauric acid, oleic acid, and glycerol
sequentially into the container prepared in step
(1), mix thoroughly, and sterilize by
autoclaving at 115°C for 15 minutes.

(6) Finally, add 20 mL of YNB solution to the
container to complete the preparation. Transfer
the dried palmitic acid into EP tubes, adding
1.8 mL of water and 0.18 mL of Tween-80,
respectively. Heat in a water bath and stir
thoroughly.

| Glycerol-fatty acid medium | X (g) | Y (g) | Z (mL) | M (ml) |
| --- | --- | --- | --- | --- |
| Glycerol: Oleic acid = 1:1 | 0 | 0 | 0.18 | 0.18 |
| Glycerol: Palmitic acid = 1:1 | 0.14 | 0 | 0 | 0.18 |
| Glycerol: Lauric acid = 1:1 | 0 | 0.12 | 0 | 0.18 |

Experimental Procedures

(1) The biosafety cabinet was irradiated with
ultraviolet light 30 minutes prior to use;

(2) Take a 50 mL centrifuge tube and add 15 mL
of glycerol-fatty acid liquid medium into the
tube;

(3) Pick a single yeast clone from the
Saccharomyces cerevisiae plate transformed with
Plasmid ① and add it into the centrifuge tube,
then incubate on a shaker at 30℃ and 200 rpm;

(4) Every 2 hours, take an aliquot of the liquid
culture of the single yeast clone transformed
with Plasmid ① and observe yeast growth by
checking for turbidity in the liquid medium.

(5) Using a 96-well plate, every 2 hours, take
100 µL of yeast suspension from each medium and
add it into the plate, with three replicates for
each group; measure OD600 using a microplate
reader and calculate the average.

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/nomv09.webp)
![](https://static.igem.wiki/teams/5564/notebook/nomv10.webp)
![](https://static.igem.wiki/teams/5564/notebook/nomv110.webp)

Analysis and Discussion of Results

Overall, yeast transformed with Plasmid ①
exhibited significantly greater growth than the
wild-type yeast W303, indicating that the
transformed yeast can effectively utilize the
glycerol-fatty acid medium for growth,
confirming the successful introduction of
Plasmid ①.

The OD values measured by the microplate reader
for yeast transformed with Plasmid ① during the
growth plateau phase across fatty acid media
followed the order: glycerol:palmitic acid = 1:1
> glycerol:oleic acid = 1:1 > glycerol:lauric
acid = 1:1, demonstrating that yeast utilize
palmitic acid and oleic acid significantly more
effectively than lauric acid. This may be
attributed to the fact that yeast cells
transformed with Plasmid ① possess an inherent
mechanism for the efficient metabolism of
long-chain fatty acids. Palmitic acid (16
carbons) and oleic acid (18 carbons) are the
predominant fatty acid components in the
phospholipids of yeast cell membranes, with
their synthesis and degradation pathways being
well established. In contrast, lauric acid, a
12-carbon medium-chain fatty acid, is not a
principal component of the yeast's endogenous
metabolism. Its shorter carbon chain may cause
the lauroyl-CoA produced upon activation to
exhibit low affinity toward yeast acyl-CoA
metabolic enzymes, such as those involved in
β-oxidation, thus impeding its efficient
incorporation into central energy metabolism or
biosynthetic pathways and resulting in decreased
utilization efficiency.

In subsequent experiments, palmitic acid and
oleic acid will be selected as the main fatty
acid components. A glycerol-to-fatty acid ratio
of 1:1 will be maintained for liquid culture of
the strain carrying plasmid ①, in order to
observe plasmid ①'s preference for different
types of fatty acid mixtures.

Notes

(1) Set up the wild-type w303 strain without
plasmid ① as a control group, to better assess
whether plasmid ① is successfully expressed.

(2) Measure the growth OD value approximately
every 2 hours to allow timely monitoring of
yeast growth rate and extent.

Growth curve determination of plasmid ① yeast
monoclonal cultures in glycerol-fatty acid media
at varying ratios

Experiment date: 2025.07.05

Experiment operator: Jiayin Sun, Yingxuan Min,
Jingning Wang

Experimental objective

Building upon Cycle 1, palmitic acid and oleic
acid were selected as the principal fatty acid
components, maintaining a glycerol:fatty acid
ratio of 1:1 for liquid culture of plasmid
①-transformed strains to assess plasmid ①
preference for different fatty acid mixtures.

Experimental Equipment and Reagents

Equipment

Biosafety cabinet, autoclave, shaker, microplate
reader, 96-well plate

Reagents

Sc-Trp, Arg, Adenine sulfate, ddH 2 O, Glycerol,
Lauric acid, Oleic acid, Palmitic acid, Tween
80, Anhydrous ethanol

Reagent Preparation

(1) Add the following components into empty
bottles;

| Glycerol-fatty acid medium | X (g) | Y (g) | Z (mL) | M (ml) |
| --- | --- | --- | --- | --- |
| Glycerol: Oleic acid = 1:1 | 0 | 0 | 0.18 | 0.18 |
| Glycerol: Palmitic acid = 1:1 | 0.14 | 0 | 0 | 0.18 |
| Glycerol: Lauric acid = 1:1 | 0 | 0.12 | 0 | 0.18 |

(2) Add X g of solid stearic acid and Y g of
lauric acid separately into two 1.5 mL
centrifuge tubes. Add 150 μL of anhydrous
ethanol to each centrifuge tube and dissolve in
a 60°C water bath. Immediately transfer to the
sterile cap of a clean 50 mL centrifuge tube.
Dry the dissolved solid material in a fume hood
until no liquid residue remains on the
centrifuge tube surface and all anhydrous
ethanol has completely evaporated.

(3) Transfer the dried palmitic acid and lauric
acid separately into EP tubes, add 1.8 mL of
water and 0.18 mL of Tween-80, then heat in a
water bath with thorough stirring to dissolve.

(4) In another EP tube, add Z mL of oleic acid,
M mL of glycerol, and 0.18 mL of Tween-80, then
place it in a water bath for heating and
thorough mixing.

(5) Preheat the solution obtained in step (1)
with continuous stirring.

(6) Add palmitic acid, lauric acid, oleic acid,
and glycerol sequentially to the container
prepared in step (1). After thorough mixing,
sterilize by autoclaving at 115°C for 15
minutes.

(7) Finally, add 20 mL of YNB solution to the
container to complete the preparation. Transfer
the dried palmitic acid to EP tubes and add 1.8
mL of water and 0.18 mL of Tween-80,
respectively. Heat in a water bath and stir
thoroughly.

| Glycerol-fatty acid medium | X (g) | Y (g) | Z (mL) | M(ml) |
| --- | --- | --- | --- | --- |
| Glycerol:Lauric acid:Oleic acid = 2:1:1 | 0 | 0.06 | 0.09 | 0.18 |
| Glycerol:Lauric acid:Palmitic acid = 2:1:1 | 0.07 | 0.06 | 0 | 0.18 |
| Glycerol:Palmitic acid:Oleic acid = 2:1:1 | 0.07 | 0 | 0.09 | 0.18 |
| Glycerol:Palmitic acid:Oleic acid: Lauric acid = 3:1:1:1 | 0.047 | 0.04 | 0.06 | 0.18 |

Experimental procedure

(1) Irradiate the biosafety cabinet with UV
light for 30 minutes prior to use;

(2) Transfer 15 mL of glycerol-fatty acid liquid
culture medium into a 50 mL centrifuge tube;

(3) Inoculate single yeast clones from
Saccharomyces cerevisiae plates transformed with
plasmid ① into the centrifuge tube and incubate
on a shaker at 30℃ and 200 rpm;

(4) Every 2 hours, transfer liquid cultures of
monoclonal yeast carrying plasmid ① and observe
the yeast growth by monitoring the turbidity of
the liquid medium.

(5) Using one 96-well plate, collect 100 µL of
culture from each medium every 2 hours and add
it into the 96-well plate. Each group includes
three biological replicates as controls. Measure
OD600 values using a microplate reader and
calculate the average.

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/nomv13.webp)
![](https://static.igem.wiki/teams/5564/notebook/nomv14.webp)
![](https://static.igem.wiki/teams/5564/notebook/nomv15.webp)

Results Analysis and Discussion

(1) Optical density (OD) values measured by
microplate reader for yeast containing plasmid ①
during the mid-logarithmic growth phase in fatty
acid media were as follows: Glycerol: palmitic
acid: oleic acid = 2:1:1 > Glycerol: palmitic
acid: oleic acid: lauric acid = 3:1:1:1 >
Glycerol: lauric acid: palmitic acid = 2:1:1 ≈
Glycerol: lauric acid: oleic acid = 2:1:1,
indicating yeast selective preference for
palmitic acid and oleic acid. Palmitic acid and
oleic acid are the two most common fatty acids
found in domestic wastewater; this engineered
yeast strain may serve as a biosafety yeast
capable of overcoming the high-fat environment
of wastewater and facilitating the bioconversion
of lipids in wastewater.

(2) In subsequent experiments, PCR and western
blot methods will be used to further verify the
normal expression of the protease encoded by
plasmid ①.

Notes

(1) Always maintain the glycerol-to-fatty acid
ratio at 1:1 to ensure the total lipid content
remains the same, thereby enabling a better
comparison of yeast preference for different
carbon sources.

(2) Measure the growth OD value approximately
every 2 hours to allow timely monitoring of
yeast growth rate and extent.

Yeast culture following plasmid ① transfection;
PCR; agarose gel electrophoresis

Experiment date: 2025.07.09

Experiment operator: Wang Quanhao, Min Yingxuan

Experimental objective

(1) Re-streak single clones for liquid yeast
culture to prepare for genomic DNA extraction
and PCR analysis.

(2) Extract two-thirds of the plasmid for use in
PCR and plasmid transfection.

(3) Verify the integration of overexpressed
enzyme genes Erg10, Erg13, and HMG1 into
Saccharomyces cerevisiae and evaluate plasmid
expression levels in engineered strains.

Experimental Equipment and Reagents

Equipment

Laminar flow cabinet, microplate reader,
analytical balance, centrifuge, fume hood,
shaker, ultrasonic homogenizer, PCR machine,
shaker, electrophoresis tank, microwave oven,
gel imaging system;

Reagents

LiAC/TE solution, sterile water; Buffer
P1、Buffer P2、Buffer P3、Buffer DW1、Wash
Solution、ElutionBuffer; Glass beads,
chloroform, methanol; 2× TransFast Taq PCR
SuperMix (+ dye), DNA template, specific
primers, 1× TAE buffer, agarose, nucleic acid
stain EB, DNA marker;

Reagent Preparation

Table 1 PCR Reaction System

| Reagent | Volume | Final Concentration |
| --- | --- | --- |
| 2×TransFast Taq PCR SuperMix(+dye) | 25μL | 1× |
| Primer 1 | 1μL | 0.2μM |
| Primer 2 | 1μL | 0.2μM |
| Template | 5μL | As required |
| DNase-Free Water | To 50μL |  |

Table 2. PCR Reaction Program

| Temperature | Time | Number of cycles |
| --- | --- | --- |
| 94℃ | 3 min | 1 cycle |
| 94℃ | 5 sec | 35 cycles |
| 52℃ | 15 sec |
| 72℃ | 30 sec |
| 72℃ | 5 min | 1 cycle |

Table 3. 2% Agarose Gel

| Reagent | Amount |
| --- | --- |
| Agarose | 1.8 g |
| 1×TAE | 90 mL |
| Nucleic Acid Dye EB | 9 μL |

Experimental Procedures

1. Yeast Culture

1.1 Irradiate the ultraclean workbench with UV
light for 30 minutes prior to use.

1.2 Take a 50 mL centrifuge tube and add 15 mL
of single dropout liquid medium.

1.3 Inoculate a single colony of Saccharomyces
cerevisiae containing plasmid ① from the culture
plate into the centrifuge tube, then incubate in
a shaker at 30℃ and 200 rpm.

1.4 Every 2 hours, transfer liquid cultures of
monoclonal yeast carrying plasmid ① and observe
the yeast growth by monitoring the turbidity of
the liquid medium.

1.5 Using one 96-well plate, collect 100 µL of
culture from each medium every 2 hours and add
it into the 96-well plate. Each group includes
three biological replicates as controls. Measure
OD600 values using a microplate reader and
calculate the average.

2. Plasmid Extraction

2.1 Confirm whether RNase A has been added to
Buffer P1.

2.2 Confirm whether anhydrous ethanol has been
added to the Wash Solution.

2.3 Inspect Buffers P2 and P3 for the presence
of precipitates.

2.4 Collect 1.5–5 mL of overnight yeast culture;
centrifuge at 8,000×g for 2 minutes to pellet
the cells, then discard the supernatant.

2.5 Add 250 μL of Buffer P1 to the pellet and
thoroughly resuspend the cells.

2.6 Add 250 μL of Buffer P2, immediately invert
the centrifuge tube gently 5–10 times to mix,
then incubate at room temperature for 2–4
minutes.

2.7 Add 350 μL of Buffer P3, immediately invert
the centrifuge tube gently 5–10 times to mix.

2.8 Centrifuge at 12,000×g for 10 minutes.
Transfer the supernatant to the adsorption
column and incubate for 3–4 minutes. Centrifuge
at 8,000×g for 30 seconds, then discard the
liquid from the collection tube.

2.9 Add 500 μL of Buffer DW1, centrifuge at
9,000×g for 30 seconds, then discard the liquid
from the collection tube.

2.10 Add 500 μL of Wash Solution, centrifuge at
9,000×g for 30 seconds, then discard the liquid
from the collection tube. Repeat step 2.10 once.

2.11 Centrifuge the empty adsorption column at
9,000×g for 1 minute.

2.12 Place the adsorption column into a clean
1.5 mL centrifuge tube, add 50–100 μL of Elution
Buffer onto the center of the adsorption
membrane, incubate at room temperature for 1
minute, then centrifuge for 1 minute. Collect
and preserve the DNA solution in the tube.

3. Genomic DNA Extraction from Saccharomyces
cerevisiae

3.1 Aliquot 200 μL of yeast culture, centrifuge
at 4000 rpm for 30 s, and discard the
supernatant;

3.2 Resuspend the cell pellet in 100 μL of
LiOAc-1% SDS solution, then incubate at 70°C for
5 minutes;

3.3 Add 300 μL of anhydrous ethanol, mix
thoroughly by repeated pipetting, centrifuge at
15000 rpm for 3 minutes, and discard the
supernatant;

3.4 Wash the precipitate with 70% ethanol
solution, centrifuge, and discard the
supernatant;

3.5 Dissolve the precipitate in 100 μL of water
and store at -20 ℃.

PCR and Agarose Gel Electrophoresis

4.1 Prepare the PCR reaction system according to
Table 1;

4.2 Set the PCR reaction program according to
Table 2 and perform PCR;

4.3 Prepare 2% agarose gel according to Table 3,
dissolve by microwave heating;

4.4 Add 9 μL of EB to the agarose gel, mix well,
pour into the casting tray with combs in place,
lay flat in the refrigerator, and allow the gel
to solidify;

4.5 Place the gel in the electrophoresis tank,
add an appropriate amount of 1×TAE
electrophoresis buffer. After loading samples,
run electrophoresis at a constant voltage of 140
V for 25 min;

4.6 After electrophoresis, observe and
photograph under UV light.

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/nomv16.webp)
![](https://static.igem.wiki/teams/5564/notebook/nomv17.webp)
![](https://static.igem.wiki/teams/5564/notebook/nomv180.webp)

Results Analysis and Discussion

All three target genes were detected with clear
bands in the positive control group PC (plasmid
①), indicating that the three target genes were
successfully cloned into the plasmid vector.

The three target genes all showed positive bands
in the target strain, suggesting that the genes
were successfully introduced into Saccharomyces
cerevisiae.

The expression levels of the three target genes
in the target yeast culture were significantly
higher than those in the negative control group,
indicating successful overexpression of the
target genes in Saccharomyces cerevisiae.

Protein Extraction from Strains Transformed with
Plasmid ①

Experiment Date: 2025.07.11

Experiment operator: Jingning Wang, Quanhao
Wang, Yingxuan Min, Jiayin Sun

Experimental objective

(1) Protein extraction from samples to prepare
for western blot analysis verifying proper
plasmid expression

Experimental Equipment and Reagents

Equipment

Biosafety cabinet, centrifuge, water bath, gel
casting plates, electrophoresis tank, vertical
electrophoresis apparatus, transfer apparatus,
destaining shaker, chemiluminescence imaging
system;

Reagents

NaOH, β-glycerophosphate sodium salt, sodium
orthovanadate, NaF, EDTA, PMSF Reagent
Preparation

Table 1. Composition of Lysis
Buffer

| Reagent | Volume | Final Concentration |
| --- | --- | --- |
| NaOH | 1ml | 0.1M |
| sodium β-glycerophosphate | 11ul | 100mM |
| sodium orthovanadate | 11ul | 100mM |
| NaF | 11ul | 5M |
| EDTA(ethylenediaminetetraacetic acid) | 3ul | 0.5M |
| PMSF(Phenylmethylsulfonyl fluoride) | 11ul | 100mM |

Experimental Procedure

Sample Preparation

1.1 Prepare yeast lysis buffer as detailed in
the table below: using NaOH as the base, add the
specified reagents per 1 mL of NaOH; protease
and phosphatase inhibitors may be added as
required by the experiment (optional);

1.2 Take 200 μL of yeast culture, centrifuge
(4000 rpm, 30 s) and discard the supernatant;

1.3 Add lysis buffer to the collected cell
pellet and lyse on ice for 10 minutes;

1.4 Centrifuge (5000 rpm, 1 min) and discard the
supernatant;

Add 80 μL of 1× loading buffer to the 1.5 mL
precipitate and mix thoroughly; incubate in a
metal bath at 100°C for 5 minutes, then store at
-20°C for subsequent use

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/nomv19.webp)
![](https://static.igem.wiki/teams/5564/notebook/nomv20.webp)

Western Blot Detection of Protein Expression
Success

Experiment Date: 2025.07.13

Experiment operator: Wang Jingning, Wang
Quanhao, Min Yingxuan, Sun Jiayin

Experiment Objective

(1) To verify the successful introduction of
ERG10, ERG13, and HMG1 enzyme genes into
Saccharomyces cerevisiae

Experimental Equipment and Reagents

Equipment

Laminar flow clean bench, centrifuge, water
bath, gel casting plates, electrophoresis tank,
vertical electrophoresis system, transfer
electrophoresis system, decolorization shaker,
chemiluminescence imaging system

Reagents

Tris, glycine, SDS, methanol, polyacrylamide,
APS, TEMED, 1.5M Tris-HCl (pH 8.8), 1.0 M
Tris-HCl (pH 6.8), KCl, NaCl, Tween-20,
ddH2O

Reagents

Table 1 Electrophoresis Buffer
Preparation

| Reagent | Amount |
| --- | --- |
| Tris | 3g |
| Glycine | 14.6g |
| SDS | 1g |
| ddH2O | Make up to 1000 mL |

Table 2 Transfer Buffer

| Reagent | Amount |
| --- | --- |
| Tris | 2.91g |
| Glycine | 14.4g |
| Methanol | 200ml |
| ddH2O | Make up to 1000 mL |

Table 3 Stacking Gel Preparation

| Reagent | Amount |
| --- | --- |
| ddH2O | 2.1 mL |
| 30% Polyacrylamide | 0.5 mL |
| 1.0M Tris-HCl (pH6.8) | 0.38 mL |
| 10%SDS | 30 μL |
| 10%APS | 30 μL |
| TEMED | 3 μL |

Table 4 Separating Gel
Preparation

| Reagent | Amount |
| --- | --- |
| ddH2O | 1.6 mL |
| 30% Polyacrylamide | 2 mL |
| 1.5M Tris-HCl (pH8.8) | 1.3 mL |
| 10%SDS | 50 μL |
| 10%APS | 50 μL |
| TEMED | 5 μL |

Table 5 5% Blocking Reagent
Preparation

| Reagent | Amount |
| --- | --- |
| Skim Milk Powder | 2.5g |
| 1×TBST | 50ml |

Table 6 Developer Solution
Preparation

| Reagent | Amount |
| --- | --- |
| Developer Solution A | 500ul |
| Developer Solution B | 500ul |

Table 7 TBST Preparation

| Reagent | Amount |
| --- | --- |
| NaCl | 8g |
| KCl | 0.2g |
| Tris | 3g |
| Tween-20 | 1ml |
| ddH2O | Make up to 1 L |

Experimental Procedure

Protein Loading and Gel Electrophoresis

1.1 Prepare 5% stacking gel and 12% resolving
gel according to Tables 4 and 5. The resolving
gel is incubated at room temperature for 20-30
min, and the stacking gel is incubated at room
temperature for approximately 5-10 min. Insert
the gels into the electrophoresis apparatus and
fill the tank with electrophoresis buffer.
During sample loading, add 10μL of protein
sample into each well, and add 4 μL of marker
into one well. Handle samples gently to avoid
bubble formation.

1.2 Electrophoresis: Electrophoresis conditions:
stacking gel at 80 V for 30 min ; Lower
separation gel 120 V, 60-80 min;

1.3 Transfer: Upon completion of
electrophoresis, remove the gel plates,
carefully detach the gel, and cut the PVDF
membrane and filter paper according to the gel
dimensions; Prior to transfer, the PVDF membrane
must be activated in methanol for approximately
20-30s. Conduct the transfer in an ice-water
mixture at a constant current of 200 mA for 2h;

1.4 Milk Blocking and Hybridization: Remove the
PVDF membrane and wash with 1×TBST on a
horizontal shaker 3 times, 10min each. Block
with 5% skim milk at room temperature on a
horizontal shaker for 2h.

1.5 After blocking for 2h, wash with TBST three
times, 5min each, then incubate with the
appropriately diluted primary antibody (dilution
ratio according to the specific antibody
datasheet, diluted in TBST). After adding the
primary antibody, place at 4℃ overnight. The
next day, wash three times with TBST on a
shaker, 10min each, then incubate with the
secondary antibody for 1h. Finally, wash again
with TBST three times, 10min each.

1.6 ECL Detection: According to Table 7 and the
Thermo Fisher ECL instructions, mix solution A
and solution B at a 1:1 volume ratio. After
mixing, incubate in the dark at room temperature
for 1-2min. Capture images using a gel imaging
system and analyze the band gray values with its
image processing software, followed by
statistical analysis.

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/nomv230.webp)

# Sapogenin - Construction of oleanolic acid

Cultivation and Cryopreservation of
Saccharomyces
cerevisiae

Experiment Date: 2025.07.21

Experiment operator: Zhao Xiaoqing, Sun Jiayin

Objectives

1. To cultivate and cryopreserve Saccharomyces
cerevisiae for subsequent preparation of
competent cells for plasmid transfection.

2. To formulate the plasmid into a solution for
convenient transfection.

Experimental Equipment and Reagents

Equipment

Shaker, refrigerator, laminar flow hood,
spectrophotometer

Reagents

YPD medium, plasmid powder, sterile water,
glycerol

Reagent Preparation

Table 1 YPD Medium(500mL)

| Reagent | Amount |
| --- | --- |
| Peptone | 10g |
| Yeast extract | 5g |
| H2O | 500mL |
| glucose | 10g |

Experimental Procedure

Cultivation of Saccharomyces
cerevisiae

(1) Irradiate the ultra-clean workbench with
ultraviolet light for 30 minutes beforehand.
Prepare YPD medium and microwave-treat it in
advance for later use;

(2) Take a 50 mL centrifuge tube and pour 5 mL
of YPD liquid medium into it;

(3) Pick a single colony of Saccharomyces
cerevisiae W303 from the culture plate and
inoculate into the tube. Incubate on a shaking
incubator (30°C, 200 rpm);

(4) After approximately 7hours, measure the
OD600. If OD600 = 0.3, add 2 mL of the culture
to 100 mL of YPD medium.

Prepare the plasmid

(1) Retrieve the plasmid powder tube and
centrifuge (4μg);

(2) Add 30μL sterile water to the tube;

(3) Transfer 10μL to an EP tube, then add 32μL
sterile water for later use;

(4) Store the remaining 20μL plasmid at -20°C.

Freeze-preserve experimental strains

Take an EP tube, add 750μL yeastl suspension and
750μL glycerol (50%) in a 1:1 ratio, mix, and
store at -80°C. Replenish the remaining yeastl
suspension in the centrifuge tube with
appropriate medium.

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/nooa01.webp)
![](https://static.igem.wiki/teams/5564/notebook/nooa02.webp)

Analysis and Discussion

Yeast exhibited robust growth and is suitable
for preparing competent cells for subsequent
experiments.

Precautions

(1) Maintain aseptic technique when isolating
single-colony yeast to prevent contamination by
extraneous microorganisms.

(2) When preparing YPD medium, avoid excessive
temperatures during autoclaving. Typically,
autoclave at 115°C for 15 minutes; higher
temperatures may cause glucose to darken.

(3) Ensure culture duration does not exceed
recommended limits. If optical density (OD)
exceeds 1.2, the strain is unsuitable for
subsequent procedures.

Preparation of Sc-Trp Medium and Competent Cells

Experiment Date: 2025.07.23

Experiment operator: Zhao Xiaoqing, Sun Jiayin

Objectives

1. Prepare Sc-Trp medium and plate it to
facilitate subsequent cultivation of
Saccharomyces cerevisiae following plasmid
transfection.

2. Prepare competent cells for plasmid
transfection.

Experimental Equipment and Reagents

Equipment

Laminar flow hood, spectrophotometer, autoclave,
shaking incubator

Reagents

SC-Trp, arginine, adenine, glucose, YNB,
ammonium sulphate, agar, sterile water, LiAC/TE
buffer

Reagent Preparation

Table 1 Sc-Trp Medium (200 mL)

| Reagent | Amount |
| --- | --- |
| Sc-Trp | 0.4g |
| L-Arginine | 0.04g |
| adenine | 0.2g |
| glucose | 4g |
| agar | 4g |
| YNB | 1.6g |
| (NH4)2SO4 | 5g |
| H2O | 200mL |

Experimental Procedure

Preparation of SC-Trp Medium

(1) Take 0.4 g SC-Trp, 0.04 g arginine, 0.2 g
adenine, and 4 g glucose; add to 60 mL water and
place in an empty bottle;

(2) Take 4 g agar and add to 120 mL water; place
in another empty bottle and add a rotor;

(3) Place both bottles alongside an empty 100 mL
bottle into an autoclave for sterilisation
(115°C, 15 minutes);

(4) Weigh 1.6 g of YNB yeast and 5 g of ammonium
sulphate into a beaker. Add 100 mL of sterilised
water and stir with a stirrer rod until
completely dissolved;

(5) Sterilise the solution by filtration through
a 0.22μm filter, then transfer to a 100mL
sterile autoclave bottle for later use;

(6) Take 20mL of the sterilised YNB solution and
add to the sterilised agar bottles. While hot,
pour the SC-Trp solution into the agar bottles
containing YNB and mix thoroughly using a
magnetic stirrer;

(7) Swiftly pour the mixture into sterile Petri
dishes to form plates. Allow to stand at room
temperature for 4 hours before refrigerating for
later use.

Preparation of Competent Cells

(1) Place sterile water in a 4°C refrigerator
for later use. Dilute LiAC/TE to 0.1 mol/L for
later use;

(2) Measure the OD600 of the Saccharomyces
cerevisiae culture from the previous day.
Proceed to the next step if OD600 = 0.8;

(3) Take 50 mL of the culture, centrifuge (3500
rpm, 2 min), and discard the supernatant;

(4) Resuspend in 50 mL of cold sterile water,
centrifuge (3500 rpm, 2 min), and discard the
supernatant;

(5) Resuspend in 1 mL 0.1 mol/L LiAC/TE buffer,
centrifuge briefly (4000 rpm, 30 s), and discard
supernatant;

(6) Resuspend in 400 μL 0.1 mol/L LiAC/TE
buffer, mix thoroughly, and refrigerate
overnight on ice.

Precautions

(1) After filtration and sterilisation, the YNB
solution must be rapidly poured into agar while
hot to prevent uneven distribution causing
abnormal solidification of the medium.

(2) Once prepared, the medium must be promptly
poured into plates to prevent premature
solidification due to prolonged exposure.

(3) Monitor the OD value of Saccharomyces
cerevisiae to prevent excessive incubation time,
which may render the sample unsuitable for
subsequent experiments.

![](https://static.igem.wiki/teams/5564/notebook/nooa03.webp)
![](https://static.igem.wiki/teams/5564/notebook/nooa04.webp)

Transfection of Saccharomyces
cerevisiae Plasmid
②

Experiment Date: 2025.07.25

Experiment operator: Zhao Xiaoqing, Sun Jiayin

Objectives

To transfect Saccharomyces cerevisiae competent
cells with a plasmid for synthesising the
intermediate compound oleanolic acid (OA).

Experimental Equipment and Reagents

Equipment

Laminar flow hood, centrifuge, 30°C incubator,
water bath, shaking incubator, drying oven.

Reagents

PEG, LiAc (1 mol/L), salmon sperm, plasmid,
yeast competent cells, YPD medium

Reagent Preparation

Table 1 YPD Medium (500 mL)

| Reagent | Amount |
| --- | --- |
| Peptone | 10g |
| Yeast extract | 5g |
| H2O | 500mL |
| glucose | 10g |

Experimental Procedure

Plasmid Transfection

(1) Add approximately 50 μL of the yeast
competent cells prepared the previous day to
each Eppendorf tube. Centrifuge (4000 rpm, 30
seconds) and discard the supernatant.

(2) Add sequentially to the EP tube: 42 μL
plasmid, 10 μL salmon sperm extract (10 mg/mL),
240 μL PEG, and 36 μL LiAc (1 mol/L). Mix
thoroughly by pipetting up and down with a 1 mL
pipette, then incubate at 30°C for 30 minutes;

(3) Add 36 μL DMSO to each EP tube. Mix
thoroughly by pipetting and heat-shock at 42°C
for 20 minutes.

Post-transfection yeast culture

(1) Remove EP tubes from water bath. Centrifuge
(3000 rpm, 20 seconds) and discard supernatant.
Resuspend in 1 mL YPD medium and incubate on
shaking incubator for 3 hours.

(2) Centrifuge (3000 rpm, 20 s), discard
supernatant, resuspend in 100 μL water, spread
culture onto plates (Sc-Trp) using glass beads,
and incubate at 30°C for approximately 3 days.

Precautions

(1) Salmon sperm must be pre-treated by boiling
for 5 minutes, then rapidly immersed in an
ice-water bath to preserve exogenous plasmids.

(2) All reagents required for plasmid
transfection must be thoroughly mixed to
maximise cell membrane permeability and enhance
transfection efficiency.

(3) Incubation times in the incubator and heat
shock duration in the water bath must be
strictly controlled to prevent reduced
transfection efficiency due to prolonged
exposure.

![](https://static.igem.wiki/teams/5564/notebook/nooa05.webp)
![](https://static.igem.wiki/teams/5564/notebook/nooa06.webp)

Yeast Monoclonal Culture Following Plasmid
Transfection

Experiment Date: 2025.07.27

Experiment operator: Zhao Xiaoqing, Sun Jiayin

Objectives

(1) To transfer successful plasmid-transfected
yeast clones, selected under nutritional
conditions, from plates to YPD medium and
incubate on a shaking incubator;

(2) To prepare YPD and LB media for subsequent
use.

Experimental Equipment and Reagents

Equipment

Laminar flow hood, autoclave, shaking incubator,
centrifuge.

Reagents

Peptone, yeast extract, sodium chloride,
glucose, sterile water.

Reagent Preparation

Table 1 LB Liquid Medium (200 mL)

| Reagent | Amount |
| --- | --- |
| peptone | 2g |
| Yeast extract | 1g |
| NaCl | 2g |
| H2O | 200mL |

Table 2 YPD Medium (500 mL)

| Reagent | Amount |
| --- | --- |
| Peptone | 10g |
| Yeast extract | 5g |
| H2O | 500mL |
| glucose | 10g |

Experimental Procedure

Monoclonal Culture of Transfected Yeast

(1) Remove the transfection-treated yeast
culture plate from the incubator and observe
yeast growth;

(2) Prepare six 50mL centrifuge tubes, each
containing 10mL YPD medium;

(3) Pick individual colonies from the
transfection plate and transfer them to the
tubes. Incubate on a shaking incubator for 24
hours.

Medium Preparation

Prepare LB and YPD liquid media according to
recipes. Sterilise in an autoclave (115°C, 15
min).

Experimental Results

As illustrated, plasmid-transfected
Saccharomyces cerevisiae exhibited robust
growth.

Results Analysis and Discussion

As shown, high yeast growth density complicated
single-colony isolation.

Solution: Reduce the amount of yeast competent
cells in the transfection system or shorten the
incubation time.

Precautions

(1) When transferring from culture plates to
centrifuge tubes, single colonies must be picked
to obtain strains with stable genetic
backgrounds.

(2) When adding medium to centrifuge tubes, to
avoid contamination from residual medium,
transfer a portion to a 50 mL centrifuge tube
for later use.

![](https://static.igem.wiki/teams/5564/notebook/nooa07.webp)
![](https://static.igem.wiki/teams/5564/notebook/nooa08.webp)
![](https://static.igem.wiki/teams/5564/notebook/nooa09.webp)
![](https://static.igem.wiki/teams/5564/notebook/nooa10.webp)

Growth Curve Measurement of Yeast Single Clones
Following Plasmid Transfection

Experiment Date: 2025.07.28

Experiment operator: Wang Jingning, Wang Quanhao

Objective

Every 12 hours, yeastl suspensions from two
single clone colonies of single plasmid yeast 3,
which exhibited higher OA production after one
day of culture, were transferred into a 96-well
plate containing Trp-deficient liquid medium.
Optical density (OD600) measurements were taken
to indirectly assess strain growth. Monitoring
was continued for 96 hours, culminating in the
plotting of the growth curve of the single clone
colonies.

Experimental Equipment and Reagents

Equipment

Class II biosafety cabinet, autoclave, shaker,
microplate reader, 96-well plate

Experimental Procedure

1、OA High-Yield Yeast Growth Curve Measurement

(1) At 10:00 AM and 10:00 PM, respectively,
remove two 3 mL centrifuge tubes containing
single clone yeast harboring a single plasmid
from the shaker after one day of liquid culture.
Observe yeast growth by checking for turbidity
in the liquid medium.

(2) Take one 96-well plate and add 100 µL of
yeast culture to each well. Establish the
Trp-deficient medium as the control group. Both
types of yeast cultures, as well as the
single-deficient medium, are set up with three
replicates each. The loading order is shown in
the following figure. Measure OD600
using a microplate reader and calculate the
average values.

![](https://static.igem.wiki/teams/5564/notebook/note040501.webp)

Experimental results

![](https://static.igem.wiki/teams/5564/notebook/note040502.webp)

Results analysis and discussion

(1) Compared to the Trp-deficient medium group,
the centrifuge tubes were visibly turbid after
shaker incubation, and the OD values measured by
the microplate reader were higher than those of
the control group, indicating substantial growth
of strain ③;

(2) Comparison of OD values for strains
incubated at 10:00 AM and 10:00 PM showed no
significant differences. The strains exhibited
slow growth in Trp-deficient medium; subsequent
data collection may occur every 24 hours;

(3) At the early cultivation stage, the average
OD value of strain 3② was higher than that of
strain 3①; continuous observation will determine
whether strain 3② is the strain with better
growth.

(4) Overall, no significant growth was observed
in the strains, possibly due to the omission of
essential nutrients during culture medium
preparation, which requires timely correction.

Precautions

(1) Establish a control group using
Trp-deficient medium to better reflect the
growth status of the yeast culture.

(2) Measure the growth OD value approximately
every 12 hours to enable timely monitoring of
the yeast growth rate and extent.

![](https://static.igem.wiki/teams/5564/notebook/nooa12.webp)

![](https://static.igem.wiki/teams/5564/notebook/nooa13.webp)

Measurement of yeast single-clone growth curves
following Plasmid ② transfection

Experiment date: 2025.08.01

Experimental personnel: Wang Jingning, Wang
Quanhao

Objective

Every 2 hours, 100 μL of culture was taken from
three single-clone colonies transfected with
Plasmid ②, cultivated in Trp-deficient liquid
medium, transferred into a 96-well plate, and
optical density (OD600) was measured to
indirectly assess strain growth. Continuous
monitoring was conducted over 96 hours, and the
growth curve of each single-clone colony was
subsequently plotted.

Experimental Equipment and Reagents

Equipment

Biosafety cabinet, autoclave, shaker, microplate
reader, 96-well plate

Experimental Procedure

1. Irradiate the biosafety cabinet with
ultraviolet light for 30 minutes in advance.

2. Add 15 mL of glycerol fatty acid liquid
medium to a 50 mL centrifuge tube.

3. Pick a single yeast clone from the culture
plate transfected with Plasmid ② and add it to
the centrifuge tube, then incubate on a shaker
at 30°C and 200 rpm.

4. Observe yeast growth by assessing the
turbidity of the liquid medium.

5. Prepare one 96-well plate and add 100 μL of
yeastl suspension to each well. Establish a
Trp-deficient media control group, as well as
three replicates each for three single-clone
yeastl suspensions and single-deficient media
controls. Measure the OD600 values
using a microplate reader and calculate the
averages.

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/nooa14.webp)

![](https://static.igem.wiki/teams/5564/notebook/nooa150.webp)

Results Analysis and Discussion

(1) The Trp-deficient media group exhibited
visibly turbid centrifuge tubes following
shaking incubation. The OD600 values measured by
the microplate reader for the three single-clone
colonies were elevated compared to the control
group, indicating substantial growth of yeast
transfected with Plasmid ②;

(2) Continuous monitoring of yeast growth showed
that yeast reached the stationary phase at
approximately 28 h.

(3) Compared to the previous growth curve,
significant strain growth was observed after
re-preparing the liquid media, confirming that
the prior media preparation lacked essential
nutrients.

Precautions

(1) Include a Trp-deficient media control group
to better assess the extent of yeast growth.

(2) Measure OD600 values approximately every 2
hours to enable timely monitoring of yeast
growth rate and extent.

Glycoflavin streaking; PCR and agarose gel
electrophoresis Verify the successful
transfection of plasmid ② enzyme gene

Experiment time: 2025.08.03

Experiment operator: Sun Jiayin, Wang Quanhao,
Min Yingxuan

Experimental Objective

(1) Monoclonal cells were prepared by
glycohydrolase streaking.

(2) To determine whether the genes of MtCPR,
CYP716A12 and β-AS were transferred into yeast,
and to detect the expression of plasmids in
yeast 1, 2 and 3.

Experimental equipment and reagents

Equipment

Clean workbench, centrifuge, micropipette and
gun head, PCR instrument, electrophoresis
instrument, gel imaging system

reagent

Plasmid 2, glycerol-free freeze-dried culture
medium, Amoxicillin LB solid agar plates, 2×
TransFast Taq PCR SuperMix (with dye), DNA
templates, specific primers, TAE buffer,
agarose, EB DNA stain, DNA markers

Reagent preparation

3.1 PCR reaction system

| Reagent | Volume | Concentration |
| --- | --- | --- |
| 2×TransFast Taq PCR SuperMix(+dye) | 25μL | 1× |
| Primer 1 | 2μL | 0.2-0.3μM |
| Primer 2 | 2μL | 0.2-0.3μM |
| Template | 5μL |  |
| DNase-Free Water | To 50μL |  |

3.2 PCR reaction procedure

| Temperature | Time | Number of cycles |
| --- | --- | --- |
| 94℃ | 3 min | 1 cycle |
| 94℃ | 5 sec | 30 cycles |
| 55℃ | 15 sec |
| 72℃ | 30 sec |
| 72℃ | 5 min | 1 cycle |

3.3 1% agarose gel

| Reagent | Amount | Number of cycles |
| --- | --- | --- |
| agarose | 0.9 g | 1 cycle |
| 1×TAE | 90 mL | 30 cycles |
| Nucleic acid dye EB | 9 μL |
| 72℃ | 30 sec |
| 72℃ | 5 min | 1 cycle |

Experimental Procedure

Glycojugate streaking

(1) The ultra-clean workbench was sterilized
with ultraviolet light 30 minutes in advance,
and the experimental table was wiped with 75%
alcohol;

(2) Mark the name of the strain, date, operator
and other information on the bottom of the
tablet with a marker pen;

(3) The bamboo stick is burned in the outer
flame of the alcohol lamp, cooled, and then
dipped directly into a small amount of yeastl
liquid (visible drops can be seen);

(4) The bamboo stick dipped in the yeastl
solution is gently drawn in a zigzag pattern on
one side of the plate (about 1/4 area, marked as
A area). The line should be dense but not
overlapping. The bamboo stick should be drawn at
an angle of about 30°-45°. After drawing the
line, discard the bamboo stick;

(5) The second marking step (Zone B) involves
passing cooled bamboo sticks 1-2 times from the
end of Zone A, extending to Zone B
(approximately half of the area) to create
sparse zigzag patterns. The third marking step
(Zones C/D) repeats the process, tracing from
the end of Zone B to Zone C (the remaining area)
with even finer lines, ultimately forming a
gradient dilution where Zone A contains
concentrated yeast and Zone D has diluted yeastl
content.

Yeast genome extraction

(1) Take 200μL yeast culture solution,
centrifuge (4000rpm, 30s) and discard the
supernatant;

(2) Resuspend the cell precipitate in 100μL
LiOAc-1%SDS solution, and incubate in 70℃ oven
for 5min;

(3) Add 300μL anhydrous ethanol, repeatedly blow
and mix, centrifuge (15000rpm, 3min) and discard
the supernatant;

(4) Wash the precipitate with 70% ethanol
solution, and discard the supernatant after
centrifugation;

(5) The precipitate was dissolved in 100μL water
and stored in a-20℃ refrigerator.

PCR

(1) The PCR reaction system was prepared
according to 3.1 in "I";

(2) Set the PCR reaction program according to
3.2 in "I" and carry out PCR;

Agarose gel electrophoresis

(1) Prepare 2% agarose gel by microwave heating
according to 3.3 in "I";

(2) Add 9 μ L EB and shake well, then pour it
into the mixing tank where the comb has been
placed. Place it flat in the refrigerator and
wait for the gel to solidify;

(3) Put the gel into the electrophoresis tank
and pour in an appropriate amount of 1× TAE
electrophoresis buffer. After sampling,
electrophoresis for 25min under constant voltage
of 140V;

(4) After electrophoresis, observe and take
photos under ultraviolet lamp;

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/nooa16.webp)
![](https://static.igem.wiki/teams/5564/notebook/nooa17.webp)
![](https://static.igem.wiki/teams/5564/notebook/nooa18.webp)
![](https://static.igem.wiki/teams/5564/notebook/nooa19.webp)

Results analysis and discussion

1. The three target genes were not detected in
the negative control group NC, indicating that
the primer amplification was specific;

2. The three target genes were detected in the
positive control group AC (plasmid 2) with clear
bands and normal molecular weight, indicating
that the three target genes were successfully
cloned into the plasmid vector;

3. MtCPR

![](https://static.igem.wiki/teams/5564/notebook/note040704.webp)

The MtCPR gene exhibited distinct bands in
yeastl strains 2 and 3 during this experiment,
consistent with the results from Experiment
7.22, indicating successful gene transfection in
these two strains. Although the banding was not
prominent in strain 1, the 7.22 experiment
revealed clear bands for this strain, confirming
successful gene transfection. The experimental
results may be attributed to either an increase
in amplified fragment length or insufficient
sample loading during electrophoresis.

4. CYP716A12

![](https://static.igem.wiki/teams/5564/notebook/note040705.webp)

The CYP716A12 gene was detected as weakly
positive in bands 1 and 2 of yeastl cultures,
consistent with the results from Experiment
7.22, indicating successful transfection of the
gene. A distinct band at 100bp was observed,
likely caused by primer dimer formation. The
absence of bands in yeastl culture 3 and the
faint banding in Experiment 7.22 suggest low
transfection efficiency in culture 3, which may
be attributed to amplified fragments being too
long for effective PCR amplification.

5. β-AS

![](https://static.igem.wiki/teams/5564/notebook/note040706.webp)

In this experiment, the β-AS gene showed no
detectable bands in yeastl strains 1, 2, and 3,
all presenting negative results. The 7.22
experimental result indicated weak positive for
strain 1, while both strains 2 and 3 remained
negative. This suggests successful but
low-efficiency transfection of the gene in
strain 1, with the negative outcomes likely
attributed to reduced PCR efficiency due to
extended amplification fragments. Both strains 2
and 3 demonstrated negative results in
subsequent experiments, indicating unsuccessful
transfection of the gene in these two yeastl
colonies.

matters need attention

1. The tail of the bamboo stick is picked out
with tweezers and then roasted.

2. Start the line from the end of the last line.
The lines should be dense but not overlapping.

3. The PCR instrument should stop working first,
then open the lid, and finally shut down.

4. Set the electrophoresis instrument to
constant voltage.

Plasmid extraction; Plasmid transfection;
Preparation of YPD and LB media; PCR and agarose
gel electrophoresis

Experiment time: 2025.08.05

Experiment operator: Sun Jiayin, Wang Quanhao,
Min Yingxuan

Objective

(1) Plasmids were extracted from overnight
cultured Ganoderma lucidum to prepare for
subsequent plasmid transfection and PCR;

(2) Saccharomyces cerevisiae was transfected
with plasmid 3 to synthesize the final product
ginsenoside Ro;

(3) Prepare YPD medium and Trp-Leu double
deficiency solid medium for standby;

(4) To determine whether the genes of three
enzymes, MtCPR, CYP716A12 and β-AS were
transferred into Saccharomyces cerevisiae and to
detect the expression of plasmids in yeast 1, 2
and 3

Experimental equipment and reagents

Equipment

laminar flow hood, shaker, 30℃ incubator, water
bath, shaker, oven, electronic balance, magnetic
stirrer, autoclave, PCR instrument,
electrophoresis system, gel imaging system,
adsorption column

reagent

Buffer P1、Buffer P2、Buffer P3、Buffer
DW1、Wash Solution、ElutionBuffer; PE, LiAc
(1mol/L), salmon sperm, plasmid, yeast receptive
state, YPD medium; Glucose, peptone, arginine,
adenine, SC-Trp-Leu; 2×TransFast Taq PCR
SuperMix (+dye), DNA template, specific primers,
1×TAE buffer, agarose, DNA dye EB, DNA Marker;

Reagent configuration

3.1 YPD medium (50 0mL)

| Reagent | Amount |
| --- | --- |
| Peptone | 10g |
| Yeast extract | 5g |
| H2O | 500mL |
| glucose | 10g |
| water H2O | 180 mL |

3.2 Trp-Leu double deficient liquid medium (180
mL)

![](https://static.igem.wiki/teams/5564/notebook/note040802.webp)

3.3 YNB solution

| Reagent | Amount |
| --- | --- |
| Yeast YNB | 1.6 g |
| Ammonium sulfate (NH4)2SO4 | 5 g |
| water H2O | 100 mL |
| glucose | 10g |
| water H2O | 180 mL |

3.4 PCR reaction system

| Reagent | Volume | Final Concentration |
| --- | --- | --- |
| 2×TransFast Taq PCR SuperMix(+dye) | 25μL | 1× |
| Primer 1 | 1μL | 0.2μM |
| Primer 2 | 1μL | 0.2μM |
| Template | 5μL | As required |
| DNase-Free Water | To 50μL |  |

3.5 PCR reaction procedure

| Temperature | Time | Number of cycles |
| --- | --- | --- |
| 94℃ | 3 min | 1 cycle |
| 94℃ | 5 sec | 35 cycles |
| 55℃ | 15 sec |
| 72℃ | 30 sec |
| 72℃ | 5 min | 1 cycle |

3.6 1% agarose gel

| Reagent | Amount |
| --- | --- |
| agarose | 0.9 g |
| 1×TAE | 90 mL |
| Nucleic acid dye EB | 9 μL |

Experimental Procedure

Viral transfection

1.1 Salmon sperm was boiled in boiling water for
5min and then rapidly placed in an ice water
mixture (on ice)

1.2 Add about 50μL of yeast receptive state
prepared the previous day to each EP tube, and
centrifuge (4000rpm, 30s) and discard the
supernatant;

1.3 Add 42μL plasmid, 10μL croaker sperm (10
mg/mL), 240μL PEG, and 36μL LiAc (1 mol/L)
sequentially into the EP tube. After repeatedly
pipetting to mix thoroughly with a 1mL pipette,
incubate the mixture in a 30℃ incubator for 30
minutes.;

1.4 Add 36μL DMSO to each EP tube, mix by
repeated blowing and then put it into a 42℃
water bath for 20min.

Yeast culture after transfection

2.1 The EP tube was taken out of the water bath,
and the supernatant was discarded after
centrifugation (3000rpm, 20s). The suspension
was resuspended in 1 mL LYPD medium and cultured
for 3h in a shaker.

2.2 After centrifugation (3000rpm, 20s), the
supernatant was discarded, and the yeastl
suspension was resuspended with 100μL water. The
yeastl suspension was spread on the plate
(Sc-Trp) with glass beads, and cultured in a 30℃
incubator for about 3 days.

Protein extraction

3.1 Check whether RNase A has been added to
buffer P1.

3.2 Check whether anhydrous ethanol has been
added to the Wash Solution.

3.3 Check for precipitation of buffer P2 and P3.

3.4 Take 1.5-5mL of the yeastl liquid that has
been cultured overnight, and collect the yeastl
body by centrifugation at 8,000×g for 2 minutes,
and discard all the culture medium.

3.5 Add 250 μL BufferP1 to the precipitate and
resuspend the yeast thoroughly.

3.6 Add 250 μL Buffer P2 and gently invert the
centrifuge tube 5-10 times immediately to mix.
Let it stand at room temperature for 2-4
minutes.

3.7 Add 350μL Buffer P3 and gently invert the
centrifuge tube 5-10 times immediately to mix.

3.8 Centrifuge at 12,000×g for 10 minutes.
Transfer the supernatant to the adsorption
column and let it stand for 3-4 minutes.
Centrifuge at 8,000×g for 30 seconds, then
discard the liquid from the collection tube.

3.9 Add 500μ L Buffer DW1 and centrifuge at
9000×g for 30 seconds. Discard the liquid in the
collection tube.

3.10 Add 500 μ L Wash Solution and centrifuge at
9000×g for 30 seconds. Pour off the liquid in
the collection tube.

3.11 Repeat 3.10 once.

3.12 The air adsorption column was centrifuged
at 9000×g for 1 min.

3.13 The adsorption column was placed in a clean
1.5mL centrifuge tube, and 50-100 μL
ElutionBuffer was added to the center of the
adsorption membrane. After 1 min at room
temperature, the centrifuge was spun for 1 min.
The DNA solution was stored in the tube.

Prepare YPD medium

4.1 Follow 3.1 of Part II.Prepare the solution;

4.2 Place the glass bottle with the solution
into the autoclave and sterilize (115℃,15min);

Preparation of Trp-Leu double deficient liquid
medium

5.1 Follow 3 of section 22. Prepare a double
deficiency solution of Trp-Leu according to 3.3.
Prepare YNB solution;

5.2 Place the glass bottle with the solution
into the autoclave and sterilize (115℃,15min);

5.3 After filtering and sterilizing the YNB
solution through a 0.22μm filter, it was
transferred to a 100mL empty high-pressure
sterilized bottle for standby;

5.4 Take 20 m L of filtered YNB solution and add
it to the Trp-Leu double deficiency solution and
mix thoroughly;

PCR and agarose gel electrophoresis

6.1 According to 3.4. Configuration of PCR
reaction system;

6.2 According to 3.5. Set the PCR reaction
program and carry out PCR;

6.3 According to 3.6. Prepare 1% agarose gel by
microwave heating and dissolution;

6.4 Add 9 μ L EB and shake well, then pour it
into the mixing tank where the comb has been
placed. Place it flat in the refrigerator and
wait for the gel to solidify;

6.5 Put the gel into the electrophoresis tank
and pour in an appropriate amount of 1× TAE
electrophoresis buffer. After sampling,
electrophoresis for 15min under a constant
voltage of 1 50 V;

6.6 After electrophoresis, observe and take
photos under ultraviolet lamp;

Experimental Result

![](https://static.igem.wiki/teams/5564/notebook/nooa200.webp)

Results analysis and discussion

1. The three target genes were clearly band in
yeast 1, yeast 2 and yeast 3, and all were
strongly positive, indicating that the three
target genes were successfully transferred into
yeast

2. The band of the NC negative control was
caused by insufficient solidification time of
the agarose gel, resulting in soft gel, proper
adhesion of the sample well, and sample leakage
during sampling (the negative control samples
used in the first two experiments did not show
the band, and the negative control samples used
in this time were the same as those in the first
two experiments);

3. A distinct band is present at 100bp, which is
caused by the formation of dimer of primer
itself;

4. The electrophoresis time was short, so the
band differentiation was slightly not obvious.

matters need attention

1. Salmon sperm need to be boiled in boiling
water for 5min in advance, and then quickly put
into ice water mixture to play the role of
protecting exogenous plasmids

2. All kinds of reagents required for particle
transfection should be mixed evenly to maximize
cell membrane permeability and improve
transfection efficiency.

3. The incubation time of the titer transfection
incubator and the heat shock time of the water
bath must be strictly controlled to prevent the
decrease of transfection efficiency after too
long time.

4. In electrophoresis, if the length of DNA is
short, the voltage can be adjusted to control
the electrophoresis speed.

Yeast culture after plasmid ②transfection; PCR;
agarose gel electrophoresis

Experiment time: 2025.08.09

Experiment operator: Wang Quanhao, Min Yingxuan

experimental objective

(1) Yeast genome extraction and PCR experiment
preparation;

(2) 2/3 of the extracted plasmid was used for
PCR and plasmid transfection;

(3) To determine whether the genes of three
enzymes expressed MtCPR, CYP716A12 and β-AS were
introduced into Saccharomyces cerevisiae, and to
detect the expression of plasmids in the
engineered yeast;

Experimental Equipment and Reagents

Equipment

Clean workbench, enzyme label instrument,
electronic balance, centrifuge, fume hood,
oscillator, ultrasonic crusher, PCR instrument,
oscillator, electrophoresis tank, microwave
oven, gel imaging system;

Reagent

LiAC/TE solution, sterile water; Buffer
P1、Buffer P2、Buffer P3、Buffer DW1、Wash
Solution、ElutionBuffer; Glass beads,
chloroform, methanol; 2×TransFast Taq PCR
SuperMix (+dye), DNA template, specific primers,
1×TAE buffer, agarose, nucleic acid dye EB, DNA
Marker;

Reagent preparation

Table 1 PCR reaction system

| Reagent | Volume | Final Concentration |
| --- | --- | --- |
| 2×TransFast Taq PCR SuperMix(+dye) | 25μL | 1× |
| Primer 1 | 1μL | 0.2μM |
| Primer 2 | 1μL | 0.2μM |
| Template | 5μL | As required |
| DNase-Free Water | To 50μL |  |

Table 2 PCR reaction program

| Temperature | Time | Number of cycles |
| --- | --- | --- |
| 94℃ | 3 min | 1 cycle |
| 94℃ | 5 sec | 35 cycles |
| 5 2℃ | 15 sec |
| 72℃ | 30 sec |
| 72℃ | 5 min | 1 cycle |

Table 3 2% agarose gel

| Reagent | Amount |
| --- | --- |
| agarose | 1.8g |
| 1×TAE | 90mL |
| Nucleic acid dye EB | 9μL |

Experimental Procedure

Plasmid extraction

1.1 Check whether RNase A has been added to
Buffer P1.

1.2 Check whether anhydrous ethanol has been
added to the Wash Solution.

1.3 Check whether buffer P2 and P3 are
precipitated.

1.4 Take 1.5-5 mL of the yeastl culture
overnight and centrifuge at 8,000×g for 2
minutes to collect the yeast and discard all the
culture medium.

1.5 Add 250 μL Buffer P1 to the precipitate and
completely suspend the yeast.

1.6 Add 250 μL Buffer P2 and gently invert the
centrifuge tube 5-10 times immediately to mix.
Let it stand at room temperature for 2-4 min.

1.7 Add 350 μL Buffer P3 and gently invert the
centrifuge tube 5-10 times immediately to mix.

1.8 Centrifuge at 12,000×g for 10 minutes.
Transfer the supernatant to the adsorption
column and let it stand for 3-4 minutes.
Centrifuge at 8,000×g for 30 seconds, then
discard the liquid from the collection tube.

1.9 Add 500 μL Buffer DW1 and centrifuge at
9000×g for 30 seconds. Pour off the liquid in
the collection tube.

1.10 Add 500 μL Wash Solution and centrifuge at
9000 x g for 30 seconds. Pour off the liquid in
the collection tube. Repeat 2.10 once.

1.11 The empty adsorption column was centrifuged
at 9000 ×g for 1 min.

1.12 Place the adsorption column into a clean
1.5mL centrifuge tube, add 50-100 μL Elution
Buffer in the center of the adsorption membrane,
and centrifuge for 1 min after room temperature
standing for 1 min. Save the DNA solution in the
tube.

Extraction of genomic DNA from Saccharomyces
cerevisiae

2.1 Take 200μL yeast culture solution and
centrifuge (4000rpm, 30s) to discard the
supernatant;

2.2 Resuspend the cell precipitate in 100μL
LiOAc-1%SDS solution, and incubate it in a 70℃
oven for 5min;

2.3 Add 300μL anhydrous ethanol, repeatedly blow
and mix, centrifuge (15000rpm, 3min) and discard
the supernatant;

2.4 Wash the precipitate with 70% ethanol
solution, and discard the supernatant after
centrifugation;

2.5 The precipitate was dissolved in 100μL water
and stored in a-20℃ refrigerator.

PCR and agarose gel electrophoresis

3.1 Prepare PCR reaction system according to
Table 1;

3.2 Set the PCR reaction program according to
Table 2 and carry out PCR;

3.3 Prepare 2% agarose gel according to Table 3
and microwave heat dissolve;

3.4 Add 9 μL EB to the agarose gel and shake it
well. Then pour it into the mixing tank where
the comb has been placed. Place it flat in the
refrigerator and wait for the gel to solidify;

3.5 Put the gel into the electrophoresis tank
and pour in an appropriate amount of 1×TAE
electrophoresis buffer. After sampling,
electrophoresis at 1 40V constant voltage for
25min;

3.6 After electrophoresis, observe and take
photos under ultraviolet lamp;

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/nooa2100.webp)

Results Analysis and Discussion

1. The three target genes were detected in the
positive control group PC (plasmid ①) with clear
bands, indicating that the three target genes
were successfully cloned into the plasmid vector

2. The positive bands of the three target genes
in the target strain suggested that the three
target genes were successfully transferred into
yeast.

3. The expression of the three target genes in
the target yeastl solution was significantly
higher than that in the negative control group,
indicating that the target genes were
successfully overexpressed in Saccharomyces
cerevisiae.

Protein extraction of yeast strain samples
transferred to plasmid ② and SDS-PAGE gel
electrophoresis; carboloyd staining

Experiment time: 2025.08.12

Experiment operator: Wang Jingning, Wang
Quanhao, Min Yingxuan, Sun Jiayin

Experimental Objective

(1) Sample protein extraction was performed by
SDS-PAGE gel electrophoresis; Coomassie
Brilliant Blue staining was used to prepare for
normal plasmid expression

(2) Determine whether the genes of three
enzymes, MtCPR, CYP716A12 and β-AS, were
transferred into Saccharomyces cerevisiae

Experimental Equipment and Reagents

Equipment

Ultra-clean workbench, centrifuge, water bath,
gel plate, electrophoresis tank, vertical
electrophoresis instrument, transfer
electrophoresis instrument, decolorization
shaker, chemiluminescence imaging system;

Reagent

NaOH, β-glycerophosphate sodium, sodium
orthovanadate, NaF, EDTA, PMSF, Tris, glycine,
SDS, Coomassie Brilliant Blue R-250, methanol,
glacial acetic acid, 30% polyacrylamide, 1.0
MTris-HCl (pH 6 .8), 1.5MTris-HCl (pH8.8),
10%APS, TEMED

Reagent preparation

Table 1 Configuration of lysate

| Reagent | Volume | Final Concentration |
| --- | --- | --- |
| NaOH | 1ml | 0.1M |
| sodium β-glycerophosphate | 11ul | 100mM |
| sodium orthovanadate | 11ul | 100mM |
| NaF | 11ul | 5M |
| EDTA(ethylenediaminetetraacetic acid) | 3ul | 0.5M |
| PMSF ( Phenylmethylsulfonyl fluoride ) | 11ul | 100mM |

Table 2 Electrophoresis buffer
configuration

| Reagent | Amount |
| --- | --- |
| Tris | 3g |
| glycocoll | 14.6g |
| SDS | 1g |
| ddH2O | Vide to 1000ml |

Table 3 Configuration of carbofuran and
brilliant blue staining solution

| Conventional R-250 staining system | |
| --- | --- |
| Reagent | Amount (100 mL) |
| Coomassie Brilliant Blue R-250（w/v） | 0.25 g |
| carbinol | 45 mL |
| glacial acetic acid | 10 mL |
| H2O | Make up to 100 mL (about 45 mL) |

Table 4 Decoloration solution
configuration

| Reagent | Amount (500 mL) |
| --- | --- |
| carbinol | 200mL |
| glacial acetic acid | 50mL |
| H2O | Supplement to 500 mL |

Table 5 Concentrated glue
configuration

| Reagent | Amount |
| --- | --- |
| ddH2O | 2.1 mL |
| 30% polyacrylamide | 0.5 m L |
| 1.0 MTris-HCl (pH 6 .8) | 0.38 m L |
| 10%SDS | 30 μ L |
| 10%APS | 30 μ L |
| TEMED | 3 μ L |
| TEMED | 3 μL |

Table 6 Separation gel
configuration

| Reagent | Amount |
| --- | --- |
| ddH2O | 1.6mL |
| 30% polyacrylamide | 2mL |
| 1.5MTris-HCl (pH8.8) | 1.3mL |
| 10%SDS | 50μL |
| 10%APS | 50μL |
| TEMED | 5μL |

Experimental Procedure

sample preparation

1.1 Yeast lysate is prepared according to Table
1: with NaOH as the benchmark, each reagent in
the table below is added to 1mL NaOH, and
protease inhibitors and phosphatase inhibitors
(non-essential) can be added according to
specific experimental requirements;

1.2 Take 200μL yeast culture solution,
centrifuge (4000rpm, 30s) and discard the
supernatant;

1.3 Add the lysis solution to the obtained
yeastl precipitate and lyse on ice for 10min;

1.4 Centrifuge (5000rpm, 1min), discard the
supernatant;

1.5 Add 80μL 1× loading buffer to the
precipitate and mix well. After metal bath
(100℃,5min), store in-20℃ refrigerator for
standby

Protein blotting and SDS-PAGE gel
electrophoresis

2.1 Configure the SDS-PAGE gel and
electrophoresis according to the method in 4.2;

2.2 Before protein loading, the sample was
bathed in hot water for 5min and centrifuged at
5000rpm for 3min. The supernatant and
precipitate were respectively loaded for
electrophoresis. After the gel electrophoresis,
remove the gel plate, carefully peel off the gel
from the glass plate, and put it into the
staining box/tray; rinse the surface of the gel
with deionized water 1 to 2 times to remove the
residual electrophoresis buffer (gentle
operation to avoid tearing the gel).

2.3 Fixation (removes SDS and protein leakage,
improves staining signal-to-noise ratio): Add
sufficient fixative solution (methanol/acetic
acid/water, typically 45% methanol + 10% acetic
acid + 45% water) to the gel-containing
container. Shake gently at room temperature for
30 minutes. The fixation process can be extended
to 1 hour or overnight at 4℃°C to enhance
staining quality.

Carma blue staining and decolorization

3.1 Discard the fixed liquid and add the newly
prepared Comass blue staining solution to
completely immerse the gel. Stain for 1-2 h at
room temperature on a horizontal shaker; if more
sensitive, stain overnight (do not exceed 24 h
to avoid increased background).

3.2 After the dyeing, discard the dye and use
the decolorizing solution (40% methanol, 10%
acetic acid, 50% water) for the first
decolorization. Shake gently at room temperature
for 30 min to 1 h until the background begins to
fade obviously.

3.3 Replace the decolorizing solution and
continue decolorization on a horizontal shaker.
Adjust the decolorizing solution based on
background coloration and band development,
repeating this process every 30-60 minutes until
the background becomes sufficiently pale and
band contrast is clear. Complete decolorization
may take several hours to a couple of days
(allow overnight exposure for continued
decolorization the next day). For faster
results, perform short decolorization cycles,
capture images immediately, then proceed with
detailed decolorization.

Gel imaging analysis

4.1 After decoloring to the desired extent,
rapidly rinse the gel with deionized water to
remove residual methanol/acetic acid. Place the
gel in a photo tray and use a gel imaging system
or flatbed scanner to photograph/scan it. It is
recommended to use either white light
transmission or reflection mode depending on the
equipment, and record exposure parameters for
comparison.

4.2 Take a photo/scans and measure the band
intensity quantitatively with image software
(e.g., ImageJ), paying attention to using the
same exposure/scanning Settings and measuring
within the unsaturated range.

Experimental Result

![](https://static.igem.wiki/teams/5564/notebook/nooa22.webp)

![](https://static.igem.wiki/teams/5564/notebook/nooa24.webp)

![](https://static.igem.wiki/teams/5564/notebook/nooa250.webp)

Results Analysis and Discussion

(1) In the supernatant and sediment groups
(labeled as Control Sup and Control Pel), no
distinct protein bands corresponding to the
indicated molecular weights of 76.8 kDa, 54.3
kDa, and 43.8 kDa were observed. This indicates
that in normal W303 yeast without genetic
manipulation, proteins matching these target
molecular weights are either absent or present
only in trace amounts undetectable by
conventional methods. These findings exclude the
possibility of interference from endogenous
yeast proteins in the experimental results.

(2)In the experimental plasmids Plasmid 2 Sup
and Plasmid 2 Pel, three distinct protein bands
were clearly visible at the positions indicated
by the arrows. The molecular weights of these
bands perfectly matched the expected target
proteins (MtCPR: 76.8 kDa; CYP716A2: 54.3 kDa;
β-As: 43.8 kDa). This result directly
demonstrates that after plasmid transfection,
yeast cells successfully transcribed and
translated the foreign genes, producing
substantial quantities of the target proteins.

Transfer of plasmid ② yeast product OA
extraction and cell cryopreservation

Experiment time: 2025.08.14

Personnel: Sun Jiayin

Experimental Objective

(1) Oleanolic acid OA was extracted from the
cells of Saccharomyces cerevisiae after 72h of
cultivation to prepare for the detection of OA
content by liquid chromatography;

(2) Brewers yeast with frozen chromosomal
plasmid ②.

Experimental Equipment and Reagents

Equipment

Ultra-clean workbench, oscillator, ultrasonic
crusher, centrifuge.

Reagent

Chloroform, methanol, 50% glycerol

Experimental Procedure

Liquid chromatography sample preparation

(1) Take 3mL of fermentation liquid in the
crushing tube, centrifuge (13000 rpm/ min,
3min);

(2) Add the culture medium, add an appropriate
amount of glass beads (diameter 0.5mm) and 1mL
extractant (chloroform: methanol =1:1), and
shake to break for 5min;

(3) After ultrasonic crushing for 30min,
centrifugation (13000 rpm/min, 3min) was
performed, and the supernatant was taken for
later use.

Saccharomyces
cerevisiae cryopreservation

(1) Take 3 EP tubes and add 750μL 50% glycerol
solution to each EP tube;

(2) Add 750μL of yeastl liquid to each EP tube,
mark it and put it in the-20℃ refrigerator for
preservation.

Determine whether OA is present in the HPLC
analysis product

(1) Gradient elution was performed using VP-ODS
C18 chromatography column (5μl,150nm×4.6nm).

(2) Separation conditions: Acetonitrile (mobile
phase A), purified water (mobile phase B);
Column temperature 35℃, injection volume 20μl,
flow rate 1.0m L/min, UV detector wavelength
203nm.

(3) Elution conditions: 0 ~ 25min, 19﹪ B, 81﹪
A, 25 ~ 50min, 29﹪ B, 71﹪ A.

Experimental Result

![](https://static.igem.wiki/teams/5564/notebook/nooa26.webp)
![](https://static.igem.wiki/teams/5564/notebook/nooa27.webp)

Results Analysis and Discussion

The absence of positive results in liquid
chromatography analysis may be attributed to the
degradation of OA caused by abnormal
photothermal conditions during product
extraction. This suggests that controlling the
temperature during ultrasonic crushing or
maintaining an appropriate organic phase ratio
in the mobile phase is necessary, as excessive
elution could prevent OA retention. Further
optimization should focus on regulating the
systems crushing temperature and selecting
suitable mobile organic phases for elution.

matters need attention

(1) When preparing the extraction solution, the
volume should be calculated in advance to
prevent insufficient dosage from repeated
preparation.

(2) In the first step of centrifugation, try to
pour out the supernatant as far as possible,
otherwise the liquid will be separated and the
cell layer may float up and affect the effect of
ultrasonic crushing.

(3) When shaking, pay attention to the moment to
see if there is any leakage of liquid. If three
tubes can not be done at the same time, the
quantity can be reduced.

Product extraction and cell cryopreservation

Experiment time: 2025.08.16

Experiment operator: Sun Jiayin, Wang Quanhao,
Min Yingxuan

Experimental Objective

(1) Cyclamen acid OA was extracted from yeast
cells for 72h to prepare for the detection of OA
content by liquid chromatography;

(2) Brewing yeast transfected by
cryopreservation.

(3) The yeast was cultured and a single clone
was determined to determine the OD value for
plasmid transfection

Experimental Equipment and Reagents

Equipment

laminar flow hood, ultrasonic homogenizer,
ultrasonic crusher, centrifuge,
spectrophotometer, 96-well plate, yeastl filter

Reagent

Chloroform, methanol, 50% glycerol, ethyl
acetate, YPD medium

Experimental Procedure

Liquid chromatography sample preparation

(1) Take 3mL of fermentation liquid in the
crushing tube, centrifuge (1 2 000 rpm/ min,
3min);

(2) Add 1mL extractant (chloroform: methanol
=1:1 or ethyl acetate) to the culture medium and
mix well by blowing, add an appropriate amount
of glass beads (0.5mm in diameter), and shake
for 5min;

(3) After 30min ultrasonic crushing,
centrifugation (12000 rpm/min, 3min) was
performed, and the supernatant was taken for
later use.

Saccharomyces
cerevisiae cryopreservation

Take 3 EP tubes and add 750μL 50% glycerol
solution to each EP tube;

Add the yeastl liquid to each EP tube until
full, mark it and put it in-20℃ refrigerator for
preservation.

Pick a single colony from the culture medium

(1) The ultra-clean workbench is irradiated with
ultraviolet lamp 30min in advance;

(2) Take 50mL centrifuge tube, pour 5mL YPD
liquid culture medium into the centrifuge tube;

(3) Pick the single clone yeast from the YXZ6
yeast culture plate and add it to the centrifuge
tube. Put it into the shaker culture
(30℃,200rpm) for about 7h;

(4) After 7 hours, measure OD600.
like OD600=0.3, then take 200μL liquid and add
it to 50mLYPD medium.

Determine whether OA is present in the HPLC
analysis product

(1) Gradient elution was performed using VP-ODS
C18 chromatography column (5μl,150nm×4.6nm).

(2) Separation conditions: Methanol (mobile
phase A), purified water (mobile phase B);
Column temperature 35℃, injection volume 20μl,
flow rate 1.0m L/min, UV detector wavelength
203nm.

(3) Elution conditions: 0 ~ 25min, 19﹪ B, 81﹪
A. 25 ~ 50min, 29﹪ B, 71﹪ A.

Experimental Result

![](https://static.igem.wiki/teams/5564/notebook/nooa290.webp)

Discussion and analysis

(1) A distinct chromatographic peak was observed
in the 23-minute region when compared with the
standard reference. This indicates that
oleanolic acid OA is indeed present in the
sample under these chromatographic conditions,
flowing out around 23.5 minutes. The presence of
oleanolic acid OA after transformation confirms
successful plasmid transfection and normal
function of the corresponding protein.

(2) The shape of the peak is basically
symmetrical, without serious tailing or forward
extension, which indicates that the
chromatography column is in good condition,
there is no serious nonspecific adsorption
between the sample and the stationary phase, and
the analysis conditions are relatively ideal.

(3) From the spectrum, the peak and the adjacent
peaks (the small peaks around 21 minutes and 25
minutes) all achieved baseline separation. This
means that the target component was well
separated and not easily interfered by the
adjacent peaks.

matters need attention

1. Trim the edge of the paper before shock to
avoid paper offset caused by EP tube offset or
leakage during shock

2. When shaking, pay attention to observe
whether the EP tube position is offset and
whether there is any leakage of liquid

3. OD of yeast on day 2600If it is greater than
0.8 but not more than 1.5, the next step can be
carried out

4. The HPLC mobile phase should be selected as a
9:1 methanol: purified water system, which can
fully elute OA without excessive elution

# Multi-step glycosylation to synthesize Ginsenoside Ro

Streak Culture of Three Strains; Preparation of
YPD, Sc-Trp-Leu Double-Deficient Solid Medium,
and Sc-Trp Liquid Medium

Experimental time: 2025.08.07

Experimental personnel: Sun Jiayin, Wang Quanhao

Objectives of the Experiment

(1) Perform streak culture on Strain 1, Strain
2, and Strain 3 from the three cryopreservation
tubes into which Plasmid ② has been transferred.

(2) Prepare YPD medium, Sc-Trp liquid medium,
and Trp-Leu double-deficient solid medium.

Experimental Equipment and Reagents

Equipment

Electronic balance, magnetic stirrer, autoclave,
aseptic operating bench, Petri dishes

Reagents

Glucose, peptone, yeast extract, arginine (Arg),
adenine, SC-Trp, SC-Trp-Leu

Reagent Preparation

Table 1 YPD Medium (500 mL)

| Reagent | Amount |
| --- | --- |
| Peptone | 10 g |
| Yeast extract | 5 g |
| 20% Glucose | 10 g |
| H2O | 500 mL |

Table 2 Sc-Trp Liquid Medium (180
mL)

| Reagent | Amount |
| --- | --- |
| SC-Trp | 0.4 g |
| Arginine | 0.04 g |
| Adenine | 0.2 g |
| 20% Glucose | 4 g |
| H2O | 180 mL |

Table 3 Trp-Leu Double-Deficient Solid
Medium (180 mL)

| Reagent | Amount |
| --- | --- |
| SC-Trp-Leu | 0.4 g |
| Arginine | 0.04 g |
| Adenine | 0.2 g |
| 20% Glucose | 4 g |
| H2O | 60 mL |
| Take another empty bottle and add a stir bar | |
| Agar | 120 g |
| H2O | 120 mL |

Table 4 YNB Solution

| Reagent | Amount |
| --- | --- |
| YNB for Yeast | 1.6 g |
| Ammonium Sulfate ((NH4)2SO4) | 5 g |
| H2O | 100 mL |

Experimental Procedure

Preparation of YPD Medium

(1) Prepare the solution according to Table 1.

(2) Place the glass bottle containing the
solution into an autoclave for autoclaving
(115°C, 15 minutes).

(3) The preparation of YPD medium is completed.

Preparation of Sc-Trp Liquid Medium

(1) Prepare the Sc-Trp solution according to
Table 2 and the YNB solution according to Table
4.

(2) Place the glass bottle with the solution
into an autoclave for autoclaving (115°C, 15
minutes).

(3) Sterilize the YNB solution by filtration
through a 0.22 μm filter, then transfer it to a
100 mL autoclaved empty bottle for later use.

(4) Take 20 mL of the filter-sterilized YNB
solution, add it to the Sc-Trp solution, and mix
thoroughly.

Preparation of Trp-Leu Double-Deficient Solid
Medium

(1) Prepare the Trp-Leu double-deficient
solution according to Table 3 and the YNB
solution according to Table 4.

(2) Place the glass bottle with the solution
into an autoclave for autoclaving (115°C, 15
minutes).

(3) Sterilize the YNB solution by filtration
through a 0.22 μm filter, then transfer it to a
100 mL autoclaved empty bottle for later use.

(4) Take 20 mL of the filter-sterilized YNB
solution, add it to the Trp-Leu double-deficient
solution, and mix thoroughly.

(5) Pour the medium into sterile Petri dishes
(approximately 15-20 mL per dish). After
standing and solidifying, store them upside down
at 4°C (to avoid the accumulation of condensed
water).

Colony Streak Culture

(1) Sterilize the biosafety cabinet with
ultraviolet light for 30 minutes in advance, and
wipe the experimental bench with 75% alcohol for
disinfection.

(2) Use a marker pen to label the bottom of the
plate with information such as strain name,
date, and operator.

(3) Pass a bamboo stick through the outer flame
of an alcohol lamp for sterilization, let it
cool down, and then directly dip a small amount
of yeast solution (a visible droplet to the
naked eye is sufficient).

(4) Gently draw a "Z" pattern with the bamboo
stick dipped in the yeast solution on one side
of the plate (about 1/4 of the area, marked as
Area A). The lines should be dense but not
overlapping, and the angle of the inoculating
tool (bamboo stick) should be approximately
30°-45° during streaking. Discard the bamboo
stick after streaking.

(5) For the second streaking (Area B), use a
cooled bamboo stick to cross 1-2 times from the
end of Area A, extend to Area B (about 1/2 of
the area), and draw a sparse "Z" pattern. For
the third streaking (Area C/D), repeat the above
steps: streak from the end of Area B to Area C
(the remaining area) with even sparser lines,
and finally form a gradient dilution (high yeast
concentration in Area A and low yeast
concentration in Area D).

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/noro01.webp)

Analysis and Discussion

The culture medium was successfully prepared,
and the streaked colonies grew normally.

Precautions

(1) Before using the biosafety cabinet,
sterilize it with ultraviolet light for 30
minutes. When starting the experiment, the first
step is to light the alcohol lamp. The flame of
the alcohol lamp in the laboratory is easy to
ignite but invisible, so be careful to avoid
scalding.

(2) After opening the bottle cap, do not place
anything above the bottle mouth, and place the
bottle cap upside down. When taking centrifuge
tubes, EP tubes, and bamboo sticks from the box,
use tweezers, and sterilize the tweezers by
passing them through the flame.

(3) YNB does not require autoclaving, nor does
it need to be treated with a microwave oven
before use; for YPD medium, perform microwave
disinfection one day before the experiment.

(4) Apply gentle force with your hand during
streaking to avoid scratching the agar. The
lines should be dense but not overlapping, and
the angle of the inoculating tool (bamboo stick)
should be approximately 30°-45° during
streaking.

Monoclonal Culture of Saccharomyces
cerevisiae
Transfected with Plasmid ② and OD Value
Determination

Experimental time: 2025.08.09

Experimental personnel: Sun Jiayin, Wang
Quanhao, Min Yingxuan

Objectives of the Experiment

Cultivate monoclonal colonies of Saccharomyces
cerevisiae transfected with Plasmid ② and
determine their OD values to prepare for plasmid
transfection.

Experimental Equipment and Reagents

Equipment

Ultra-clean workbench, absorbance meter, 96-well
plate

Reagents

YPD medium

Experimental Procedure

Picking Monoclonal Colonies from the Medium

(1) Turn on the ultraviolet lamp of the
ultra-clean workbench for 30 minutes in advance
for sterilization; prepare YPD medium and
perform microwave treatment in advance for later
use.

(2) Take a 50 mL centrifuge tube and pour 5 mL
of liquid YPD medium into it.

(3) Pick a monoclonal colony of Saccharomyces
cerevisiae (transfected with Plasmid ②) from the
culture plate, add it to the centrifuge tube,
and place the tube in a shaker for cultivation
(30°C, 200 rpm) for approximately 9 hours.

(4) After 9 hours, determine the OD₆₀₀ value. If
the OD₆₀₀ value is 0.3, take 2 mL of the yeast
solution and add it to 100 mL and 50 mL of YPD
medium respectively (to explore which system
yields better transfection efficiency).

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/noro03.webp )

Analysis and Discussion

After 9 hours, the OD value of the yeast reached
0.3, and its growth status was excellent.

Precautions

(1) For the initial 100 mL YPD medium, resuspend
the yeast cells with 800 μL of 0.1 M LiAc/TE
later.

(2) For the initial 50 mL YPD medium, resuspend
the yeast cells with 400 μL of 0.1 M LiAc/TE
later.

Preparation of Sc-Trp-Leu Medium: Competent
Cells

Experimental time: 2025.08.11

Experimental personnel: Sun Jiayin, Wang
Quanhao, Min Yingxuan

Objectives of the Experiment

(1) Prepare Sc-Trp medium and pour it into
plates to lay the foundation for the cultivation
of Saccharomyces cerevisiae after subsequent
plasmid transfection.

(2) Prepare competent cells to get ready for
plasmid transfection.

Experimental Equipment and Reagents

Equipment

Ultra-clean workbench, microplate reader,
high-temperature and high-pressure autoclave,
shaker

Reagents

SC-Trp-Leu, arginine, adenine, glucose, YNB
(Yeast Nitrogen Base), ammonium sulfate
((NH₄)₂SO₄), agar, sterile water

Reagent Preparation

Table 1 Sc-Trp-Leu Medium (200
mL)

| Reagent | Amount |
| --- | --- |
| Sc-Trp-Leu | 0.4g |
| L-Arginine | 0.04g |
| adenine | 0.2g |
| glucose | 4g |
| agar | 4g |
| YNB | 1.6g |
| (NH4)2SO4 | 5g |
| H2O | 200mL |

Experimental Procedure

Preparation of Sc-Trp-Leu Medium

(1) Take 0.4 g of Sc-Trp-Leu, 0.04 g of
arginine, 0.2 g of adenine, and 4 g of glucose,
add 60 mL of water, and place the mixture in an
empty bottle.

(2) Take 4 g of agar, add 120 mL of water, place
the mixture in another empty bottle, and add a
magnetic stir bar.

(3) Put the two aforementioned bottles and one
100 mL empty bottle into a high-temperature and
high-pressure autoclave for autoclaving (115°C,
15 minutes).

(4) Weigh 1.6 g of YNB for yeast and 5 g of
ammonium sulfate, place them in a beaker, add
100 mL of autoclaved water, and add a magnetic
stir bar to stir until completely dissolved.

(5) Sterilize the solution by filtration through
a 0.22 μm filter, then transfer it to a 100 mL
autoclaved empty bottle for later use.

(6) Take 20 mL of the filter-sterilized YNB
solution and add it to the autoclaved bottle
containing agar. While the mixture is still hot,
pour the SC-Trp solution into the agar bottle
with YNB added, and mix thoroughly using a
magnetic stirrer.

(7) Quickly pour the mixed solution into sterile
Petri dishes to make plates. Let them stand at
room temperature for 4 hours, then store them in
a refrigerator for later use.

Preparation of Competent Cells

(1) Place sterile water in a 4°C refrigerator
for later use, and dilute LiAc/TE (Lithium
Acetate/Tris-EDTA Buffer) to a concentration of
0.1 mol/L for later use.

(2) Determine the OD₆₀₀ value of the
Saccharomyces cerevisiae (transfected with
Plasmid ②) cultured the previous day. If the
OD₆₀₀ value is 0.8, proceed to the next step of
the experiment.

(3) Take 50 mL of the yeast solution, centrifuge
it (3500 rpm, 2 minutes), and discard the
supernatant.

(4) Resuspend the yeast pellet with 50 mL of
cold sterile water, centrifuge again (3500 rpm,
2 minutes), and discard the supernatant.

(5) Resuspend the pellet with 1 mL of 0.1 mol/L
LiAc/TE, centrifuge rapidly (4000 rpm, 30
seconds), and discard the supernatant.

(6) Resuspend the pellet with 400 μL of 0.1
mol/L LiAc/TE, mix well, and store it on ice in
the refrigerator overnight.

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/noro04.webp)

Precautions

(1) The filter-sterilized YNB solution must be
quickly poured into the agar while it is still
hot to prevent uneven distribution of YNB, which
may cause abnormal solidification of the medium.

(2) After the medium is prepared, it should be
quickly poured into plates to avoid premature
solidification due to prolonged standing.

(3) Monitor the OD value of Saccharomyces
cerevisiae to prevent failure to proceed to the
next step of the experiment due to excessive
culture time.

Plasmid③ transfection and glycerol stock plate
coating

Experiment time: 2025.08.12

Experimental personnel: Sun Jiayin, Wang
Quanhao, Min Yingxuan

Objectives of the Experiment

(1) Saccharomyces cerevisiae was transfected
with plasmid 3 to synthesize the final product
ginsenoside Ro;

(2) Glycochocera coating plate, easy to pick
single colony for plasmid extraction.

Experimental Equipment and Reagents

Equipment

laminar flow hood, shaker, 30℃ incubator, water
bath, shaker, oven, electronic balance, magnetic
stirrer, autoclave, PCR instrument,
electrophoresis system, gel imaging system,
adsorption column

Reagents

Buffer P1、Buffer P2、Buffer P3、Buffer
DW1、Wash Solution、ElutionBuffer; PEG, LiAc
(1mol/L), salmon sperm, plasmid, yeast receptive
state, YPD medium;

Experimental Procedure

Viral transfection

(1) Salmon sperm was boiled in boiling water for
5min and then rapidly placed in an ice water
mixture (on ice)

(2) Add about 50 μL of yeast receptive strain
that has been transplanted with plasmid ②
prepared the previous day into each EP tube, and
centrifuge (4000rpm, 30s) to discard the
supernatant;

(3) Add 42μL plasmid, 10μL croaker sperm (10
mg/mL), 240μL PEG, and 36μL LiAc (1 mol/L)
sequentially into the EP tube. After repeatedly
pipetting to mix thoroughly with a 1mL pipette,
incubate in a 30℃ incubator for 30 minutes.

(4) Add 36μL DMSO to each EP tube, mix by
repeated blowing and place it in a 42℃ water
bath for 20min.

Yeast culture after transfection

(1) The EP tube was taken out of the water bath,
and the supernatant was discarded after
centrifugation (3000rpm, 20s). The suspension
was resuspended with 1 mL YPD medium and
cultured in a shaker for 3 h.

(2) After centrifugation (3000rpm, 20s), the
supernatant was discarded, and the yeast
suspension was resuspended with 100μL water. The
yeast suspension was spread on the plate
(Sc-Trp) with glass beads, and cultured in a 30℃
incubator for about 3 days.

Glycozyme smear

Single colonies of glycerobacterium were picked
and streaked on ampicillin LB medium

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/noro05.webp)
![](https://static.igem.wiki/teams/5564/notebook/noro06.webp)

Precautions

(1) Salmon sperm need to be boiled in boiling
water for 5min in advance, and then quickly put
into ice water mixture to play the role of
protecting exogenous plasmid.

(2) All kinds of reagents required for particle
transfection should be mixed evenly to maximize
the permeability of cell membrane and improve
the transfection efficiency.

(3) The incubation time of the titer
transfection incubator and the heat shock time
of the water bath must be strictly controlled to
prevent the decrease of transfection efficiency
after too long time.

Preparation of Sc-Trp-Leu solid medium;
Preparation of Sc-Trp liquid medium; Cultivation
of Saccharomyces
cerevisiae (with plasmid 2);
Cultivation of glycerophages; Extraction of W303
genome

Experiment time: 2025.08.13

Experimental personnel: Wang Quanhao, Min
Yingxuan

Objectives of the Experiment

(1) Prepare Sc-Trp-Leu solid culture medium and
Sc-Trp liquid culture medium for standby;

(2) Cultivate yeast for wine making (with
plasmid 2) and measure OD value for preparation
of receptive state;

(3) Cultivate glycerol stock for plasmid
extraction;

(4) W303 genome was extracted for PCR negative
control;

Experimental Equipment and Reagents

Equipment

High pressure sterilizer, super clean workbench,
centrifuge, shaker, enzyme labeling instrument,
96 plate, conical flask;

Reagents

SC-Trp, arginine, adenine, glucose, ammonium
sulfate, agar, sterile water, Sc-Trp-Leu liquid
medium, YNB solution, YPD medium, LiOAc-1%SDS
solution, anhydrous ethanol, 70% ethanol;

Reagent Preparation

Table 1 Sc-Trp liquid culture medium
(60 mL)

| Reagent | Amount |
| --- | --- |
| Sc-Trp | 0.4 g |
| L-Arginine | 0.04 g |
| adenine | 0.2 g |
| glucose | 4 g |
| H2O | 60 mL |

Table 2 Sc-Trp-Leu double deficiency
solid medium (200 mL)

| Reagent | Amount |
| --- | --- |
| Sc-Trp-Leu double deficiency solution | 60 mL |
| YNB solution | 20 mL |
| Add rotor | |
| agar | 120 g |
| Water H2O | 120 mL |

Experimental Procedure

Culture preparation

(1) Prepare Sc-Trp liquid culture medium
according to Table 1 in Section 2 and place it
in an empty bottle;

(2) Take 4g agar and add 120mL water to another
empty bottle;

(3) Place the two bottles into the autoclave and
sterilize (115℃,15min);

(4) Prepare the solution according to Table 2 in
section 2 while hot, and mix it thoroughly with
a magnetic stirrer;

(5) The mixture was poured into a sterile petri
dish to make a plate. After 5 hours at room
temperature, it was put in the refrigerator for
storage;

Brewth yeast (with plasmid 2) culture

(1) The ultra-clean workbench is irradiated with
ultraviolet lamp 30min in advance;

(2) Take 50mL centrifuge tube, pour 10mL YPD
liquid culture medium into the centrifuge tube;

(3) A single colony was picked from the yeast ①
culture plate containing plasmid 2 and added to
a centrifuge tube. The culture was placed in a
shaker (30℃,200rpm) for about 7h;

(4) After 7 hours, 3 mL of yeast solution was
inoculated into 100 mL YPD medium to measure
whether the OD value was between 0.2 and 0.3;

Glycojugate culture

(1) Take a 50 mL centrifuge tube and add 15 mL
glycerol-fatty acid liquid culture medium.

(2) Pick a single colony from plate 2 and 3 of
glycerophages and add it to a centrifuge tube.
Place it in a shaker culture (30℃,200 rpm);

W303 genome extraction

(1) Take 200 μL yeast culture and centrifuge to
discard the supernatant;

(2) The cell was precipitated and resuspended in
100 μL LiOAc-1%SDS solution, and incubated in a
70℃ oven for 5min;

(3) Add 300 μL anhydrous ethanol, repeatedly
blow and mix, centrifuge (12000rpm, 3min) and
discard the supernatant;

(4) Wash the precipitate with 70% ethanol
solution, and discard the supernatant after
centrifugation;

(5) The precipitate was dissolved in 100 μL
water and stored in a -20℃ refrigerator.

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/noro07.webp)
![](https://static.igem.wiki/teams/5564/notebook/noro08.webp)

Precautions

(1) Turn on the oven to warm up in advance when
extracting the genome;

Yeast monoclonal culture after protomer
transfection

Experiment time: 2025.08.15

Experimental personnel: Wang Jingning, Wang
Quanhao

Objectives of the Experiment

Three single clone colonies of dual plasmid
yeast 3 were picked from the plate, transferred
to Trp-Leu double deficiency medium, and
cultured on a shaker;

Experimental Equipment and Reagents

Equipment

Ultra-clean workbench, autoclave, shaker,
centrifuge.

Reagents

Peptone, yeast extract, sodium chloride,
glucose, sterile water.

Reagent Preparation

Table 1 Trp-Leu double deficiency
medium (500mL)

| Reagent | Amount |
| --- | --- |
| Sc-Trp-Leu | 0.4g |
| L-Arginine | 0.04g |
| adenine | 0.2g |
| Glucose | 4g |
| H2O | 180mL |

Experimental Procedure

Plasmid ②③ dual plasmid yeast 3 single clone
double deficiency culture medium culture

(1) Take the yeast ②③ double plasmid 3 culture
plate out of the yeast incubator and observe the
growth of yeast;

(2) Take 350mL centrifuge tubes and pour 15 mL
Trp-Leu deficiency medium into each centrifuge
tube;

(3) Three single clones were picked from yeast
plasmid 3 plate, transferred to the above
centrifuge tube, and cultured on a shaker;

(4) Subsequently, yeast growth was monitored
every 12h.

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/noro09.webp)

Analysis and Discussion

**The yeast growth was monitored every 12h,
and the growth had far exceeded the
logarithmic phase of the obvious yeast
growth curve, and was close to the plateau
phase. The monitoring interval time was too
long, and the growth rate of yeast exceeded
the expected rate**

Precautions

(1) When transferring from the culture plate to
the centrifuge tube, a single clone must be
selected to obtain a strain with stable genetic
background.

(2) When pouring the medium into the centrifuge
tube, in order to avoid contamination by the
remaining medium, part of the medium can be
transferred to a 50mL centrifuge tube for
reserve.

(3) The growth curve should be tested in time,
and the interval time should not be too long

Determination of growth curve of yeast
monoclonal culture after transfection with
plasmid ② + ③

Experimental time: 2025.08.17

Experimental personnel: Wang Jingning, Wang
Quanhao

Objectives of the Experiment

Every 2h, three single clone colonies of plasmid
② cultured in Trp-Leu deficient liquid medium
were transferred into 96-well plate with 100ul
yeast solution. The absorbance value was
measured to indirectly judge the growth status
of the strain. The continuous monitoring was
carried out for 96h, and finally the growth
curve of the single clone colony was drawn.

Experimental Equipment and Reagents

Equipment

Laminar flow cabinet, autoclave, shaker,
microplate reader, 96-well plate

Experimental Procedure

(1) The ultra-clean workbench is irradiated with
ultraviolet lamp 30min in advance;

(2) Take 50mL centrifuge tube and pour 15mL
glycerol fatty acid liquid culture medium into
the centrifuge tube;

(3) Pick three single clone yeast from the
culture plate of Saccharomyces cerevisiae with
the transferred plasmid ② + ③ and add it to the
centrifuge tube, and put it into the shaking bed
culture (30℃,200rpm);

(4) Observe the growth of yeast to see if the
liquid culture medium becomes cloudy;

(5) Take one 96-well plate, add 100ul of yeast
fluid into each well, set the Trp deficiency
culture medium control group, and set three
groups of repeated controls for three monoclonal
yeast fluid and single deficiency culture
medium. Measure the OD600 value with a
microplate reader and calculate the average
value.

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/noro110.webp)

Analysis and Discussion

(1) Trp-Leu defect culture medium After shaking
culture, the centrifuge tube was obviously
turbid. The OD value of three single clone
colonies measured under the microplate reader
was also increased compared with the control
group. The yeast growth degree of the plasmid ②+
③ was considerable after transplantation.

(2) The yeast growth was continuously monitored,
and the yeast reached the plateau period in
about 30h;

Precautions

(1) Trp-leu defect medium control group was set
up to better reflect the growth degree of yeast
fluid.

(2) The growth OD value was measured every 2h as
far as possible to monitor the growth rate and
degree of yeast in time.

Glycoflavin streaking; PCR and agarose gel
electrophoresis to verify the successful
transfection of plasmid ② + ③ enzyme gene

Experimental time: 2025.08.18

Experimental personnel: Sun Jiayin, Wang
Quanhao, Min Yingxuan

Objectives of the Experiment

(1) Monoclonal cells were prepared by
glycohydrolase streaking.

(2) The genes of UGT73P40, UGT73F3 and Pn022859
were identified to determine whether they were
transferred into yeast, and the expression of
plasmids in yeast strains was detected.

Experimental Equipment and Reagents

Equipment

Clean workbench, centrifuge, micropipette and
gun head, PCR instrument, electrophoresis
instrument, gel imaging system

Reagents

Plasmid 2, glycerol-free freeze-dried culture
medium, Amoxicillin LB solid agar plates, 2x
TransFast Taq PCR SuperMix (with dye), DNA
templates, specific primers, TAE buffer,
agarose, EB DNA stain, and DNA markers

Reagent Preparation

Table 1 PCR reaction system

| Reagent | Volumz | Concentration |
| --- | --- | --- |
| 2×TransFast Taq PCR SuperMix(+dye) | 25μL | 1× |
| Primer 1 | 2μL | 0.2-0.3μM |
| Primer 2 | 2μL | 0.2-0.3μM |
| Template | 5μL |  |
| DNase-Free Water | To 50μL |  |

Table 2 PCR reaction procedures

| Temperature | Time | Number of cycles |
| --- | --- | --- |
| 94℃ | 3 min | 1 cycle |
| 94℃ | 5 sec | 30 cycles |
| 55℃ | 15 sec |
| 72℃ | 30 sec |
| 72℃ | 5 min | 1 cycle |

Table 3 1% agarose gel

| Reagent | Amount |
| --- | --- |
| agarose | 0.9 g |
| 1×TAE | 90 mL |
| Nucleic acid dye EB | 9 μL |

Experimental Procedure

Glycojugate streaking

(1) The ultra-clean workbench was sterilized
with ultraviolet light 30 minutes in advance,
and the experimental table was wiped and
disinfected with 75% alcohol;

(2) Mark the name of the strain, date, operator
and other information on the bottom of the
tablet with a marker pen;

(3) The bamboo stick is burned in the outer
flame of the alcohol lamp, cooled, and then
dipped directly into a small amount of yeast
liquid (visible drops can be seen);

(4) The bamboo stick dipped in yeast solution is
gently drawn in a zigzag pattern on one side of
the plate (about 1/4 area, marked as A area).
The line should be dense but not overlapping.
The angle of the bamboo stick is about 30°-45°
when drawing the line. Discard the bamboo stick
after drawing the line;

(5) The second marking step (Zone B) involves
passing cooled bamboo sticks 1-2 times from the
end of Zone A, extending to Zone B
(approximately half of the area) to create
sparse zigzag patterns. The third marking step
(Zones C/D) repeats the process, extending from
Zone B's end to Zone C (the remaining area),
with even finer lines. This creates a gradient
dilution pattern where Zone A contains
concentrated yeast while Zone D has sparse yeast
distribution.

Yeast genome extraction

(1) Take 200μL yeast culture solution and
centrifuge (4000rpm, 30s) to discard the
supernatant;

(2) Resuspend the cell precipitate in 100μL
LiOAc-1%SDS solution, and incubate it in a 70℃
oven for 5min;

(3) Add 300μL anhydrous ethanol, repeatedly blow
and mix, centrifuge (15000rpm, 3min) and discard
the supernatant;

(4) Wash the precipitate with 70% ethanol
solution, and discard the supernatant after
centrifugation;

(5) The precipitate was dissolved in 100μL water
and stored in a-20℃ refrigerator.

PCR

(1) PCR reaction system was prepared according
to 3.1 in Section 2;

(2) Set the PCR reaction program according to
3.2 in Section 2 and carry out PCR;

Agarose gel electrophoresis

(1) Prepare 2% agarose gel according to 3.3 in
Section 2 by microwave heating;

(2) Add 9 μL EB and shake well, then pour it
into the mixing tank where the comb has been
placed. Place it flat in the refrigerator and
wait for the gel to solidify;

(3) Put the gel into the electrophoresis tank
and pour in an appropriate amount of 1x TAE
electrophoresis buffer. After sampling,
electrophoresis for 25min under constant voltage
of 140V;

(4) After electrophoresis, observe and take
photos under ultraviolet lamp;

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/noro130.webp)

Analysis and Discussion

(1) The three target genes were not detected in
the negative control group NC, indicating that
the primer amplification was specific;

(2) The three target genes were detected in the
positive control group PC (plasmid ② + ③ ) with
clear bands and normal molecular weight,
indicating that the three target genes were
successfully cloned into the plasmid vector;

UGT73P40

![](https://static.igem.wiki/teams/5564/notebook/note050804.webp)

In this experiment, the UGT73P40 gene showed
obvious bands in yeast 2 and yeast 3, indicating
tthat he gene was successfully transferred in
these two colonies; the band was not obvious in
yeast 1, indicating that the gene was
successfully transferred, and the results of
this experiment may be caused by the increase of
amplified fragment length or insufficient sample
addition during electrophoresis;

UGT73F3

![](https://static.igem.wiki/teams/5564/notebook/note050805.webp)

In this experiment, the UGT73F3 gene was clearly
visible in three monoclonal yeast strains, which
was strongly positive and basically consistent
with the previous results, indicating that the
gene was successfully transferred.

Pn022859

![](https://static.igem.wiki/teams/5564/notebook/note050806.webp)

The experimental results showed weak positive
for yeast 1, positive for yeast 2 and yeast 3,
indicating that the gene may have been
successfully transfected in yeast 1, but the
transfection amount was low. The negative result
in this study may be caused by the increase of
amplification fragment length and the decrease
of PCR efficiency.

Precautions

(1) The tail of the bamboo stick is picked out
with tweezers and then roasted.

(2) Start the line from the end of the last
line. The lines should be dense but not
overlapping.

(3) The PCR instrument should stop working
first, then open the lid, and finally shut down.

(4) Set the electrophoresis instrument to
constant voltage.

Protein extraction of yeast strain samples
transferred to plasmid ② + ③ ; SDS-PAGE gel
electrophoresis; Coomassie Brilliant Blue
staining

Experimental time: 2025.08.22

Experimental personnel: Wang Jingning, Wang
Quanhao, Min Yingxuan, Sun Jiayin

Objectives of the Experiment

(1) Sample protein extraction was performed by
SDS-PAGE gel electrophoresis; Coomassie
Brilliant Blue staining was used to prepare for
normal plasmid expression.

(2) Determine whether the genes of three
enzymes, MtCPR, CYP716A12 and β-AS, were
transferred into Saccharomyces cerevisiae.

Experimental Equipment and Reagents

Equipment

Ultra-clean workbench, centrifuge, water bath,
gel plate, electrophoresis tank, vertical
electrophoresis instrument, transfer
electrophoresis instrument, decolorization
shaker, chemiluminescence imaging system;

Reagents

NaOH, β-glycerophosphate sodium, sodium
orthovanadate, NaF, EDTA, PMSF, Tris, glycine,
SDS, Coomassie Brilliant Blue R-250, methanol,
glacial acetic acid, 30% polyacrylamide, 1.0M
Tris-HCl (pH6.8),1.5M Tris-HCl (pH 8.8),10% APS,
TEMED.

Reagent Preparation

Table 1 Configuration of lysate

| Reagent | Volume | Final Concentration |
| --- | --- | --- |
| NaOH | 1ml | 0.1M |
| sodium β-glycerophosphate | 11ul | 100mM |
| sodium orthovanadate | 11ul |
| NaF | 11ul |
| EDTA(ethylenediaminetetraacetic acid) | 3ul | 0.5M |
| PMSF(Phenylmethylsulfonyl fluoride) | 11ul | 100mM |

Table 2 Electrophoresis buffer
configuration

| Reagent | Amount |
| --- | --- |
| Tris | 3g |
| glycocoll | 14.6g |
| SDS | 1g |
| ddH2O | Vide to 1000ml |

Table 3 Configuration of carmine and
brilliant blue staining solution

| Conventional R-250 staining system | |
| --- | --- |
| Reagent | Amount (100 mL) |
| Coomassie Brilliant Blue R-250(w/v) | 0.25 g |
| carbinol | 45 mL |
| glacial acetic acid | 10 mL |
| H2O | Make up to 100 mL (about 45 mL) |

Table 4 Decoloration solution
configuration

| Reagent | Amount (500 mL) |
| --- | --- |
| carbinol | 200 mL |
| glacial acetic acid | 50 mL |
| H2O | Make up to 500 mL |

Table 5 Concentrated glue
configuration

| 5% Concentrated Gel | |
| --- | --- |
| Reagent | Amount |
| ddH2O | 2.1 mL |
| 30% polyacrylamide | 0.5 mL |
| 1.0M Tris-HCl (pH6.8) | 0.38 mL |
| 10%SDS | 30 μL |
| 10%APS | 30 μL |
| TEMED | 3 μL |

Table 6 Separation gel
configuration

| 12% separation gel | |
| --- | --- |
| Reagent | Amount |
| ddH2O | 1.6 mL |
| 30% polyacrylamide | 2 mL |
| 1.5M Tris-HCl (pH8.8) | 1.3 mL |
| 10%SDS | 50 μL |
| 10%APS | 50 μL |
| TEMED | 5 μL |

Experimental Procedure

Sample preparation

(1) Prepare yeast lysate according to Table 1:
with NaOH as the benchmark, add each reagent in
the table below to 1mL NaOH, and add protease
inhibitors and phosphatase inhibitors
(non-essential) according to specific
experimental requirements;

(2) Take 200μL yeast culture solution,
centrifuge (4000rpm, 30s) and discard the
supernatant;

(3) Add the lysis solution to the obtained yeast
precipitate and lyse on ice for 10min;

(4) Centrifuge (5000rpm, 1min), discard the
supernatant;

(5) Add 80μL 1x loading buffer to the
precipitate and mix well. After metal bath
(100℃,5min), store in-20℃ refrigerator for
standby

Protein blotting and SDS-PAGE gel
electrophoresis

(1) Configure the SDS-PAGE gel and
electrophoresis according to the method in 4.2;

(2) Before protein loading, the sample was
bathed in hot water for 5min and centrifuged at
5000rpm for 3min. The supernatant and
precipitate were respectively loaded for
electrophoresis;

(3) After the gel electrophoresis, remove the
gel plate, carefully peel off the gel from the
glass plate, and put it into the staining
box/tray; rinse the surface of the gel with
deionized water 1 to 2 times to remove the
residual electrophoresis buffer (gentle
operation to avoid tearing the gel);

(4) Fixation (removes SDS and protein leakage,
improves staining signal-to-noise ratio): Add
sufficient fixative solution (methanol/acetic
acid/water, typically 45% methanol + 10% acetic
acid + 45% water) to the gel-containing
container. Shake gently at room temperature for
30 minutes. The fixation process can be extended
to 1 hour or overnight at 4℃°C to enhance
staining quality.

Carma blue staining and decolorization

(1) Discard the fixed liquid and add the newly
prepared Comass blue staining solution to
completely immerse the gel. Stain for 1-2 h at
room temperature on a horizontal shaker; if more
sensitive, stain overnight (do not exceed 24 h
to avoid increased background).

(2) After the dyeing, discard the dye and use
the decolorizing solution (40% methanol, 10%
acetic acid, 50% water) for the first
decolorization. Shake gently at room temperature
for 30 min~1 h until the background begins to
fade obviously.

(3) Replace the decolorizing solution and
continue decolorization on a horizontal shaker.
Adjust the decolorizing solution based on
background coloration and band development,
repeating this process every 30-60 minutes until
the background becomes sufficiently pale and
band contrast is clear. Complete decolorization
may take several hours to two days (allow
overnight exposure for continued processing).
For faster results, perform short decolorization
cycles, capture images immediately, then proceed
with detailed processing.

Gel imaging analysis

(1) After decoloring to the desired extent,
rapidly rinse the gel with deionized water to
remove residual methanol/acetic acid. Place the
gel in a photo tray and use a gel imaging system
or flatbed scanner for photography/scanning. It
is recommended to use white light transmission
or reflection mode depending on the equipment,
and record exposure parameters for comparison;

(2) Take a photo/scans and measure the band
intensity quantitatively with image software
(e.g. ImageJ), paying attention to using the
same exposure/scanning Settings and measuring
within the unsaturated range.

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/noro160.webp)

Analysis and Discussion

(1) In the supernatant and sediment groups
(labeled as Control Sup and Control Pel), no
distinct protein bands corresponding to the
indicated molecular weights of 81.5 kDa, 61.1
kDa, and 55.2 kDa were observed. This indicates
that in normal W303 yeast without genetic
manipulation, proteins matching these target
molecular weights are either absent or present
only in trace amounts undetectable by
conventional methods. These findings exclude the
possibility of interference from endogenous
yeast proteins in the experimental results.

(2) In the experimental plasmids Plasmid 3 Sup
and Plasmid 3 Pel, three distinct protein bands
were clearly visible at the positions indicated
by the arrows. The molecular weights of these
bands perfectly matched the expected target
proteins (Pn022859:81.5 kDa, UGT-73P40:61.1 kDa,
and UGT73F3: 55.2 kDa). This result directly
demonstrates that after plasmid transfection,
yeast cells successfully transcribed and
translated the foreign genes carried on the
plasmids, synthesizing substantial amounts of
the target proteins.

The final product of transplanted plasmid ② + ③
yeast was ginsenoside Ro extraction and cell
cryopreservation

Experimental time: 2025.08.24

Experimental personnel: Sun Jiayin, Wang
Jingning, Wang Quanhao, Min Yingxuan

Objectives of the Experiment

(1) Ginsenoside Ro was extracted from the cells
of Saccharomyces cerevisiae at 72h to prepare
for the detection of Ro content by liquid
chromatography;

(2) Brewer's yeast with frozen chromosomal
plasmid ② + ③ .

Experimental Equipment and Reagents

Equipment

Ultra-clean workbench, oscillator, ultrasonic
crusher, centrifug

Reagents

Chloroform, methanol, 50% glycerol

Experimental Procedure

Liquid chromatography sample preparation

(1) Take 3mL of fermentation liquid in the
crushing tube, centrifuge (13000 rpm/min, 3min);

(2) Add the culture medium, add an appropriate
amount of glass beads (diameter 0.5mm) and 1mL
extractant (chloroform: methanol =1:1), and
shake to break for 5min;

(3) After ultrasonic crushing for 30min,
centrifugation (13000 rpm/min, 3min) was
performed, and the supernatant was taken for
later use.

Saccharomyces
cerevisiae cryopreservation

(1) Take 3 EP tubes and add 750μL 50% glycerol
solution to each EP tube;

(2) Add 750μL of yeast solution to each EP tube,
mark it and put it in the-20℃ refrigerator.

HPLC analysis to determine whether Ro is present
in the product

(1) Gradient elution was performed using VP-ODS
C18 column (5μl,150nmx4.6nm).

(2) Separation conditions:

Acetonitrile (mobile phase A), purified water
(mobile phase B); Column temperature 35℃,
injection volume 20μl, flow rate 1.0mL/min, UV
detector wavelength 203nm.

(3) Elution conditions:

0 ~ 25min, 19﹪B, 81﹪A。25 ~ 50min, 29﹪B,
71﹪A。

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/noro17.webp)

![](https://static.igem.wiki/teams/5564/notebook/noro18.webp)

![](https://static.igem.wiki/teams/5564/notebook/noro190.webp)

Analysis and Discussion

The liquid chromatography analysis showed a
positive result.

Precautions

(1) When preparing the extraction solution, the
volume should be calculated in advance to
prevent insufficient dosage from repeated
preparation.

(2) In the first step of centrifugation, try to
pour out the supernatant as far as possible,
otherwise the liquid will be separated and the
cell layer may float to affect the effect of
ultrasonic crushing.

(3) When shaking, pay attention to the moment to
see if there is any leakage of liquid. If three
tubes can not be done at the same time, the
quantity can be reduced.

Rough enzyme extraction and in vitro
transformation of active components IVa and R1

Experimental time: 2025.09.01

Experimental personnel: Min Yingxuan, Wang
Jingning

Objectives of the Experiment

(1) The crude enzyme was extracted from the
yeast cells that were transferred to plasmid ② +
③ to prepare for in vitro experiments;

(2) Using R1 and IVa as substrates and UDPG as
cofactor, crude enzymes were used to transform
the active ingredients;

(3) To detect whether the product of the in
vitro transformation experiment contains the
target product Ro.

Experimental Equipment and Reagents

Equipment

Ultra-clean workbench, ultrasonic crusher,
centrifuge, constant temperature water bath,
vortex oscillator, liquid chromatograph

Reagents

Methanol, ethyl acetate, Panax notoginseng
saponin R1, ginseng saponin IVa, ginsenoside Ro,
uridine diphosphate glucose UDPG, dimethyl
sulfoxide DMSO, Tween 80, tris hydrochloride
trimethylolammonium salt Tris-Hcl (PH=8.5),
sterile water.

Reagent Preparation

Table 1 Preparation of reaction
substrate solution

| Reagent | Amount | Solvent | Amount | Concentration |
| --- | --- | --- | --- | --- |
| Triptansin R1 | 9.33 mg | DMSO | 0.1ml | 100mM |
| Buckthorn saponin IVa | 7.95mg | DMSO | 0.1ml | 100mM |
| UDPG Glucose diphosphoadenosine | 3.15mg | DMSO | 0.1ml | 50mM |

Table 2 Preparation of reaction
system

| Reagent | Amount |
| --- | --- |
| Bath solution (containing RI or IVa) | 0.15ul |
| UDPG Glucose diphosphoadenosine | 30ul |
| Tris-HCl(pH=8.5) | 15ul |
| Rough enzyme extract | 251.85ul |
| Tewen 80 | 3ul |

Experimental Procedure

Extraction of target protein from engineered
yeast

(1) Pick the W303 strain on YPD medium and the
single colony with good plasmid ② + ③
transfection in Sc-Trp-Leu double deficiency
medium respectively, and carry out liquid
culture in YPD medium and Sc-Trp-Leu double
deficiency medium respectively.

(2) Cultivation After 20h, when OD600 reaches
1.8~2, take 1ml of the yeast liquid with a
pipette and re-inoculate it into the liquid
culture medium to cultivate until OD600 reaches
1.8~2. Collect 15ml of the yeast liquid,
centrifuge at 4000rpm for 10min, and collect the
precipitate.

(3) Wash the obtained yeast with 1mlPBS once,
centrifuge at 10000rpm for 2min, and then
suspend the precipitate with 1m IPBS.

(4) Finally, the yeast cells were broken with an
ultrasonic cell crusher. The temperature was
controlled in an ice bath at a low temperature
(0-4℃) and ultrasonic crushing was performed for
20min.

(5) After cell suspension is broken, it becomes
obviously clear. The obtained cell suspension is
the whole protein solution and can be directly
used for enzymatic reaction.

Construction of enzymatic reaction system

(1) As shown in Table 1, Dissolution of R1 and
IVa: Dissolve notoginsenoside R1 and
chikusetsusaponin IVa in DMSO (dimethyl
sulfoxide) solution. Specifically, 9.33 mg of
notoginsenoside R1 is dissolved and made up to
0.1 mL with DMSO, and 7.95 mg of
chikusetsusaponin IVa is dissolved and made up
to 0.1 mL with DMSO, thus preparing 100 mM
system solutions.

(2) As shown in Table 2, System preparation
(total system volume 300 μL) Experimental group:
(Crude enzyme extract of strains transfected
with plasmids ② + ③ ) Add 251.85 μL of crude
enzyme solution, 15 μL of Tris-HCl (pH 8.5), 30
μL of 50 mM UDPG, 0.15 μL of 100 mM substrate
(R1 or IVa), and 3 μL of Tween 80 in sequence,
then gently pipette to mix well. Control group
(crude enzyme extract of w303 strain): Replace
the crude enzyme solution of the experimental
group with the crude enzyme solution of w303
Saccharomyces cerevisiae, while other components
and their dosages are exactly the same.

(3) Control of reaction conditions: All reaction
tubes are placed in a 20°C constant temperature
water bath for reaction in the dark for 20
hours. During this period, gently invert and mix
once every 5 hours to prevent substrate
precipitation.

Reaction product treatment

(1) Enzyme inactivation and centrifugation:
After the reaction is completed, place the
reaction tube in a 99°C water bath and heat for
10 minutes to inactivate the enzyme activity,
then immediately cool to room temperature in an
ice bath. Centrifuge at 12000 rpm for 15
minutes, and transfer the supernatant
(containing reaction products) to a new
centrifuge tube.

(2) Extraction and concentration: Add an equal
volume of ethyl acetate, vortex and oscillate
for 1 minute, let it stand for phase separation
(about 5 minutes), and pipette the upper ethyl
acetate phase (containing saponin products) to a
new tube. Repeat the extraction twice, combine
all ethyl acetate phases, place them in a 50°C
water bath, evaporate to dryness slowly, and
dissolve the residual solid with 300 μL of
methanol.

(3) Filtration and purification: Filter the
methanol solution through a 0.45 μm organic
phase filter membrane, collect the filtrate into
a high-performance liquid chromatography
injection vial, store at 4°C, and wait for
detection.

Product detection and result analysis

High-performance Liquid Chromatography (HPLC)
Injection: Inject 20 μL of the filtrates from
the experimental group, control group, and the
standard solutions (R1, IVa, Ro) respectively
into the liquid chromatography injection valve,
and record the chromatograms.

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/noro20.webp)
![](https://static.igem.wiki/teams/5564/notebook/noro21.webp)
![](https://static.igem.wiki/teams/5564/notebook/noro22.webp)
![](https://static.igem.wiki/teams/5564/notebook/noro23.webp)
![](https://static.igem.wiki/teams/5564/notebook/noro24.webp)
![](https://static.igem.wiki/teams/5564/notebook/noro25.webp)
![](https://static.igem.wiki/teams/5564/notebook/noro26.webp)
![](https://static.igem.wiki/teams/5564/notebook/noro27.webp)
![](https://static.igem.wiki/teams/5564/notebook/noro28.webp)
![](https://static.igem.wiki/teams/5564/notebook/noro29.webp)

Analysis and Discussion

When compared with the reference standard, the
sample group exhibited a sharp and symmetrical
main chromatographic peak within 59-60 minutes,
showing high consistency with the standard. This
indicates that the transplanted enzyme
demonstrated significant activity in vitro
experiments, capable of converting substrates
IVA and R1 into the final product Ro. In vitro
experiments demonstrated that the transplanted
glycosyltransferase into Saccharomyces
cerevisiae exhibited enzymatic activity.
Meanwhile, the peak area of the sample group
derived from R1 conversion was observed to
decrease compared to that from IVA conversion.
Our results suggest that the conversion
efficiency from R1 to Ro is significantly lower
than that from IVA to Ro. To address this, we
aim to optimize enzyme selectivity through
directed evolution to reduce intermediate
product R1 formation.

Precautions

(1) The temperature of ultrasonic crushing was
controlled at 0-4℃ to prevent excessive
temperature from damaging the structure of crude
enzyme protein and reducing enzyme activity.

(2) When preparing the substrate reaction
system, the amount of DMSO and Tween 80 should
be strictly controlled. High concentration of
DMSO may cause denaturation of protein spatial
structure. Tris-Hcl can be added to control the
PH of the reaction system.

(3) In HPLC liquid chromatography detection,
methanol was often evaporated at room
temperature to prevent excessive temperature and
saponin Ro desaccharification and degradation.

# Enhancing glucose supply to improve product yield

Cultivation and Cryopreservation of Yeast

Experimental time: 2025.09.01

Experimental personnel: Zhao Xiaoqing, Sun
Jiayin

Objectives of the Experiment

1. Cultivate and freeze-store Saccharomyces
cerevisiae to prepare for subsequent plasmid
transfection in the production of competent
cells.

2. Prepare the plasmid as a solution for
subsequent transfection.

Experimental Equipment and Reagents

Equipment

Shaker, refrigerator, laminar flow hood,
spectrophotometer

Reagents

YPD medium, plasmid powder, sterile water,
glycerol

Reagent Preparation

Table 1 YPD Medium(500mL)

| Reagent | Amount |
| --- | --- |
| Peptone | 10g |
| Yeast extract | 5g |
| H2O | 500mL |
| glucose | 10g |

Experimental Procedure

Cultivation of Saccharomyces
cerevisiae

(1) Irradiate the ultra-clean workbench with
ultraviolet light for 30 minutes beforehand.
Prepare YPD medium and microwave-treat it in
advance for later use;

(2) Take a 50 mL centrifuge tube and pour 5 mL
of YPD liquid medium into it;

(3) Pick a single colony of Saccharomyces
cerevisiae W303 from the culture plate and
inoculate into the tube. Incubate on a shaking
incubator (30°C, 200 rpm);

(4) After approximately 7 hours, measure the
OD600. If OD600 = 0.3, add 2 mL of the culture
to 100 mL of YPD medium.

Prepare the plasmid

(1) Retrieve the plasmid powder tube and
centrifuge (4μg);

(2) Add 30μL sterile water to the tube;

(3) Transfer 10μL to an EP tube, then add 32μL
sterile water for later use;

(4) Store the remaining 20μL plasmid at -20°C.

Freeze-preserve experimental strains

Take an EP tube, add 750μL bacterial suspension
and 750μL glycerol (50%) in a 1:1 ratio, mix,
and store at -80°C. Replenish the remaining
bacterial suspension in the centrifuge tube with
appropriate medium.

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/noud01.webp)
![](https://static.igem.wiki/teams/5564/notebook/noud02.webp)
![](https://static.igem.wiki/teams/5564/notebook/noud03.webp)

Analysis and Discussion

Yeast exhibited robust growth and is suitable
for preparing competent cells for subsequent
experiments.

Precautions

(1) Maintain aseptic technique when isolating
single-colony yeast to prevent contamination by
extraneous microorganisms.

(2) When preparing YPD medium, avoid excessive
temperatures during autoclaving. Typically,
autoclave at 115°C for 15 minutes; higher
temperatures may cause glucose to darken.

(3) Ensure culture duration does not exceed
recommended limits. If optical density (OD)
exceeds 1.2, the strain is unsuitable for
subsequent procedures.

Preparation of Sc-Trp medium and competent cells

Experiment time: 2025.09.04

Experiment operator: Zhao Xiaoqing, Sun Jiayin

Objectives of the Experiment

1. Prepare Sc-Trp medium and plate it to prepare
for the subsequent cultivation of Saccharomyces
cerevisiae after plasmid transfection.

2. Prepare the competent cells for plasmid
transfection.

Experimental Equipment and Reagents

Equipment

Ultra-clean workbench, absorbance meter, high
temperature and high pressure sterilizer, shaker

Reagents

Ultra-clean workbench, absorbance meter, high
temperature and high pressure sterilizer, shaker

Reagent Preparation

Table 1 Sc-Trp culture medium
(200mL)

| Reagent | Amount |
| --- | --- |
| Sc-Trp | 0.4g |
| L-Arginine | 0.04g |
| adenine | 0.2g |
| glucose | 4g |
| agar | 4g |
| YNB | 1.6g |
| (NH4)2SO4 | 5g |
| H2O | 200mL |

Experimental Procedure

Sc-Trp medium configuration

(1) Take 0.4g SC-Trp, 0.04g arginine, 0.2g
adenine and 4g glucose, add 60mL water, and put
it in an empty bottle;

(2) Take 4g agar and add 120mL water into
another empty bottle, and add a rotor;

(3) Put the above two bottles and an empty 100mL
bottle into the autoclave for high-pressure
sterilization (115℃,15min);

(4) Weigh 1.6g yeast with YNB, 5g ammonium
sulfate, place it in a beaker, add 100mL
autoclaved water, put in a rotor to stir until
completely dissolved;

(5) After sterilization by filtration through
0.22μm filter, the solution was transferred to a
100mL empty high-pressure sterilized bottle for
standby;

(6) Take 20mL of filtered YNB solution and add
it to the high-pressure agar bottle. Pour SC-Trp
solution into the agar bottle with YNB while
hot, and use a magnetic stirrer to mix
thoroughly;

(7) Pour the mixture into a sterile petri dish
to make a plate. After 4 hours at room
temperature, put it in the refrigerator for
storage.

Preparation of competent cells

(1) Place sterile water in a 4℃ refrigerator for
standby, and dilute LiAC/TE to 0.1mol/L for
standby;

(2) Determine the OD600 of yeast culture the
previous day, if OD600=0.8, then proceed to the
next experiment;

(3) Take 50mL of bacterial liquid, centrifuge
(3500rpm, 2min) and discard the supernatant;

(4) Resuspend in 50mL cold sterile water,
centrifuge (3500rpm, 2min), and discard the
supernatant;

(5) Resuspend with 1mL 0.1mol/L LiAC/TE, and
centrifuge rapidly (4000rpm, 30s), then discard
the supernatant;

(6) Resuspend in 400μL 0.1mol/L LiAC/TE, mix
well, and overnight in the refrigerator on ice.

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/noud04.webp)
![](https://static.igem.wiki/teams/5564/notebook/noud05.webp)

Analysis and Discussion

(1) The filtered and sterilized YNB solution
should be poured into the agar quickly while hot
to prevent abnormal solidification of the
culture medium caused by uneven distribution of
YNB.

(2) After the culture medium is prepared, it
should be poured into the plate quickly to
prevent the culture medium from solidifying in
advance for too long.

(3) Observe the OD value of yeast to prevent the
culture time from being too long to enter the
next experiment.

Plasmid ④transfection; yeast culture after
transfection

Experiment time: 2025.09.05

Experiment operator: Wang Quanhao

Objectives of the Experiment

1. The prepared competent cells were used for
plasmid transfection;

2. Transfection of plasmids ④ into yeast to
utilize fatty acids;

3. Prepare YPD medium and Trp single deficiency
solid medium for standby.

Experimental Equipment and Reagents

Equipment

Laminar flow hood, shaker, 30℃ incubator, water
bath, shaker, oven, electronic balance, magnetic
stirrer, autoclave

Reagents

PEG, LiAc (1mol/L), salmon sperm, plasmid, yeast
receptive state, YPD culture, PEG, DMSO Glucose,
peptone, arginine, adenine, SC-Trp;

Reagent Preparation

Table 1 YPD medium (500 mL)

| Reagent | Amount |
| --- | --- |
| Peptone | 10g |
| Yeast extract | 5g |
| H2O | 500mL |
| glucose | 10g |

Tabel 2 Trp single lacks solid medium
(180 mL)

| Reagent | Amount |
| --- | --- |
| SC-Trp | 0.4 g |
| arginine Arg | 0.04 g |
| adenine | 0.2 g |
| 20% glucose | 4 g |
| Water H2O | 60 mL |
| Take another empty bottle and add a rotor | |
| agal-agal | 120 g |
| Water H2O | 120 mL |

Table 3 YNB medium configuration

| Component | Consumption (100mL) |
| --- | --- |
| YNB for yeast culture | 1.6g |
| (NH4)2SO4 | 5g |
| H2O | 100mL |

Experimental Procedure

Plasmid transfection

(1) Salmon sperm was boiled in boiling water for
5 minutes, and then quickly placed in an ice
water mixture (on ice);

(2) Add about 50 μL yeast receptive strain
prepared the previous day into each EP tube, and
centrifuge (4000rpm, 30s) to discard the
supernatant;

(3) Add 42μL plasmid, 10μL croaker sperm
(10mg/mL), 240μL PEG, and 36μL LiAc (1mol/L)
into the EP tube in sequence. After repeatedly
mixing by pipetting with a 1mL pipette, incubate
at 30℃ incubator for 30min;

(4) Add 36μL DMSO to each EP tube, mix by
repeated blowing and then put it into a 42℃
water bath for 20min.

Yeast culture after transfection

(1) Take the EP tube from the water bath,
centrifuge (3000rpm, 20s) and discard the
supernatant. Resuspend in 1 mL YPD medium and
culture in a shaker for 3h;

(2) Centrifugation (3000rpm, 20s) After
discarding the supernatant, resuspend with 100μL
water, spread the bacterial liquid with glass
beads (Sc-Trp), and cultivate in a 30℃ incubator
for about 3 days.

Preparation of Trp single deficiency solid
medium

(1) Prepare Trp single solution according to (2)
in "II", and prepare YNB solution according to
(3);

(2) Put the glass bottle with solution into the
autoclave and sterilize (115℃,15min);

(3) After sterilization of YNB solution through
0.22μm filter, it was transferred to a 100 mL
empty high-pressure sterilized bottle for
standby;

(4) Take 20 mL of filtered YNB solution and add
it to the Trp double deficiency solution and mix
thoroughly;

(5) Pour the medium into a sterile petri dish
(about 15-20mL per plate). After solidification,
invert it and store at 4°C (avoid condensation
water accumulation).

Preparation of YPD medium

Prepare PYD medium according to 3.1 in Section
2, and the other steps are the same as 4. in
Section 3. Sterilize with ultraviolet light as a
liquid culture medium.

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/noud06.webp)
![](https://static.igem.wiki/teams/5564/notebook/noud07.webp)

Analysis and Discussion

(1) Salmon sperm need to be boiled in boiling
water for 5min in advance, and then quickly put
into ice water mixture to play the role of
protecting exogenous plasmids.

(2) All kinds of reagents required for particle
transfection should be mixed evenly to maximize
the permeability of cell membrane and improve
the transfection efficiency.

(3) The incubation time of the titer
transfection incubator and the heat shock time
of the water bath must be strictly controlled to
prevent the decrease of transfection efficiency
after too long time.

PCR and agarose gel electrophoresis after
plasmid ④ transfection

Experiment time: 2025.09.07

Experiment operator: Wang Quanhao, Min Yingxuan

Objectives of the Experiment

1. Prepare competent cells for plasmid
transfection;

2. 2/3 of the extracted plasmid was used for PCR
and plasmid transfection;

3. To determine whether the genes of FBP-ase1,
PGM2 and UGP1, which were expressed, were
transferred into Saccharomyces cerevisiae, and
to detect the expression of plasmids in the
engineered bacteria.

Experimental Equipment and Reagents

Equipment

Clean workbench, enzyme label instrument,
electronic balance, centrifuge, fume hood,
oscillator, ultrasonic crusher, PCR instrument,
oscillator, electrophoresis tank, microwave
oven, gel imaging system;

Reagents

LiAC/TE solution, sterile water;

Buffer P1、Buffer P2、Buffer P3、Buffer
DW1、Wash Solution、ElutionBuffer;

Glass beads, chloroform, methanol;

2xTransFast Taq PCR SuperMix (+dye), DNA
template, specific primers, 1xTAE buffer,
agarose, nucleic acid dye EB, DNA Marker;

Reagent Preparation

Table 1 PCR reaction system

| Reagent | Volume | Final Concentration |
| --- | --- | --- |
| 2×TransFast Taq PCR SuperMix(+dye) | 25μL | 1× |
| Primer 1 | 1μL | 0.2μM |
| Primer 2 | 1μL | 0.2μM |
| Template | 5μL | As required |
| DNase-Free Water | To 50μL |  |

Table 2 PCR reaction program

| Temperature | Time | Number of cycles |
| --- | --- | --- |
| 94℃ | 3 min | 1 cycle |
| 94℃ | 5 sec | 35 cycles |
| 52℃ | 15 sec |
| 72℃ | 30 sec |
| 72℃ | 5 min | 1 cycle |

Table 3 2% agarose gel

| Reagent | Amount |
| --- | --- |
| agarose | 1.8 g |
| 1xTAE | 90 mL |
| Nucleic acid dye EB | 9 μL |
| 72℃ | 30 sec |
| 72℃ | 5 min |

Experimental Procedure

Protein extraction

(1) Check whether RNase A has been added to
buffer P1.

(2) Check whether anhydrous ethanol has been
added to the Wash Solution.

(3) Check for precipitation of buffer P2 and P3.

(4) Take 1.5-5 mL of the overnight culture,
centrifuge at 8,000 ×g for 2 minutes to collect
the bacteria, and discard all the culture
medium.

(5) Add 250 μL Buffer P1 to the precipitate and
resuspend the bacteria thoroughly.

(6) Add 250 μL Buffer P2 and gently invert the
centrifuge tube 5-10 times immediately to mix.
Let it stand at room temperature for 2-4
minutes.

(7) dd 350 μL Buffer P3 and gently invert the
centrifuge tube 5-10 times immediately to mix.

(8) Centrifuge at 12,000 ×g for 10 minutes.
Transfer the supernatant to the adsorption
column and let it stand for 3-4 minutes.
Centrifuge at 8,000 ×g for 30 seconds, then
discard the liquid from the collection tube.

(9) Add 500 μL Buffer DW1 and centrifuge at 9000
×g for 30 seconds. Pour off the liquid in the
collection tube.

(10) Add 500 μL Wash Solution and centrifuge at
9000 ×g for 30 seconds. Pour off the liquid in
the collection tube.

(11) Repeat (2)(10) once

(12) The air adsorption column was centrifuged
at 9000 ×g for 1 min.

(13) Place the adsorption column into a clean
1.5mL centrifuge tube, add 50-100 μL Elution
Buffer in the center of the adsorption membrane,
and centrifuge for 1 min after room temperature
standing for 1 min. Save the DNA solution in the
tube.

PCR and agarose gel electrophoresis

(1) The PCR reaction system was prepared
according to Table 1;

(2) Set the PCR reaction program according to
Table 2 and carry out PCR;

(3) Prepare 2% agarose gel according to Table 3
and dissolve it by microwave heating;

(4) Add 9 μL EB to the agarose gel and shake it
well. Then pour it into the mixing tank where
the comb has been placed. Place it flat in the
refrigerator and wait for the gel to solidify;

(5) Put the gel into the electrophoresis tank
and pour in an appropriate amount of 1x TAE
electrophoresis buffer. After sampling,
electrophoresis for 25min under constant voltage
of 140V;

(6) After electrophoresis, observe and take
photos under ultraviolet lamp.

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/noud08.webp)

![](https://static.igem.wiki/teams/5564/notebook/noud090.webp)

Analysis and Discussion

(1) The three target genes were detected in the
positive control group PC (plasmid ④) with clear
bands, indicating that the three target genes
were successfully cloned into the plasmid
vector.

(2) The positive bands of the three target genes
in the target strain suggested that the three
target genes were successfully transferred into
yeast

(3) The three target genes were not detected in
the negative control group NC, indicating that
the primer amplification was specific.

Protein extraction of bacterial strain samples
transferred to plasmid ④

Experiment time: 2025.09.09

Experiment operator: Wang Jingning, Wang
Quanhao, Min Yingxuan, Sun Jiayin

Objectives of the Experiment

1. Sample protein extraction was prepared for
western blot test of normal plasmid expression;

Experimental Equipment and Reagents

Equipment

Ultra-clean workbench, centrifuge, water bath,
gel plate, electrophoresis tank, vertical
electrophoresis instrument, transfer
electrophoresis instrument, decolorization
shaker, chemiluminescence imaging system;

Reagents

NaOH, β-glycerophosphate sodium, sodium
prontovantanate, NaF, EDTA, PMSF

Reagent Preparation

Table 1 Configuration of lysate

| Reagent | Volume | Final Concentration |
| --- | --- | --- |
| NaOH | 1ml | 0.1M |
| sodium β-glycerophosphate | 11ul | 100mM |
| sodium orthovanadate | 11ul | 100mM |
| NaF | 11ul | 5M |
| EDTA(ethylenediaminetetraacetic acid) | 3ul | 0.5M |
| PMSF(Phenylmethylsulfonyl fluoride) | 11ul | 100mM |

Experimental Procedure

Sample preparation

(1) Prepare yeast lysate according to the
following table: with NaOH as the base, add each
reagent in the table below to 1mL NaOH, and add
protease inhibitors and phosphatase inhibitors
(non-essential) according to specific
experimental requirements;

(2) Take 200μL yeast culture solution,
centrifuge (4000rpm, 30s) and discard the
supernatant;

(3) Add the lysis solution to the obtained
bacterial precipitate and lyse on ice for 10min;

(4) Centrifuge (5000rpm, 1min), discard the
supernatant;

(5) Add 80μL 1x loading buffer to the
precipitate and mix well. After metal bath
(100℃,5min), store in-20℃ refrigerator for
standby

Western Blot to detect successful expression of
protein

Experiment time: 2025.09.11

Experiment operator: Wang Quanhao, Min Yingxuan

Objective of the Experiment

Determine whether the genes of FBP-ase1, PGM2
and UGP1 were transferred into Saccharomyces
cerevisiae.

Experimental Equipment and Reagents

Equipment

Ultra-clean workbench, centrifuge, water bath,
gel plate, electrophoresis tank, vertical
electrophoresis instrument, transfer
electrophoresis instrument, decolorization
shaker, chemiluminescence imaging system;

Reagent Preparation

Table 1 Electrophoresis buffer
configuration

| Reagent | Amount |
| --- | --- |
| Tris | 3g |
| Glycocoll | 14.6g |
| SDS | 1g |
| ddH2O | Vide to 1000ml |

Table 2 Transmembrane buffer

| Reagent | Amount |
| --- | --- |
| Tris | 2.91g |
| glycocoll | 14.4g |
| carbinol | 200ml |
| ddH2O | Vide to 1000ml |

Table 3 Concentrated glue
configuration

| Reagent | Amount |
| --- | --- |
| ddH2O | 2.1 mL |
| 30% polyacrylamide | 0.5 mL |
| 1.0M Tris-HCl (pH6.8) | 0.38 mL |
| 10%SDS | 30 μL |
| 10%APS | 30 μL |
| TEMED | 3 μL |

Table 4 Separation gel
configuration

| Reagent | Amount |
| --- | --- |
| ddH2O | 1.6 mL |
| 30% polyacrylamide | 2 mL |
| 1.5M Tris-HCl (pH8.8) | 1.3 mL |
| 10%SDS | 50 μL |
| 10%APS | 50 μL |
| TEMED | 5 μL |

Table 5 5% sealant configuration

| Reagent | Amount |
| --- | --- |
| skim milk powder | 2.5g |
| 1×TBST | 50ml |

Table 6 Developer configuration

| Reagent | Amount |
| --- | --- |
| Developer A solution | 500ul |
| Developing solution B | 500ul |

Table 7 TBST configuration

| Reagent | Amount |
| --- | --- |
| NaCl | 8g |
| KCl | 0.2g |
| Tris | 3g |
| Tween-20 | 1ml |
| ddH2O | Vacuum to 1L |

Experimental Procedure

Protein blotting and gel electrophoresis

(1) Prepare the 5% concentration gel and 12%
separation gel according to Tables 4 and 5.
Allow the separation gel to chill at room
temperature for 20-30 minutes, while the
concentration gel should remain at room
temperature for approximately 5-10 minutes.
Place both gels into an electrophoresis tank
filled with electrophoresis buffer. During
loading, add 10 μL of protein sample to each
well, and include 4 μL marker in one well.
Handle carefully to avoid bubble formation
during the process;

(2) Electrophoresis: Electrophoresis conditions:
80 V for the upper layer of concentrated gel for
30min; 120 V for the lower layer of separation
gel for 60 ~ 80 min;

(3) Blotting: After electrophoresis is complete,
remove the gel plate and gently lift the gel.
Cut the corresponding PVDF membrane and filter
paper according to the required dimensions.
Prior to blotting, activate the PVDF membrane
with methanol for approximately 20-30 seconds.
Perform the blot using an ice-cold water mixture
at a constant current of 200mA for 2 hours;

(4) Milk sealing and hybridization: Take out the
PVDF film, wash it with 1x TSBT solution for 3
times in a horizontal shaker, each time for 10
min; seal it with 5% skim milk and seal it for
2h in a horizontal shaker at room temperature;

(5) After 2-hour incubation, wash with TBST
three times (5 minutes each). Incubate with
diluted primary antibody (dilution ratio follows
the antibody manufacturer's instructions) using
TBST solution. Place in a 4°℃ fridge overnight.
The next day, perform three TBST washes (10
minutes each) on a shaker before secondary
antibody incubation for 1 hour. Finally, conduct
three TBST washes (10 minutes each);

(6) AECL Colorimetry: As specified in Table 7,
follow the ECL Colorimetry Manual (Thermo Fisher
Scientific) to prepare Solution A and Solution B
in a 1:1 volume ratio. After mixing both
solutions, allow them to stand at room
temperature under light protection for 1-2
minutes. Capture images using the gel imaging
system and analyze the grayscale values of each
band through dedicated software. Finally,
perform statistical analysis on the processed
data.

Experimental Results

![](https://static.igem.wiki/teams/5564/notebook/noud12.webp)

![](https://static.igem.wiki/teams/5564/notebook/noud13.webp)

![](https://static.igem.wiki/teams/5564/notebook/noud140.webp)

Analysis and Discussion

Plasmid ④ contains and can express the three
genes FBP-ase1, PGM2 and UGP1 with clear bands.
The positive results show a significant contrast
with the negative controls, indicating reliable
results. The strains transformed with plasmid ④
can effectively utilize UDPG, providing an
efficient gluconeogenesis pathway that
facilitates normal growth and carbon source
utilization.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one
iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5564/banner/engineering.webp)

Fatty Acid Utilization
OA to CE
CE to Ro
UGT73F3 Engineering
UDPG Pool Expansion

![](https://static.igem.wiki/teams/5564/engineering/engcycle0.webp)
![](https://static.igem.wiki/teams/5564/engineering/engcycle4.webp)
![](https://static.igem.wiki/teams/5564/engineering/engcycle1.webp)
![](https://static.igem.wiki/teams/5564/engineering/engcycle2.webp)
![](https://static.igem.wiki/teams/5564/engineering/engcycle3.webp)

* Cycle 1: Peroxisomal Localization
* Cycle 2: Mixed Substrate Optimization

* Cycle 1: Enzyme Discovery
* Cycle2: Computational Validation
* Cycle3: UDP-GlcA Test

* Cycle 1: In Vivo Assembly
* Cycle 2: In Vitro Activity

* Cycle 1: machine learning models
* Cycle 2: Top Mutant Screening

* Cycle: Overexpression of the key enzymes

# Utilizing Fatty Acids

Objective: To connect the fatty-acid catabolic pathway
with the Ro biosynthetic pathway so that Saccharomyces cerevisiae
can use fatty acids as a carbon source to synthesize Ro.

Cycle 1: Peroxisomal Localization

In S. cerevisiae, fatty acids are naturally catabolized
via β-oxidation in the peroxisome to generate acetyl-CoA. From the literature, we learned
that acetyl-CoA cannot freely pass through the peroxisomal
membrane, whereas the subsequent steps of the mevalonate (MVA) pathway take place
in the cytosol. Therefore, resolving how to utilize fatty acids is essential.

Design:

The literature indicates that metabolites produced from acetyl-CoA through the
MVA pathway-such as mevalonate (MVA), isopentenyl pyrophosphate (IPP), and
dimethylallyl pyrophosphate (DMAPP)-can freely cross the peroxisomal membrane.
Therefore, we introduced the key MVA enzymes ERG10, ERG13, and HMG1 into S.
cerevisiae and targeted their expression to the peroxisome.

Build:

We designed plasmids driven by constitutive promoters and carrying a tryptophan
selection marker. To ensure that the proteins localize to
the peroxisome, we fused the genes of interest to the peroxisomal
targeting signal 1 (PTS1) via a flexible linker.

![](https://static.igem.wiki/teams/5564/engineering/eng010.webp)

Test:

Using equimolar carbon amounts of lauric acid, oleic acid, and palmitic acid as
fatty-acid carbon sources-with a constant glycerol carbon source added-we monitored the
growth curves of Saccharomyces cerevisiae to assess fatty-acid
utilization. The results showed that, after transformation with plasmid 1,
the yeast's ability to utilize fatty acids was markedly improved; however, its
utilization of lauric acid was weaker than that of palmitic acid and oleic acid.

![](https://static.igem.wiki/teams/5564/engineering/eng020.webp)

Learn:

Based on this anomalous observation, we considered that lauric acid may not
efficiently enter yeast cells and is therefore difficult to utilize. Literature indicates
that:

(1) Palmitic acid is one of the predominant saturated fatty acids in the
phospholipids of S. cerevisiae cell membranes. Accordingly, yeast
fatty-acid transporters (e.g., Fat1p) exhibit high affinity for fatty
acids of C14-C18 chain length.

(2) Faa1p and Faa4p are the key enzymes primarily responsible for activating
long-chain fatty acids in yeast. Their active-site architecture is best suited to C12-C18
fatty acids, with typically highest activity toward C14-C16.

(3) Medium-chain fatty acids possess stronger detergent effects than long-chain
fatty acids and can disrupt membrane integrity more effectively. When
environmental lauric acid concentrations are high, this imposes greater membrane
toxicity on yeast, thereby inhibiting cell growth.

Cycle 2: Mixed Substrate Optimization

Design:

We redesigned the fatty-acid carbon source, favoring mixed
(combinational) fatty-acid inputs. To verify whether the oleic acid + palmitic
acid pair is the optimal combination, we formulated four mixed fatty-acid media and measured
the growth curves.

Build:

Same as Cycle 1.

Test:

Using four mixed fatty-acid carbon sources, we monitored the
growth curves of Saccharomyces cerevisiae to evaluate
its fatty-acid utilization.

![](https://static.igem.wiki/teams/5564/engineering/eng030.webp)

Learn:

With mixed fatty-acid carbon sources, Saccharomyces
cerevisiae showed the fastest growth when oleic acid + palmitic
acid were used as the mixture, suggesting that this medium is better suited for
S. cerevisiae to produce ginsenoside Ro.

# Constructing the OA→CE Metabolic Pathway

Objective: To screen and build the optimal metabolic
route to achieve the conversion from OA to CE.

Cycle 1: Enzyme Discovery

Design:

Through gene family analysis, we constructed a phylogenetic
tree covering Csl enzymes from the genomes of ginseng, notoginseng, and soybean,
and screened Csl enzymes capable of catalyzing the conversion of OA to CE.

Build:

We first obtained the genome files of ginseng, notoginseng, and soybean from
NCBI. After genome annotation, we extracted protein-coding sequence
FASTA files. Using Pn022859 as the reference
sequence, we searched for candidate Csl enzymes in the three species with BLAST and HMM
models. We then performed domain and motif analyses of the
candidates using NCBI CD-Search and MEME Suite to determine the distribution of functional
domains and motifs. Next, we merged the candidate Csl FASTA files from the three species,
removed low-similarity and overly short sequences, and built a phylogenetic tree using the
neighbor-joining (NJ) method to classify the candidates into gene families
and subfamilies.

![](https://static.igem.wiki/teams/5564/model/dry-main-figure0-1.webp)

Test:

By constructing a phylogenetic tree, we identified 21
Csl enzyme sequences belonging to the same family as Pn022859. Further subfamily
delineation revealed that the closest evolutionary
relatives to PN022859 are EVM0017102 and EVM0010403, which are Csl sequences from Panax
ginseng.

![](https://static.igem.wiki/teams/5564/engineering/eng050.webp)

Learn:

Gene family classification clusters genes by sequence similarity; however,
whether closely related genes necessarily share similar functions remains to be determined.

Cycle2: Computational Validation

Design:

To investigate whether genes closely related to Pn022859 in the phylogenetic tree
possess similar functions-or even superior catalytic efficiency-we validated the sequences
in the same family as Pn022859 using molecular docking and molecular
dynamics.

Build:

We first used AlphaFold 3 to construct three-dimensional models for the 21 Csl
candidate sequences in the same family as Pn022859. The 3D structure of the Csl substrate OA
was obtained from PubChem. We performed molecular docking between each candidate Csl enzyme
and OA using AutoDock Vina, selected the top five Csl enzymes by best binding affinity, and
then conducted molecular dynamics simulations with GROMACS to verify whether these Csl
enzymes in the same family as Pn022859 exhibit similar or even better catalytic performance
toward OA.

Test:

We validated the above with molecular docking and molecular dynamics simulations.
The docking results showed that Pm022859 binds OA well (Best Affinity = -12.6 kcal/mol), and
we identified four Csl enzymes with better binding than PN022859: EVM0004671 from ginseng
(Best Affinity = -12.6 kcal/mol) and three from soybean-XM\_041006423 (-12.7 kcal/mol),
XM\_003536208 (-13.0 kcal/mol), and NM\_001365113 (-13.2 kcal/mol). Based on the docking
results, there remains room to improve the Csl enzyme used for the OA→CE reaction. We
therefore carried out MD simulations in GROMACS for the top-five Csl-OA docked complexes.
The Pn022859 complex shows the smallest radius-of-gyration (Rg)
distribution among the complexes, indicating favorable dynamic stability. For
details, see the Model page.

![](https://static.igem.wiki/teams/5564/engineering/eng060.webp)

![](https://static.igem.wiki/teams/5564/engineering/eng070.webp)

Learn:

At the dry-lab level, we screened Csl sequences functionally similar to Pn022859
and, using molecular docking and molecular dynamics simulations, verified Pn022859's strong
catalytic performance in the OA→CE conversion. We found that Pn022859 exhibited superior
in-silico performance, indicating that this reaction step still has room for further
optimization.

Cycle3: UDP-GlcA Test

Design:

Through dry-lab screening, we identified Pn022859 as the key enzyme for
converting OA to CE. This step requires UDP-GlcA (uridine diphosphate glucuronic acid);
based on the literature, we selected the UDP-glucose dehydrogenase AtUGDH and transformed it
into Saccharomyces cerevisiae.

Build:

We designed two plasmids driven by constitutive promoters, each carrying a
leucine selection marker.

![](https://static.igem.wiki/teams/5564/engineering/eng080.webp)

Test:

On day 3 post-transformation, we harvested cells, extracted intracellular
contents, and quantified R1 by high-performance liquid chromatography
(HPLC).

![](https://static.igem.wiki/teams/5564/engineering/eng100.webp)

Learn:

After introducing AtUGDH, R1 levels decreased rather than increased. We
hypothesize that the OA→CE step consumes UDP-GlcA, depleting UDP-glucose (UDPG) and thereby
reducing downstream glycosylation efficiency. Therefore, we chose not to introduce AtUGDH at
this step.

# Construction of the CE-to-Ro Metabolic Pathway

Objective: Screening Glycosyltransferases and Optimizing
the Metabolic Pathway

Cycle 1: In Vivo Assembly

Design:

Based on literature research, we selected two glycosyltransferases, UGT73P40 and UGT73F3, from Panax
notoginseng and co-expressed them with PN022859 in Saccharomyces cerevisiae.

Bulid:

The genes for UGT73P40, UGT73F3, and PN022859 were codon-optimized,
synthesized, and assembled into a single expression
plasmid. This plasmid was driven by constitutive promoters and carried a leucine selection
marker for stable transformation and maintenance in the yeast chassis.

![](https://static.igem.wiki/teams/5564/engineering/eng090.webp)

Figure 1. Schematic representation of the plasmid
pRS315-UGT73P40-UGT73F3-PN022859

Test:

Three days post-transformation, intracellular metabolites were extracted from
Saccharomyces cerevisiae and analyzed by high-performance liquid
chromatography (HPLC). The results showed
that the ginsenoside Ro standard peak appeared at t = 59 min. Since
Ro is a trisaccharide saponin, the appearance of an additional peak
at t = 85 min in the sample chromatogram is likely attributable to
a disaccharide ginsenoside byproduct.

![](https://static.igem.wiki/teams/5564/engineering/eng110.webp)

Figure 2. HPLC peak of ginsenoside Ro
standard

![](https://static.igem.wiki/teams/5564/engineering/eng120.webp)

Figure 3. HPLC peak of ginsenoside Ro extracted from
engineered S.cerevisiae

Learn:

To explain this unexpected result, we hypothesize that it may stem from compatibility issues after transferring the Panax notoginseng
glycosyltransferase system into Saccharomyces cerevisiae. In
plants, glycosylation levels of these enzymes are generally low, whereas yeast exhibits
comparatively higher glycosylation activity. Consequently, the transferred
glycosyltransferases may undergo hyperglycosylation, which could
reduce their enzymatic activity and ultimately prevent the formation of the trisaccharide
ginsenoside Ro.

Cycle 2: In Vitro Activity

Objective:Assessment of Glycosyltransferase Activity in
Saccharomyces cerevisiae.

Design:

Crude intracellular enzymes were extracted from Saccharomyces
cerevisiae and incubated in vitro with the respective
substrates R1 or IVa. The formation of the final product, ginsenoside Rod to determine whether the enzymatic conversion was
successful.The plasmid design was the same as described in Cycle 1.

Bulid:

We cultured the engineered yeast strain from Cycle 1, lysed the cells, and
prepared active crude enzyme extracts. Parallel in vitro reaction
systems were established, each containing the enzyme extract supplemented with
either substrate R1 or IVa and the necessary cofactor UDP-glucose.

Test:

The results showed that the conversion efficiency of R1 to
ginsenoside Ro was significantly lower than that of IVa.

![](https://static.igem.wiki/teams/5564/engineering/eng130.webp)

Figure 4. HPLC peak of ginsenoside Ro using
chikusetsusaponin IVa (IVa) as substrate

![](https://static.igem.wiki/teams/5564/engineering/eng140.webp)

Figure 5. HPLC peak of ginsenoside Ro using
zingibrosideR1(R1) as substrate

Learn:

In vitro experiments demonstrated that the glycosyltransferases expressed in
Saccharomyces cerevisiae are enzymatically active. However, in vivo
assays revealed substantial accumulation of disaccharide byproducts, suggesting that substrate specificity issues may lead to intermediate buildup. The
results indicated that the conversion efficiency from R1 to ginsenoside
Ro is considerably lower than that from IVa to Ro. Based
on these findings, we aim to apply directed evolution to the enzyme
to alter its substrate specificity and thereby reduce the formation of the intermediate R1.

# Single-point fully saturated computational screening for enhanced activity of UGT73F3

Objective:To identify potential activity-enhancing
mutants via '497x19 single-point saturation mutation + machine learning prediction +
molecular docking validation', thereby defining targets for subsequent validation.

Cycle 1: machine learning models

Objective:Training and Evaluating Mutation Effect
Predictors

Design:

Employ dataset S10988 (10,510; 5,255 positive/negative) for five-fold
cross-validation, with S2814 (5,978; 1,118/4,860) as independent test set; Features
comprised 1,280-dimensional sequence embedding differences (WT-Mut, dESM) for ESM1v/ESM2b,
combined with AlphaFold3 structure mapping within a 10 Å neighbourhood of mutation sites;
models included GCN/GAT (structure + sequence), MLP (sequence differences only), SVM/RF
(comparative baseline).

Build:

DGL implements GCN (GraphConvx2) and GAT (GATConvx2, multi-head attention) +
global average pooling; MLP architecture: 1280→512→1024→2 (ReLU); SVM/RF grid search; binary
classification cross-entropy + Adam(1e-4) + early stopping.

Test:

On S10988 cross-validation, graph models and MLP achieved AUC≈0.96-0.99,
Accuracy/F1≈0.94-0.95; On independent set S2814, AUC≈0.62-0.70, specificity TNR>0.86-0.95,
but sensitivity TPR≈0.21-0.35 (e.g., AF3-GCN TPR=0.24).

Learn:

Graph model cross-validation yields excellent results, yet sensitivity markedly
declines on independent datasets, indicating generalisation challenges (potentially due to
strong structural/contextual dependencies). Decision: Retain the multi-model comparison
framework for computational screening of all UGT73F3 variants, proceeding to the next round.
Decision: Retain the multi-model comparison framework for computational screening of all
UGT73F3 variants, proceeding to the next round.

![](https://static.igem.wiki/teams/5564/engineering/eng150.webp)

Figure 6. Workflow for predicting mutations by
dESM.

Cycle 2: Top Mutant Screening

Objective:Full-sequence single-point mutant screening +
molecular docking verification

Design:

Generate 9,443 single-point mutations of UGT73F3; score using trained model and
select Top-100; perform 5 independent dockings per mutation to assess energy stability.

Build:

AutoDock Vina 1.2.x batch script automatically configures grids, subdirectories,
and logging; extracts lowest-energy conformations; converts PDBQT to MOL2 using Open Babel
3.x; aggregates duplicate binding energies.

Test:

Multiple docking runs confirmed energy stability; S175P demonstrated optimal
performance with an average binding energy of –8.64 kcal·mol⁻¹, indicating highest potential
activity.

![](https://static.igem.wiki/teams/5564/engineering/eng160.webp)

Figure 7. Docking result of the S175P mutant as
rendered with PyMOL

![](https://static.igem.wiki/teams/5564/engineering/eng170.webp)

Learn:

S175P identified as current top candidate (top hit); next step: conduct molecular
dynamics (MD) simulations to validate stability and binding mode.

# Increase UDP-glucose (UDPG) availability

Cycle: Overexpression of the key enzymes

Objective:The biosynthesis of ginsenoside Ro involves
multiple glycosylation steps. By increasing
intracellular UDP-glucose (UDPG) availability, the efficiency of these
glycosylation reactions can be enhanced, leading to higher Ro production.

Design:

Literature reports indicate that yeast possesses a native
glycerol-to-glucose metabolic pathway. To increase intracellular UDP-glucose (UDPG)
levels, we overexpressed the key enzymes FBPase1, PGM2, and UGP1. The genes
were assembled into a plasmid
driven by constitutive promoters and carrying a tryptophan
selection marker for stable expression in Saccharomyces cerevisiae.

Bulid:

The genes encoding the key enzymes FBPase1, PGM2, and UGP1 were codon-optimized
for Saccharomyces cerevisiae, synthesized, and assembled into a
single expression plasmid. This plasmid, based on the pRS314 backbone, was driven by strong
constitutive promoters and carried a tryptophan selection marker.

![](https://static.igem.wiki/teams/5564/engineering/eng180.webp)

Figure 8. Schematic representation of the plasmid
pRS314-FBPase1-PGM2-UGP1

Test:

![](https://static.igem.wiki/teams/5564/engineering/eng190.webp)

Figure 9. PCR verification of the three genes
encoded on the plasmid

![](https://static.igem.wiki/teams/5564/engineering/eng200.webp)

Figure 10. Verification of the three plasmid-encoded
proteins by Western blot using tag-specific antibodies

Learn:

By introducing the key enzymes responsible for converting glycerol to
glucose-derived UDP-glucose (UDPG), intracellular UDPG
levels in Saccharomyces cerevisiae can be increased, facilitating
the glycosylation of intermediates to produce the final product, ginsenoside Ro. To explore
optimal strategies, we plan to investigate the best glycerol-to-medium
ratio. Specifically, we will measure intracellular UDPG levels under different
glycerol concentrations and correlate these data with yeast growth curves to identify the
optimal cultivation conditions.

# References:

1.Wang, Z., Su, C., Zhang, Y., Shangguan, S., Wang, R., & Su, J. (2024). Key
enzymes involved in the utilization of fatty acids by Saccharomyces
cerevisiae: a review. Frontiers in microbiology, 14, 1294182.
https://doi.org/10.3389/fmicb.2023.1294182

2.Qiu, F., Kang, N., Tan, J., Yan, S., Lin, L., Cai, L., Goodman, J. M., & Gao,
Q. (2023). Fatty Acyl Coenzyme A Synthetase Fat1p Regulates Vacuolar Structure and
Stationary-Phase Lipophagy in Saccharomyces
cerevisiae.
Microbiology spectrum, 11(1), e0462522. https://doi.org/10.1128/spectrum.04625-22

3.Tang, H., Wang, S., Wang, J., Song, M., Xu, M., Zhang, M., Shen, Y., Hou, J., &
Bao, X. (2016). N-hypermannose glycosylation disruption enhances recombinant protein
production by regulating secretory pathway and cell wall integrity in Saccharomyces
cerevisiae. Scientific reports, 6, 25654.
https://doi.org/10.1038/srep25654

4.Faergeman, N. J., Black, P. N., Zhao, X. D., Knudsen, J., & DiRusso, C. C.
(2001). The Acyl-CoA synthetases encoded within FAA1 and FAA4 in Saccharomyces
cerevisiae function as components of the fatty acid
transport system linking import, activation, and intracellular Utilization. The Journal of
biological chemistry, 276(40), 37051-37059. https://doi.org/10.1074/jbc.M100884200

5.Li, Z., Chen, Y., Liu, D., Zhao, N., Cheng, H., Ren, H., Guo, T., Niu, H.,
Zhuang, W., Wu, J., & Ying, H. (2015). Involvement of glycolysis/gluconeogenesis and
signaling regulatory pathways in Saccharomyces
cerevisiae biofilms
during fermentation. Frontiers in microbiology, 6, 139.
https://doi.org/10.3389/fmicb.2015.00139

6.Mukherjee, M., Blair, R. H., & Wang, Z. Q. (2022). Machine-learning guided
elucidation of contribution of individual steps in the mevalonate pathway and construction
of a yeast platform strain for terpenoid production. Metabolic engineering, 74, 139-149.
https://doi.org/10.1016/j.ymben.2022.10.004

{% endblock %}


{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their
significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5564/banner/results.webp)

* OVERVIEW
* Peroxisomal Mevalonate Pathway Reconstitution
* Oleanane-Type Triterpenoid Synthesis
* Ginsenoside Ro Multi-Enzyme Glycosylation System
* UDP-Glucose Supply Module
* Future Perspectives
* Reference

# OVERVIEW

We employed a metabolic engineering strategy to deconstruct the biosynthesis of ginsenoside Ro into
four independent
modules. First, in the Peroxisomal Mevalonate Pathway Reconstruction
module, we targeted ERG10, ERG13, and HMG1 to the
peroxisome, thereby enabling efficient utilization of fatty acid-derived acetyl-CoA. Second, within
the Oleanane-Type Triterpenoid Synthesis module, we introduced β-AS , CYP716A12 and MtCPR ,among other elements, to generate the oleanolic
acid precursor. Third, in the Ginsenoside Ro Multi-Enzyme Glycosylation
System, we integrated a suite of
glycosyltransferases to achieve the selective glycosylation of Ro.
Finally, in the UDP-Glucose Supply Module, we expanded the donor sugar pool by overexpressing
FBPase1, PGM2, and
UGP1.Through the synergistic operation of these four modules, we successfully
reconstituted the biosynthesis of
ginsenoside Ro.

# Peroxisomal Mevalonate Pathway Reconstitution

In the peroxisomal mevalonate (MVA) pathway reconstruction module, we engineered plasmids to express
Acetoacetyl-CoA
thiolase (Erg10), Hydroxymethylglutaryl-CoA synthase (Erg13), and Hydroxymethylglutaryl-CoA
reductase (HMG1) within the
peroxisome by incorporating a peroxisomal targeting signal 1 (PTS1). This
design established the metabolic route from
acetyl-CoA to mevalonate (MVA), thereby enabling the utilization of
acetyl-CoA derived from β-oxidation for MVA
biosynthesis.

![](https://static.igem.wiki/teams/5564/results/res001.webp)

Figure 1. Schematic representation of the plasmid
pRS314-Erg10-Erg13-HMG1

![](https://static.igem.wiki/teams/5564/results/res002a.webp)
![](https://static.igem.wiki/teams/5564/results/res002b.webp)
![](https://static.igem.wiki/teams/5564/results/res002c.webp)

Figure 2. PCR verification of the three genes encoded on the
plasmid
(A) Erg10, showing the expected band size of 390 bp.
(B) Erg13, showing the expected band size of 1201 bp.
(C) HMG1, showing the expected band size of 366 bp.

![](https://static.igem.wiki/teams/5564/results/res003.webp)

Figure 3. Western blot verification of the three plasmid-encoded proteins,
detected with tag-specific antibodies

![](https://static.igem.wiki/teams/5564/results/res004.webp)

Figure 4. Growth curve of S.cerevisiae on
different single-fatty-acid media before and after plasmid transformation, monitored
over time

![](https://static.igem.wiki/teams/5564/results/res005.webp)

Figure 5. Growth curve of plasmid-transformed S.cerevisiae on
different mixed-fatty-acid media,monitored over time

# Oleanane-Type Triterpenoid Synthesis

In the oleanane-type triterpenoid synthetic module, we successfully reconstituted the pathway in
Saccharomyces
cerevisiae by heterologously expressing β-amyrin synthase (β-AS) from
Glycyrrhiza glabra, cytochrome P450 monooxygenase
(CYP716A12) from Medicago truncatula, and cytochrome P450 reductase 1 (MtCPR). Functional
expression of these enzymes
enabled the complete conversion from 2,3-oxidosqualene(2,3-OSQ) to β-amyrin and
subsequently to oleanolic acid (OA),
providing the key triterpenoid precursor OA for the biosynthesis of ginsenoside Ro.

![](https://static.igem.wiki/teams/5564/results/res006.webp)

Figure 6. Schematic representation of the plasmid
pRS314-βAS-CYP716A12-MtCPR

![](https://static.igem.wiki/teams/5564/results/res007a0.webp)
![](https://static.igem.wiki/teams/5564/results/res007b0.webp)
![](https://static.igem.wiki/teams/5564/results/res007c0.webp)

Figure 7. PCR verification of the three genes encoded on the
plasmid
(A) MtCPR, showing the expected band size of 403 bp.
(B) CYP716A12, showing the expected band size of 328 bp.
(C) β-AS, showing the expected band size of 378 bp.

![](https://static.igem.wiki/teams/5564/results/res008.webp)

Figure 8. SDS-PAGE analysis of the three plasmid-encoded proteins
visualized by Coomassie Brilliant Blue staining

![](https://static.igem.wiki/teams/5564/results/res009.webp)

Figure 9. Growth curve of plasmid-transformed S.cerevisiae on
SC-Trp selective medium, monitored over time

![](https://static.igem.wiki/teams/5564/results/res010.webp)

Figure 10. High-performance liquid chromatography (HPLC) analysis of the
oleanolic acid (OA) product peak

# Ginsenoside Ro Multi-Enzyme Glycosylation System

In the multi-enzyme ginsenoside Ro glycosylation system, we constructed plasmids for heterologous
expression in
Saccharomyces cerevisiae of UGT73P40, UGT73F3, and Pn022859 from Panax notoginseng. This
reconstituted the ginsenoside
Ro glycosylation module, enabling a multi-step glycosylation network converting oleanolic acid to
ginsenoside Ro.

![](https://static.igem.wiki/teams/5564/results/res011.webp)

Figure 11. Schematic representation of the plasmid
pRS315-UGT73P40-UGT73F3-Pn022859

![](https://static.igem.wiki/teams/5564/results/res012a.webp)
![](https://static.igem.wiki/teams/5564/results/res012b.webp)
![](https://static.igem.wiki/teams/5564/results/res012c.webp)

Figure 12. PCR verification of the three genes encoded on the
plasmid
(A) UGT73P40, showing the expected band size of 400 bp.
(B) UGT73F3, showing the expected band size of 612 bp.
(C) Pn022859, showing the expected band size of 387 bp.

![](https://static.igem.wiki/teams/5564/results/res013.webp)

Figure 13. SDS-PAGE analysis of the three plasmid-encoded proteins
visualized by Coomassie Brilliant Blue staining

![](https://static.igem.wiki/teams/5564/results/res014.webp)

Figure 14. Growth curve of plasmid-transformed S.cerevisiae in
SC-Trp-Leu selective medium, monitored over time

![](https://static.igem.wiki/teams/5564/results/res015.webp)

Figure 15. High-performance liquid chromatography (HPLC) analysis of
ginsenoside Ro produced by S.cerevisiae

# UDP-Glucose Supply Module

In the UDP-glucose supply module, we constructed plasmids for overexpression in S.cerevisiae of
fructose-1,6-bisphosphatase (FBPase1), phosphoglucomutase (PGM2), and UTP-glucose-1-phosphate
uridylyltransferase
(UGP1). This enhances sugar phosphate flux and UDP-Glc biosynthesis, providing sufficient sugar
donors for the
glycosylation reactions of ginsenoside Ro.

![](https://static.igem.wiki/teams/5564/results/res016.webp)

Figure 16. Schematic representation of the plasmid
pRS314-FBPase1-PGM2-UGP1

![](https://static.igem.wiki/teams/5564/results/res017a.webp)
![](https://static.igem.wiki/teams/5564/results/res017b.webp)
![](https://static.igem.wiki/teams/5564/results/res017c.webp)

Figure 17. PCR verification of the three genes encoded on the
plasmid
(A) FBPase1, showing the expected band size of 355 bp.
(B) PGM2, showing the expected band size of 381 bp.
(C) UGP1, showing the expected band size of 360 bp.

![](https://static.igem.wiki/teams/5564/results/res018.webp)

Figure 18. Verification of the three plasmid-encoded proteins by Western
blot using tag-specific antibodies

# Future Perspectives

Future work will focus on scaling up and optimization. We plan to implement
glycerol/fatty acid feeding strategies and
fine-tune pH, oxygen levels, and feed rates to transfer the pathway into bioreactor fermentation. Adaptive laboratory
evolution or dynamic regulation strategies, such as inducible systems, will be explored to enhance robustness on
non-glucose carbon sources. Additionally, we aim to screen more UGT
variants to achieve higher titers. Finally, the
pathway could be extended to other oleanane-type saponins, such as
rhamnosylated derivatives, to broaden its
applications. Overall, this project opens multiple avenues for synthetic biology,
green chemistry, and collaborations
with biotechnology companies.

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