iGEM 2025 — Team subcat-union

{% 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 %} {% from
"/components/article-content.html" import articleContent %} {% call
articleContent("NOTEBOOK", [{"content": "title 1","anchor": "#title\_1"},
{"content": "title 2","anchor": "#title\_2"}, {"content": "title 3","anchor":
"#title\_3"}], true) %}

Main Timeline:

Each
month is depicted with a grid system. Each square in the
picture below
corresponds to a 3-day period.

January：

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Recruiting of new members |  |  |  |  |  |  |  |  |  |  |

February：

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Recruiting of new members |  |  |  |  |  |  |  |  |  |  |
| Research the literature |  |  |  |  |  |  |  |  |  |  |

March：

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Design of project subject |  |  |  |  |  |  |  |  |  |  |
| Safety training |  |  |  |  |  |  |  |  |  |  |

April：

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Determine the project topic |  |  |  |  |  |  |  |  |  |  |
| Formulation of project planning |  |  |  |  |  |  |  |  |  |  |

May：

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Formulation of project planning |  |  |  |  |  |  |  |  |  |  |
| Theoretical study |  |  |  |  |  |  |  |  |  |  |

June：

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Theoretical study |  |  |  |  |  |  |  |  |  |  |

July:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Star the experiment |  |  |  |  |  |  |  |  |  |  |
| Experimental data analysis |  |  |  |  |  |  |  |  |  |  |
| Discuss and solve the problem |  |  |  |  |  |  |  |  |  |  |
| Education |  |  |  |  |  |  |  |  |  |  |
| Expert Interview Guide |  |  |  |  |  |  |  |  |  |  |

August:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Building the Team Wiki |  |  |  |  |  |  |  |  |  |  |
| Continuing the experiment |  |  |  |  |  |  |  |  |  |  |
| Discuss and solve the problem |  |  |  |  |  |  |  |  |  |  |

September:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Data integration |  |  |  |  |  |  |  |  |  |  |
| Building the Team Wiki |  |  |  |  |  |  |  |  |  |  |
| Education |  |  |  |  |  |  |  |  |  |  |
| Arrangement and analysis of experimental data |  |  |  |  |  |  |  |  |  |  |

October:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Project arrangement and preparation |  |  |  |  |  |  |  |  |  |  |

**Safety training is required before
the
experiment begins**

**Stage one****：****Construction of plasmid**

**Task: PCR**

**Goal****：****Obtain the target gene**

**Participants:** Yue Cao, Yuqin
Zhang, Yutong
Du, Jian Tang, Zilan Qin, Jingwei Wu, Yunke Lu, Lizhuo Chen, Daoyang Zhang

**Materials****：**Forward/Reverse Primers, Agar
2%， Template
plasmid, H2O, Buffer, Enzymes， Base pairs（ATCG）

**Procedure****：**

Put 25
μL 2\*PCR mix into each PCR tubes, then add 22 μL ddwater. Then add 1 μL Forward Primer and 1 μL
Reverse
Primer into the tubes. After that, add 1 μL template plasmid into each PCR tubes.

Put
those total 14 PCR tubes load into thermal cycle and start PCR.

The
PCR cycle starts with 95°C 3 minutes Then move as 95°C for 30 seconds, 55°C for 30 seconds and
72°C for 1.5
minutes. This cycle repeats 33 times.Finally, 72°C for 10min.

**Task:** **Verified the PCR
product by
electrophoresis, purified the target DNA fragment, and performed bacterial
transformation using the
heat-shock method**.

**Goal****：**Construction of plasmid.

**Participants:** Yue Cao, Yuqin
Zhang, Yutong
Du, Jian Tang, Zilan Qin, Jingwei Wu, Yunke Lu, Lizhuo Chen, Daoyang Zhang

**Procedure****：**

**Agarose gel
electrophoresis
verification**

Mix agarose and ATE buffer
together, with a ratio of 1：100

Sol by heat and dissolve in
the
microwave oven and add nucleic acid dye when it cools down to 50-60℃

Pour into the mold to cooling
and
solidification

Place 8μl PCR the mold
cavities
and then electrophoresis 180V for 20 minutes

Finally put the gel in to the
Gel
imager，ultraviolet
irradiation to observe
DNA that has reached a specific length

**Column-based DNA
gel
Purification.**

Cut
out the gel block containing the target fragment from the agarose gel and weigh it.

Add
Buffer B2 into the gel block and metal bath55℃.

Transfer sol solution
into the
adsorption column and 8000Xg centrifugation for 1 minute.

Add
Wash Buffer and 9000Xg centrifugation for 1 minute, pour the solution in the tube.

Repeat
the previous step.

Let
empty adsorption column in the centrifuge for 1 minute.

Put
adsorption column into a clean 1.5mL centrifuge tube and add 30μl Elution Buffer into the center
of the
adsorption film. centrifugation for 1 minute.

Finally preserve DNA
solution.

**DNA
homologous recombination and transformation.**

Prepare
solutions.

Mix
5μl 2×ClonExpress Mix, 1μl aga, 1μl sall, 3μl pom together.

Heat
at 50 ℃ for 15 minutes. Then ice bath for 4 minutes.

Preparation for
antibiotic plates
(LB+1:1000 Kanamycin)

Thaw competent *E.
coli*
cells on the ice.

Add 10μL recombinant
product
(plasmid) into the competent cells, gently flick vessel wall, gently mix it by using the pipette
tip.
Incubate on ice for 30 minutes, stationary.

42℃ heat shocks for
30s in order
to create channels and pores in the cell membrane that connect the cell interior to the external
environment.

Remove immediately,
incubate on
ice for 2 minutes to reduce the temperature.

 Add 900μL LB culture
medium.
Incubate at 37℃ with shaking for one hour.

Centrifuge and discard
800μL of
the supernatant.

Separate the thallus
in the
remaining 200μL of LB medium.

Evenly spread the
sample on
antibiotic plates by using a sterile cell spreader.

Task: Plasmid Verification

**Goal****：**Verify whether the plasmid was
successfully
constructed.

**Participants:** Yue Cao, Yuqin
Zhang, Yutong
Du, Jian Tang, Zilan Qin, Jingwei Wu, Yunke Lu, Lizhuo Chen, Daoyang Zhang

**Result:**

![](https://static.igem.wiki/teams/5741/images/notebook-1.webp)

Figure
1

**Stage two****：****Protein expression and
verification**

**Goal****：**The
protein expression was induced and verified by SDS-page.

**Task:** Protein extraction,
purification, and
verification result

**Participants:** Yue Cao, Yuqin
Zhang, Yutong
Du, Jian Tang, Zilan Qin, Jingwei Wu, Yunke Lu, Lizhuo Chen, Daoyang Zhang

**Procedure****：**

**Cultivate bacterial
samples for
protein production:**

Select
the correct colonies and culture them to an OD of 0.5.

Prepare 4 conical
flasks.

Add
100ml of LB culture medium in 2 flasks and 100ml of soywhey culture medium in 2 flasks.

Add
IPTG(1mM) in 1 flask of LB culture and 1 flask of soywhey culture.

Add
galactose(1mM) another flask of LB culture and another flask of soywhey culture.

Place
it in the shaking incubator maintain 25°C, shake for 20 hours.

Collect the strains
cultured last
night, get 50ml from each and pour them into a tube.

Through a process of
centrifugation and pouring out the supernatant, obtain a sediment of *E.coli*.

Add
4ml of lysis buffer and 40 μL of lysozyme. Place the tube within ice
or keep it
under a low temperature for 30 min.

Due to
an ultrasonication to the bacteria formula and once again.

Place
it in the centrifuge and preserve the supernatant to extract specifically the protein.

Then with pipette transfer the Nickel
column in to
the tube with the protein.

**Purify the protein
mixture:**

For
each tube of formula, prepare 8 of 2ml tubes, a 10ml tubes and a gravity flow column,
respectively note the
eight 2ml tubes as w1 w2 w3 w4 e1 e2 e3 e4, and it should be noted with the type of formula
dealing with the
bacteria a day before.

While
the volve remain closed, transfer the formula into the gravity flow column.

Remaining under a low
temperature,
open the volve and collect them in a 10ml tube.

While
the liquid part is totally leaked within, pick up the gravity flow column, place it respectively
on the
related first four 2ml tubes (w1-4). Wash 4 times with washing buffer.

Wash
the same column four more times using the elution buffer, and collect the filtered liquid in
four other EP
tubes.

**Verification**

Into a
PCR tube, add 20μl protein liquid

Load
the sample in the protein gel.

Run
electrophoresis: run at 80V for 20 minutes, then switch to 180V and run for an additional 40
minutes.

Observed by Coomassie
brilliant
blue staining.

**Result:**

![](https://static.igem.wiki/teams/5741/images/notebook2.webp)

Figure
2

**Stage three****：**Functional Verification of the
Carbohydrate
Utilization Module.

**Task:**Determination of
strain growth curve after introducing carbohydrate utilization module

**Participants:** Yue Cao, Yuqin
Zhang, Yutong
Du, Jian Tang, Zilan Qin, Jingwei Wu, Yunke Lu, Lizhuo Chen, Daoyang Zhang

**Procedure****：**

Wild-type strain and
S1 strain
were cultured in LB medium for 24h.

The
cultured bacteria were collected by centrifugation, and the supernatant was discarded.

Use
sterile water to resuspend the strain, determine the OD600, and position the final
concentration
of the strain as OD600=0.2.

The
two strains were cultured in LB and soy whey media respectively, and OD600 was determined after
24
hours.

**Stage four****：**Construction of galactose
self-induction
system.

**Task:** Checked GFP brightness to
find the
best promoter.

**Participants:** Yue Cao, Yuqin
Zhang, Yutong
Du, Jian Tang, Zilan Qin, Jingwei Wu, Yunke Lu, Lizhuo Chen, Daoyang Zhang.

**Procedure****：**

PgalP-a-EGFP,
PgalP-b-EGFP,
PgalP-c-EGFP strain were cultured in LB medium for 24h.

The
cultured bacteria were collected by centrifugation, and the supernatant was discarded.

Use
sterile water to resuspend the strain, determine the OD600, and position the final
concentration
of the strain as OD600=0.2.

The
fluorescence intensity of the strain was measured at 0, 2, 4, 24, 36 and 48 hours after culture.
(The
excitation wavelength of GFP is 488 nm and the emission wavelength is 507 nm.)

**Stage five****：**Construction and verification of 3'-SL
synthesis
module and combine it
with the above two systems.

**Task:** Verified 3-module
co-transformation.

**Participants:** Yue Cao, Yuqin
Zhang, Yutong
Du, Jian Tang, Zilan Qin, Jingwei Wu, Yunke Lu, Lizhuo Chen, Daoyang Zhang.

**Procedure****：**

Bacterial Colony
Selection and
Preliminary Culture

-Configuration of the
PCR system(5
µL mix，water,0.1 µL primer F
and
R,template)

-Set
PCR procedure.

-Select individual
colonies for
colony PCR.

-Prepare the bacterial
solution.

-Pour
1 µL of the bacterial solution into a 10 µL PCR tubes.

**Stage six****：**The
synthetic amount of 3'-SL was determined by HPLC

**Task:** Tested if the three modules
worked

**Participants:** Yue Cao, Yuqin
Zhang, Yutong
Du, Jian Tang, Zilan Qin, Jingwei Wu, Yunke Lu, Lizhuo Chen, Daoyang Zhang.

**Procedure****：**

After
streaking and activating the fermentation strain, a single colony was picked and inoculated into
5 mL of LB
liquid medium (with 5 μL Chl and Amp) and then cultured at 37°C and 200 r/min for 8-12 hours.
The culture
was transferred to 100 mL of Soybean Yellow Pulp Water at an initial OD600 of 0.1 and
cultured at
37°C and 200 r/min for approximately 8 hours. The culture was centrifuged at 4°C and 5000 rpm
for 10
minutes. The bacterial cells were resuspended in 24 mL of Tris-HCl buffer (50 mmol/L, pH 7.0,
containing 250
mg Neu5Ac). A substrate solution with a concentration of 10 g/L (1 mL of 250 g/L lactose) was
added to start
the reaction.

HPLC
analysis method for the product: differential refractive index detector, detector temperature
35°C; Rezex
ROA-Organic Acid H+ (8%) chromatographic column, column temperature 60°C; mobile phase: 5 mmol/L
H2SO4
solution, flow rate 0.6 mL/min; injection volume 10 μL.

![](https://static.igem.wiki/teams/5741/images/notebook3.webp)

Figure
3

{% endcall %} {% 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 %} {% from
"/components/article-content.html" import articleContent %} {% call
articleContent("ENGINEERING", [{"content": "Overview","anchor": "#title\_1"},
{"content": "Cycle 1: Construction and verification of carbohydrate utilization module.","anchor": "#title\_2"},
{"content": "Cycle 2: Construction and verification of galactose self-induction module.","anchor": "#title\_3"},
{"content": "Cycle 3: Construction and verification of 3'-SL synthesis module.","anchor": "#title\_4"},
{"content": "Construction and verification of three plasmid co-transformed strains","anchor": "#title\_5"},
{"content": "References","anchor":"#title\_9"}]) %}

# **Overview**

Breast milk
oligosaccharides
(HMO) are very important to the health of infants, among which 3'- sialyllactose (3'-SL)
has attracted
much attention because of its various benefits in promoting brain development and
enhancing immunity.
With the global demand for high-quality infant formula increasing, the market demand for
3'-SL is
expected to rise sharply [ 1 ]. However,
the
traditional extraction and chemical synthesis methods are inefficient, costly and may
cause pollution,
so it is difficult to meet the market demand [ 2 ]. The
purpose of this
project
is to construct an Escherichia coli chassis which can use cheap soy whey as
culture medium and
galactose
in soy whey to realize self-induced expression system, so as to produce 3'-SL
efficiently and
economically. This study not only provides a new solution for the industrial
production of 3'-SL but
also provides a useful reference for the biological manufacture of other high
value-added chemicals,
which have remarkable economic benefits and environmental protection
significance.

**Our
design
consists of
three main cycles:**

**Cycle 1:
Construction and
verification of carbohydrate utilization module.** (pCOLAtrc(lost O)-sacC-aga,
[BBa\_25TFWJDX](https://registry.igem.org/parts/bba-25tfwjdx))

**Cycle 2:
Construction and
verification of galactose self-induction module.**

pCOM4-PgalP-a-EGFP,
[BBa\_2572ULRB](https://registry.igem.org/parts/bba-2572ulrb)

pCOM4-PgalP-b-EGFP,
[BBa\_25NRQBKP](https://registry.igem.org/parts/bba-25nrqbkp)

pCOM4-PgalP-c-EGFP,
[BBa\_25Q32CXO](https://registry.igem.org/parts/bba-25q32cxo)

pCOM4-PgalP-GalP,
[BBa\_25M3YRM6](https://registry.igem.org/parts/bba-25m3yrm6)

**Cycle
3:** **Construction
and
verification of 3'-SL synthesis module.**

pET-CSS-SaiT,
[BBa\_251TW3T1](https://registry.igem.org/parts/bba-251tw3t1)

![](https://static.igem.wiki/teams/5741/images/engi-1.webp)

# **Cycle 1: Construction and verification of carbohydrate utilization module.**

**Design:**

The carbon
sources in soy whey
are mainly sucrose, stachyose and raffinose. In order to utilize these carbohydrates, it
is necessary
for the strain to express extracellular sucrase(Sacc) and α -galactosidase(Aga), so as
to hydrolyze the
carbon sources that bacteria cannot use (sucrose, stachyose and raffinose) into carbon
sources that
bacteria can use (glucose, fructose and galactose).
Therefore, we
first need to
introduce Sacc and Aga into *E.coli*, so that *E.coli* can survive in soy
whey.

![](https://static.igem.wiki/teams/5741/images/engi-2.webp)

**Figure
1
The plasmid map
of pCOLAtrc(lost O)-scaC-aga**

**Build:**

Agaa and
Sacc
can convert
carbon sources that E.coli can't use into available carbon sources (such as glucose,
fructose and
galactose). After introducing them into bacteria, we initially obtained the strains that
could be
successfully constructed by antibiotic screening and further verified them by DNA
sequencing. It was
found that the DNA fragments were consistent with expectations (*AGA* (2193bp)
and *SACC*
(1509bp), the sequencing results showed that the construction of our strains was
successful.

![](https://static.igem.wiki/teams/5741/images/engi-3.webp)

**Figure
2.
Constructing carbohydrate utilization module.**

**Test:**

**1.**  **SDS-PAGE of
carbohydrate
utilization module**

After
confirming the success
of the strain construction, we want to further verify whether these two genes can
express the protein
normally. We have carried out experimental tests in soy whey medium. The experimental
group used IPTG to
induce protein expression. The experimental results showed that the strain could grow
normally and
express Aga and Sacc proteins under the induction of IPTG in soy whey medium. After
introducing this
module, the strain can grow normally in soy whey and express protein normally under the
induction of
IPTG.

![](https://static.igem.wiki/teams/5741/images/engi-4.webp)

**Figure
3
SDS-PAGE of
carbohydrate utilization module.**

**2.** **Strain
growth
determination**

After
confirming that the
protein can be successfully induced to express, we want to know whether these two
proteins can function
normally. That is, whether *Escherichia coli* can grow normally in soy whey after
Aga and Sacc
are introduced. Therefore, We tested the growth of strains with carbohydrate utilization
module and
without carbohydrate utilization module in soy whey. The experimental results show that
the strain can
grow normally in soy whey after introducing protein, but the growth of the strain
without introducing
protein is obviously inhibited. This result shows that
the
strain can grow normally
in soy whey after containing carbohydrate utilization module.

![](https://static.igem.wiki/teams/5741/images/engi-5.webp)

**Figure
4.
Growth ability of strains under different culture conditions.**

# **Cycle 2: Construction and verification of galactose self-induction module.**

**Design:**

At
present, our bacteria need to use exogenous IPTG for protein induction, IPTG has
potential cytotoxicity
and high cost, so we wonder if we can build a module. After the module is introduced,
bacteria can
spontaneously induce protein expression by using galactose in soy whey. On the one hand,
this can reduce
the cost, on the other hand, it can avoid introducing chemical components that may have
side effects.
Galactose can not only be used as a nutrient for normal cell growth, but also as an
inducer to induce
protein expression, so how to induce protein expression as much as possible on the basis
of maintaining
bacterial growth is extremely critical.
In order
to improve the
induced yield of protein with galactose as inducer, we constructed three promoters
regulated by
galactose, and constructed EGFP fluorescent protein downstream of the promoters. By
observing the
expression intensity of fluorescent protein, we can quickly obtain the induced yield of
protein. Through
screening, we can find the promoter with the strongest inducing ability when galactose
is used as a
protein inducer. After selecting the most suitable promoter, we further want to replace
EGFP gene with
galactose transporter. After adding galactose inducer, the promoter is regulated by
galactose, which
strongly induces the expression of galactose transporter downstream.
Galactose
transporter
transports galactose to 3'-SL module and activates the synthesis of 3'-SL.

![](https://static.igem.wiki/teams/5741/images/engi-6.webp)

**Figure
5
The plasmid
maps. A.** **pCOM4-PgalP-a-EGFP.B.**
**pCOM4-PgalP-b-EGFP.**

**C.**
**pCOM4-PgalP-c-EGFP.D.**
**pCOM4-PgalP-GalP.**

**Build:**

In
order to obtain *E.coli* with different promoters, we obtained DNA fragments of
different
promoters by PCR and connected them with plasmid vectors by homologous recombination.
After obtaining
the complete plasmid, we transformed the plasmid into *E.coli*, and carried out
preliminary
screening by antibiotics(Figure 6A). Subsequently, we selected monoclonal colonies from
the plate and
verified them by PCR(Figure 6B). After the PCR verification was successful, we further
confirmed that
the strain construction was successful through DNA sequencing(Figure 6C).

![](https://static.igem.wiki/teams/5741/images/engi-7.webp)

**Figure
6
Construction and
Verification of Promoter Screening Module.**

**A.
Antibiotic screening
plate. B.** **Agarose gel
electrophoresis
verification. C.**
**The
results of**
**DNA
sequencing
verification.**

After
selecting the most
suitable promoter, we further want to replace EGFP gene with galactose transporter.
After adding
galactose inducer, the promoter is regulated by galactose, which strongly induces the
expression of
galactose transporter downstream. Galactose transporter transports galactose to 3'-SL
module and
activates the synthesis of 3'-SL. Therefore, we first replaced EGFP gene with galactose
transporter
*GALP* by molecular biology. Through antibiotic screening (Figure 7A), colony PCR
verification
(Figure 7B), and DNA sequencing (Figure 7C), we confirmed that the plasmid construction
was
correct.

![](https://static.igem.wiki/teams/5741/images/engi-8.webp)

**Figure
7
Construction of pCOM4-PgalP-GalP. A. Antibiotic screening plate.
B.** **Agarose gel electrophoresis
verification.
C.** **The
results
of** **DNA sequencing
verification.**

**Test:**

**1. The expression
level of
EGFP was verified by SDS-PAGE.**

After
confirming the success
of the strain construction, we want to further verify whether these genes can express
the protein
normally.Three strains were cultured in LB medium, and IPTG and galactose were
used as inducers
to induce protein expression, and the protein expression level was verified by SDS-PAGE.
Through the
experimental results, we can find that EGFP protein can be expressed normally under the
culture
conditions with IPTG and galactose as inducers.

![](https://static.igem.wiki/teams/5741/images/engi-9.webp)

**Figure
8
SDS-PAGE
of****galactose
self-induction module.**

**2.Measurement of
EGFP
fluorescence intensity**

By
measuring the fluorescence intensity, we quickly measured the induction intensity of the
promoter under
the condition of galactose induction. Through the experimental results, we can find that
the
fluorescence intensity of the blank control group and the control group has hardly
changed with the
increase of time. However, with the increase of time, the fluorescence intensity of EGFP
increased with
different strains containing different promoters. Among them, pGalp-a promoter has the
strongest ability
to induce protein expression, so we chose pGalp-a promoter for the follow-up
experiment.

![](https://static.igem.wiki/teams/5741/images/engi-10.webp)

**Figure
9
Fluorescence
intensity determination.**

**3.The expression
level of
Galactose transporter was verified by SDS-PAGE.**

After the
successful
construction of the strain, we also verified the protein expression level of the strain
by SDS-PAGE.
Through the experimental results **(**Figure 10), we can find that Galp protein can
be expressed
normally under the induction of IPTG and galactose.

![](https://static.igem.wiki/teams/5741/images/engi-11.webp)

**Figure
10.
The result of
SDS-PAGE.**

# **Cycle 3:** **Construction and verification of 3'-SL synthesis module.**

**Design:**

 After
confirming that bacteria
can survive in soy whey and can spontaneously induce protein production by using
nutrients in soy whey,
the next step is to obtain the synthesis module of 3'-SL.CMP-Neu5Ac Synthase (CSS) and
α2,3-sialyltransferase (α2,3-SiaT) are the key enzymes for 3'-SL synthesis. CSS
catalyzes the formation
of CMP-Neu5Ac, and free N-acetylneuraminic acid (Neu5Ac) combines with CTP (cytidine
triphosphate) under
the action of CSS to form CMP-Neu5Ac. As an activated form of sialic acid, CMP-Neu5Ac is
the donor of
subsequent sialylation reaction. α2,3-SiaT is responsible for transferring Neu5Ac in
CMP-Neu5Ac to
galactose residue of acceptor lactose to form 3'-SL.
We
introduced these two
enzymes into *E.coli*, enabling *E.coli* to synthesize 3'-SL.

![](https://static.igem.wiki/teams/5741/images/engi-12.webp)

**Figure
11****The plasmid map of pET-CSS-SaiT**

**Build:**

First of
all,
we obtained DNA
fragments of different promoters by PCR and connected them with plasmid vectors by
homologous
recombination. After obtaining the complete plasmid, we transformed the plasmid into
*E.coli*,
and carried out preliminary screening by antibiotics (Figure 12A). Subsequently, we
selected monoclonal
colonies from the plate and verified them by PCR (Figure 12B). After the PCR
verification was
successful, we further confirmed that the strain construction was successful through DNA
sequencing
(Figure 12C).

![](https://static.igem.wiki/teams/5741/images/engi-13.webp)

**Figure
12.
Construction and
verification of 3'-SL synthesis module. A. Antibiotic screening plate.
B.** **Agarose gel
electrophoresis
verification. C.**
**The
results of**
**DNA
sequencing
verification.**

**Test:**

**SDS-PAGE was used
to
verify the protein expression level**

After the
successful
construction of the strain, we also verified the protein expression level of the strain
by SDS-PAGE.
Through the experimental results **(**Figure 13), we can find that two proteins can
be expressed
normally under the induction of IPTG and galactose.

![](https://static.igem.wiki/teams/5741/images/engi-14.webp)

**Figure
13. The
result of SDS-PAGE.**

# **Construction and verification of three plasmid co-transformed strains**

## **Build:**

After all
the
three modules
are constructed, we need to co-transform the plasmids of the three modules into the same
bacteria to
make the bacteria have complete functions. After the three plasmids were transformed
into bacteria, we
also made a preliminary screening through antibiotics to obtain the strains that could
be successfully
constructed (Figure 14A). Then we further verified it by colony PCR (Figure 14B) and DNA
sequencing
(Figure 14C), and the experimental results showed that our co-transformed strain was
constructed
correctly.

![](https://static.igem.wiki/teams/5741/images/engi-15.webp)

**Figure
14.
Construction and verification of three plasmid co-transformed strains. A.
Antibiotic screening
plate. B.** **Agarose gel electrophoresis
verification.
C.** **The
results
of** **DNA sequencing
verification.**

## **Test:**

After
transferring the plasmid
of three modules, we first want to determine whether the co-transformed bacteria can
survive normally in
soy whey. Therefore, we cultured the co-transformed bacteria in LB and soy whey
respectively and
determined the growth curve. Through the determination of growth curve, we found that
bacteria can grow
normally in Soy whey after plasmid co-transformation, and the growth ability is
completely consistent
with that of bacteria cultured in LB medium. Next, we need to determine the ability of
the strain to
produce 3'-SL in soy whey.

![](https://static.igem.wiki/teams/5741/images/engi-16.webp)

**Figure
15.****Determination
of strain
growth curve after co-transformation****.**

After
confirming the correct
construction of the strain, we need to measure the yield of 3'-SL in the next
step. After
streaking and
activating the fermentation strain, a single colony was picked and inoculated into 5 mL
of LB liquid
medium (with 5 μL Chl and Amp) and then cultured at 37°C and 200 r/min for 8-12 hours.
The culture was
transferred to 100 mL of soy whey at an initial OD600 of 0.1 and cultured at
37°C and 200
r/min for approximately 8 hours. The culture was centrifuged at 4°C and 5000 rpm for 10
minutes. The
bacterial cells were resuspended in 24 mL of Tris-HCl buffer (50 mmol/L, pH 7.0,
containing 250 mg
Neu5Ac or 500 mg Neu5AC). A substrate solution with a concentration of 10 g/L (1 mL of
250 g/L lactose)
was added to start the reaction.
The
results of HPLC showed
that when adding 250 mg/L of Neu5Ac, *E.coli* could produce more than 80 mg/L of
3'-SL (Figure
16A) in 16 hours, and the conversion rate of 3'-SL was over 30% (Figure 16B). When
adding 500 mg/L of
Neu5Ac, *E.coli* can produce more than 80mg/L of 3'-SL (Figure 16C) in 24 hours,
and the
conversion rate of 3'-SL is over 40% (Figure 16D).

![](https://static.igem.wiki/teams/5741/images/engi-17.webp)

**Figure
16.
Determination of synthetic yield of 3'-SL by HPLC.**

**A.3'-SL
yield
(Neu5AC 250 mg) B. 3'-SL conversion rate (Neu5AC 250 mg)**

**C.3'-SL
yield
(Neu5AC 500 mg) D. 3'-SL conversion rate (Neu5AC 500 mg)**

## **Learn:**

In
conclusion, our experiment can not only synthesize high-value 3'-SL but also control the
water pollution
caused by soy whey, which is of great significance in protecting the ecological
environment. There are
also some areas that need to be optimized in our experiment. At present, the conversion
rate of 3'-SL is
about 40%. We can further optimize the promoter or other biological elements in the
future, so as to
further improve the conversion rate and increase the protein yield. Secondly, we can
apply the
construction strain to other products. By replacing the 3'-SL synthesis module, we can
realize the
efficient production of other high value-added products. Finally, if our products need
to be put into
industrial production in the future, we need to optimize the fermentation process, and
further research
is needed to maintain the stability of strains and ensure the stable supply and quality
consistency of
raw materials of soy whey. And we also need to optimize the product separation and
purification process
to develop an efficient and low-cost product separation and purification process
suitable for industrial
production.

# **References**

---

[1] PALUR D
S, PRESSLEY S R,
ATSUMI S 2023. Microbial production of human milk oligosaccharides. Molecules
[J], 28.

[2] SCHELCH S, ZHONG C,
PETSCHACHER B, et al.
2020. Bacterial sialyltransferases and their use in biocatalytic cascades for
sialo-oligosaccharide
production. Biotechnology Advances [J], 44: 107613.

{% endcall %} {% endblock %}

{% extends "layout.html" %} {% block title %}Experiments{% endblock %} {% block
lead %}Describe the research, experiments, and protocols you used in your
project. It is designed to provide sufficient information for other teams to
replicate our work.{% endblock %} {% block page\_content %} {% from
"/components/article-content.html" import articleContent %} {% call
articleContent("EXPERIMENTS", [
{"content": "Preparation of culture medium","anchor": "#title\_1"},
{"content": "Construction and verification of carbohydrate utilization module","anchor": "#title\_2"},
{"content": "Construction and verification of galactose self-induction module","anchor": "#title\_3"},
{"content": "Construction and verification of 3'-SL synthesis module","anchor": "#title\_4"},
{"content": "Construction and verification of three plasmid co-transformed strains","anchor": "#title\_5"}]) %}

# **1.Preparation of culture medium**

**Goal:** Preparation of
bacterial growth
and culture environment

**Materials:**

**·**Yeast
extract

·NaCl

·Agar
2%

·Gloves

·Lab
coat

·Bacteria culture
tube

   ·Graduated cylinder
and
beaker

·ddH2O

·Tryptone

**Procedure:**

**LB
Medium:** Weigh 2.5 g tryptone, 1.25 g yeast extract, 2.5 g NaCl. Add distilled water
to 250 mL.
Sterilize at 121°C for 30 min.

For
solid medium, add 2% agar powder before sterilization. Sterilize at 121°C for 30 min

# **2.Construction** **and verification of carbohydrate utilization module**

![](https://static.igem.wiki/teams/5741/images/expirements1.webp)

Figure
1
Schematic diagram
of
plasmid construction of module 1

**2.1.
Construction
of
pCOLAtrc(lost O)-scac-aga**

**Goal:** Construct a module,
which can make
bacteria survive in yellow serous water after introduction.

**A.**
Amplification of
target gene band
by PCR

**Materials:**

·Pipette and
tips

·PCR
tube

·EP
tube

·ddwater

·Wash
Buffer

·Forward
Primer

·Reverse
Primer

·Enzymes

·Base
pairs (ATCG）

·PCR
Mix

·Template
plasmid

·Absorption
column

·Collection
tube

**Procedures:**

  a. Put 25 μL 2\*PCR mix
into each
PCR tubes, then add 22 μL ddwater. Then add 1 μL Forward Primer and 1 μL Reverse Primer into the
tubes.
After that, add 1 μL template plasmid into each PCR tubes.

  b. Put those total 14
PCR tubes
load into thermal cycle and start PCR.

The
PCR cycle starts with 95°C 3 minutes

Then
move as 95°C for 30 seconds, 55°C for 30 seconds and 72°C for 1.5 minutes. This cycle repeats 33
times.

Finally, 72°C for
10min.

**B.**
Agarose gel
electrophoresis
verification

**Goal:** Verify whether the PCR
product is
correct**.**

**Materials:**

Agarose

TAE
Buffer

Nucleic acid
dye

DNA
Loading Buffer

DNA
marker

Gel
preparation

**Procedures:**

Mix
agarose and ATE buffer together, with a ratio of 1：100

Sol
by heat and dissolve in the microwave oven and add nucleic acid dye when it cools down to
50-60℃

Pour
into the mold to cooling and solidification

Place 8μl PCR the
mold
cavities
and then electrophoresis 180V for 20 minutes

Finally put the
gel in
to the Gel
imager，ultraviolet
irradiation to observe
DNA that has reached a specific length

**C.**
Column-based DNA gel
Purification.

**Goal:**
Separation and
purification of
target gene.

**Materials:**

Gel
Extraction Kit

**Procedures:**

Cut
out the gel block containing the target fragment from the agarose gel and weigh it.

Add
Buffer B2 into the gel block and metal bath55℃

Transfer sol
solution
into the
adsorption column and 8000Xg centrifugation for 1 minute

Add
Wash Buffer and 9000Xg centrifugation for 1 minute, pour the solution in the tube.

Repeat the
previous
step

Let
empty adsorption column in the centrifuge for 1 minute

Put
adsorption column into a clean 1.5mL centrifuge tube and add 30μl Elution Buffer into the center
of the
adsorption film. centrifugation for 1 minute.

Finally preserve
DNA
solution

 **D.** DNA
homologous
recombination

**Goal:**
Plasmids were obtained
by
homologous recombination.

**Materials**

2×ClonExpress Mix
15μl

Aga
1μl

Sacc
1μl

PCR
tube

EP
tube

ddH2O

**Procedure:**

(1)   Prepare
solutions.

Mix
5μl 2×ClonExpress Mix, 1μl aga, 1μl sall, 3μl pom together.

(2)
Heat at 50 ℃ for 15 minutes. Then ice bath for 4 minutes.

 **E.**Plasmid
transformation.

**Goal:**
Transform plasmid into
competent
***E. coli*** cells.

**Materials**:

Competent *E. coli*
cells（*E.coli*
DH5a/BL21）

LB culture medium

Peptone, yeast extract, NaCl
(for
making LB agar)

Kanamycin

Petri dishes

Sterile cell spreader

Transformed bacterial colony
plates

DH5α competent Escherichia
coli
(for transformation)

LB liquid medium containing
appropriate antibiotics

Shaking incubator

**Procedure:**

Preparation for
antibiotic plates
(LB+1:1000 Kanamycin)

Thaw
competent *E. coli* cells on the ice.

Add
10μL recombinant product (plasmid) into the competent cells, gently flick vessel wall, gently
mix it by
using the pipette tip. Incubate on ice for 30 minutes, stationary.

42℃
heat shocks for 30s in order to create channels and pores in the cell membrane that connect the
cell
interior to the external environment.

Remove
immediately, incubate on ice for 2 minutes to reduce the temperature.

 Add 900μL LB culture
medium.
Incubate at 37℃ with shaking for one hour.

Centrifuge and
discard
800μL of
the supernatant.

Separate the
thallus
in the
remaining 200μL of LB medium.

Evenly
spread the sample on antibiotic plates by using a sterile cell spreader.

 **F.**Colony
PCR.

**Goal:** To
verify
whether the
strain has been successfully constructed through colony PCR.

**Materials**:

Polymerase chain reaction
primers

Polymerase chain reaction
buffer
(to maintain pH and provide an ionic environment)

DNA polymerase (e.g., Taq
polymerase)

Sterile distilled
water

Microcentrifuge tubes

1% agarose

0.5× or 1× TAE buffer

DNA loading dye (1:5
ratio)

DNA marker (molecular weight
standard)

DNA dye (e.g., YeaRed,
1:10,000
dilution)

Gel tray and comb

Electrophoresis tank and
power
supply

Gel imager

**Procedure:**

 Bacterial Colony
Selection and
Preliminary Culture

-Configuration of
the
PCR system
（5 µL mix，water,0.1 µL primer F
and
R,template）

-Set
PCR procedure.

-Select individual
colonies for
colony PCR.

-Prepare the
bacterial
solution.

-Pour
1 µL of the bacterial solution into a 10 µL PCR tubes.

**2.2.SDS-PAGE was
used to verify
the protein expression level**

**Goal:** Induce the production
of target
protein and verify it.

**A.** Induced expression of
protein. Verify the
expression of protein.

**Materials**:

IPTG

Galactose

30%
Acr-Bis

Lower
gel buffer

Upper
gel buffer

10%
Gel polymerization catalyst

TEMED
Substitute

Tris-SDS-
Glycine

**Procedure:**

-Select the correct
colonies and
culture them to an OD of 0.5.

Prepare 4 conical
flasks.

Add 100ml of LB culture
medium in
2 flasks and 100ml of soywhey culture medium in 2 flasks.

Add IPTG(1mM) in 1 flask of
LB
culture and 1 flask of soywhey culture.

Add galactose(1mM)another
flask
of LB culture and another flask of soywhey culture.

Place it in the shaking
incubator
maintain 25°C, shake for 20 hours.

Collect the strains
cultured last
night, get 50ml from each and pour them into a tube.

Through a process
of
centrifugation and pouring out the supernatant, obtain a sediment of *E.coli*.

Add
4ml of lysis buffer and 40 μL of lysozyme.Place the tube within ice
or keep it
under a low temperature 30 min.

Due to
an ultrasonication to the bacteria formula and once again.

Place
it in the centrifuge and preserve the supernatant to extract specifically the protein.

Then
with pipette transfer the Nickel column in to the tube with the protein.

**Purify the
protein
mixture:**

For
each tube of formula, prepare 8 of 2ml tubes, a 10ml tubes and a gravity flow column,
respectively note the
eight 2ml tubes as w1 w2 w3 w4 e1 e2 e3 e4, and it should be noted with the type of formula
dealing with the
bacteria a day before.

While
the volve remain closed, transfer the formula into the gravity flow column.

Remaining under a
low
temperature,
open the volve and collect them in a 10ml tube.

While
the liquid part is totally leaked within, pick up the gravity flow column, place it respectively
on the
related first four 2ml tubes (w1-4). Wash 4 times with washing buffer.

Wash
the same
column four more times using the elution buffer, and collect the filtered liquid in four other
EP
tubes.

**Verification**

Into a
PCR tube, add 20μl protein liquid

Load
the sample in the protein gel.

Run
electrophoresis: run at 80V for 20 minutes, then switch to 180V and run for an additional 40
minutes.

Observed by
Coomassie
brilliant
blue staining.

**2.3.
Determination
of strain
growth curve after introducing carbohydrate utilization module**

**Goal:**
Verify that the
carbohydrate
utilization module is useful.

**Materials**:

LB
medium

Soy
whey (Contains sucrose (11.73 g/L), stachyose (4.25 g/L), raffinose (2.67 g/L), galactose (0.42
g/L),
glucose (0.36 g/L), fructose (0.31 g/L))

*E.coli* BL21

S1 strain (*Escherichia
coli* introduced with carbohydrate utilization module)

**Procedure:**

Wild-type strain
and
S1 strain
were cultured in LB medium for 24h.

The
cultured bacteria were collected by centrifugation, and the supernatant was discarded.

Use
sterile water to resuspend the strain, determine the OD600, and position the final
concentration
of the strain as OD600=0.2.

The
two strains were cultured in LB and soy whey media respectively, and OD600 was determined after
24
hours.

# **3.Construction** **and verification of galactose self-induction module**

**3.1.
Construction
of
pCOM4-PgalP-a-EGFP, pCOM4-Pgal** **P-b-EGFP,
pCOM4-PgalP-c-EGFP**

![](https://static.igem.wiki/teams/5741/images/expirements2.webp)

Figure 2 Schematic
diagram of
plasmid construction of module 2

**Goal:** Different promoters
were
constructed for subsequent screening to obtain the promoter with the strongest protein induction
ability.

**A.**
Amplification of target
gene band by PCR

**Materials**: Basic materials are
consistent
with 2.1-A. Using specific primers
and DNA
templates

**Procedure:** The experimental
scheme is
consistent with 2.1-A.

**B.** Agarose
gel
electrophoresis verification

**Materials**: Basic materials are
consistent
with 2.1-B

**Procedure:**
The
experimental
scheme is consistent with 2.1-B.

**C.**
Column-based
DNA gel
Purification.

**Materials**: Basic materials are
consistent
with 2.1-C.

**Procedure:**
The
experimental
scheme is consistent with 2.1-C.

**D.**DNA homologous recombination

**Materials**: Basic materials are
consistent
with 2.1-D.

**Procedure:**
The
experimental
scheme is consistent with 2.1-D.

**E.**Plasmid transformation.

**Materials**: Basic materials are
consistent
with 2.1-E.

**Procedure:**
The
experimental
scheme is consistent with 2.1-E.

**F.**Colony PCR.

**Materials**: Basic materials are
consistent
with 2.1-F.

**Procedure:**
The
experimental
scheme is consistent with 2.1-F.

**3.2. SDS-PAGE
was
used to
verify the protein expression level**

**Goal:** Induce the production
of target
protein and verify it.

**A.** Induced expression of
protein. Verify the
expression of protein.

**Materials**: Basic materials are
consistent
with 2.2-A. Using specific primers
and DNA
templates.

**Procedure:** The experimental
scheme is
consistent with 2.2-A.

**3.3. The
promoter
with the
strongest induction ability was screened by fluorescence intensity**

**Goal:** The most suitable
promoter was
obtained by fluorescence intensity screening.

**Materials**:

Strain
containing PgalP-a-EGFP

Strain
containing PgalP-b-EGFP

Strain
containing PgalP-c-EGFP

IPTG

Galactose

LB
medium

**Procedure:**

PgalP-a-EGFP,
PgalP-b-EGFP,
PgalP-c-EGFP strain were cultured in LB medium for 24h.

The
cultured bacteria were collected by centrifugation, and the supernatant was discarded.

Use
sterile water to resuspend the strain, determine the OD600, and position the final
concentration
of the strain as OD600=0.2.

The
fluorescence intensity of the strain was measured at 0, 2, 4, 24, 36 and 48 hours after culture.
(The
excitation wavelength of GFP is 488 nm and the emission wavelength is 507 nm.)

**3.4.
Construction
of
pCOM4-PgalP-GalP**

**Goal:** After screening the
best promoter,
EGFP gene was replaced by galactose transporter *GALP*.

**A.**
Amplification of target
gene band by PCR

**Materials**: Basic materials are
consistent
with 2.1-A. Using specific primers
and DNA
templates

**Procedure:** The experimental
scheme is
consistent with 2.1-A.

**B.** Agarose
gel
electrophoresis verification

**Materials**: Basic materials are
consistent
with 2.1-B

**Procedure:**
The
experimental
scheme is consistent with 2.1-B.

**C.**
Column-based
DNA gel
Purification.

**Materials**: Basic materials are
consistent
with 2.1-C.

**Procedure:**
The
experimental
scheme is consistent with 2.1-C.

**D.**DNA homologous recombination

**Materials**: Basic materials are
consistent
with 2.1-D.

**Procedure:**
The
experimental
scheme is consistent with 2.1-D.

**E.**Plasmid transformation.

**Materials**: Basic materials are
consistent
with 2.1-E.

**Procedure:**
The
experimental
scheme is consistent with 2.1-E.

**F.**Colony PCR.

**Materials**: Basic materials are
consistent
with 2.1-F.

**Procedure:** The
experimental scheme is consistent with 2.1-F.

**3.5. SDS-PAGE
was
used to
verify the protein expression level**

**Goal:** Induce the production
of target
protein and verify it.

**A.** Induced expression of
protein. Verify the
expression of protein.

**Materials**: Basic materials are
consistent
with 2.2-A. Using specific primers
and DNA
templates.

**Procedure:** The experimental
scheme is
consistent with 2.2-A.

# **4.Construction** **and verification of 3'-SL synthesis module**

![](https://static.igem.wiki/teams/5741/images/expirements3.webp)

Figure 3 Schematic
diagram of
plasmid construction of module 3

**4.1.
Construction
of
pET-CSS-SaiT**

**Goal:** Constructing the
Synthesis Module
of 3'-SL.

**A.**
Amplification of target
gene band by PCR

**Materials**: Basic materials are
consistent
with 2.1-A. Using specific primers
and DNA
templates

**Procedure:** The experimental
scheme is
consistent with 2.1-A.

**B.** Agarose
gel
electrophoresis verification

**Materials**: Basic materials are
consistent
with 2.1-B

**Procedure:**
The
experimental
scheme is consistent with 2.1-B.

**C.**
Column-based
DNA gel
Purification.

**Materials**: Basic materials are
consistent
with 2.1-C.

**Procedure:**
The
experimental
scheme is consistent with 2.1-C.

**D.**DNA homologous recombination

**Materials**: Basic materials are
consistent
with 2.1-D.

**Procedure:**
The
experimental
scheme is consistent with 2.1-D.

**E.**Plasmid transformation.

**Materials**: Basic materials are
consistent
with 2.1-E.

**Procedure:**
The
experimental
scheme is consistent with 2.1-E.

**F.**Colony PCR.

**Materials**: Basic materials are
consistent
with 2.1-F.

**Procedure:** The
experimental scheme is consistent with 2.1-F.

**4.2. SDS-PAGE
was
used to
verify the protein expression level**

**Goal:** Induce the production
of target
protein and verify it.

**A.** Induced expression of
protein. Verify the
expression of protein.

**Materials**: Basic materials are
consistent
with 2.2-A. Using specific primers
and DNA
templates.

**Procedure:** The experimental
scheme is
consistent with 2.2-A.

# **5. Construction and verification of three plasmid co-transformed strains**

![](https://static.igem.wiki/teams/5741/images/expirements4.webp)

Figure 4 Schematic
diagram of
 Plasmid
co-transformation and
verification

**5.1.
The Construction
of Co-transformed Plasmids**

**Goal:** Three plasmids
responsible for
different modules were transformed into a bacterium together, which enabled the bacterium to
grow in soy
whey and spontaneously induce the synthesis of 3'-SL.

**A.** Construction of
Co-transformed
Strain

**Materials**:

Three
modules were successfully constructed,
other materials
are
consistent with 2.1-E.

**Procedure:**
The
experimental
scheme is consistent with 2.1-E.

**B.**Colony PCR.

**Materials**: Basic materials are
consistent
with 2.1-F.

**Procedure:**
The
experimental
scheme is consistent with 2.1-F.

**5.2. The
synthetic
amount of
3'-SL was determined by HPLC**

**Goal:** The yield of 3'-SL was
determined
by HPLC.

**Materials**:

Co-transformed
strain

LB
liquid medium

Chloromycetin

Tris-HCl
buffer

Neu5Ac

Lactose

High
performance liquid chromatograph and related reagents

**Procedure:**

After
streaking and activating the fermentation strain, a single colony was picked and inoculated into
5 mL of LB
liquid medium (with 5 μL ChI and Amp), and then cultured at 37°C and 200 r/min for 8-12 hours.
The culture
was transferred to 100 mL of Soybean Yellow Pulp Water at an initial OD600 of 0.1 and cultured
at 37°C and
200 r/min for approximately 8 hours. The culture was centrifuged at 4°C and 5000 rpm for 10
minutes. The
bacterial cells were resuspended in 24 mL of Tris-HCl buffer (50 mmol/L, pH 7.0, containing 250
mg Neu5Ac).
A substrate solution with a concentration of 10 g/L (1 mL of 250 g/L lactose) was added to start
the
reaction.

HPLC
analysis method for the product: differential refractive index detector, detector temperature
35°C; Rezex
ROA-Organic Acid H+ (8%) chromatographic column, column temperature 60°C; mobile phase: 5 mmol/L
H2SO4
solution, flow rate 0.6 mL/min; injection volume 10 μL.

{% endcall %} {% 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 %} {% from
"/components/article-content.html" import articleContent %} {% call
articleContent("RESULTS", [
{"content": "Overview","anchor": "#title\_1"},
{"content": "Construction and verification of carbohydrate utilization module","anchor": "#title\_2"},
{"content": "Construction and verification of galactose self-induction module","anchor": "#title\_3"},
{"content": "Construction and verification of 3'-SL synthesis module","anchor": "#title\_4"},
{"content": "Construction and verification of three plasmid co-transformed strains","anchor": "#title\_5"},
{"content": "Future plans","anchor": "#title\_6"},
{"content": "References","anchor": "#title\_7"}
]) %}

# **Overview**

Breast
milk oligosaccharides (HMO) are very important to the health of infants, among which 3'-
sialyllactose
(3'-SL) has attracted much attention because of its various benefits in promoting brain
development and
enhancing immunity. With the global demand for high-quality infant formula increasing, the
market demand for
3'-SL is expected to rise sharply.Breast milk oligosaccharide (HMO) is the third richest solid
component in
breast milk after lactose and lipid in mature breast milk, and its content is about 5 to 15
g/L[1].

In
recent years, it has been widely concerned because of its impact on human health and its
application in
high-quality infant formulas. At present, more than 200 HMOs with different structures have been
identified,
including fucosylation, non-fucosylation and sialylation[2]. Among them, sialylated HMO
accounts for 12-14%, and 3'- sialyllactose (3'-SL) is its representative monomer. Its structure
is
Neu5Ac-α2,3Gal-β1,4Glc, which is composed of N- acetylneuraminic acid and lactose
units[3].

Studies have shown
that 3'-SL has
prebiotic activity, which can play a role by inhibiting pathogen adhesion, preventing
necrotizing
enterocolitis, enhancing immune function and promoting brain development[4]. The European Union has approved
3'-SL sodium salt as a new resource of infant formula (EU 2023/1582), and it has been added to
Nestle BEBA
formula at a dosage of 1-5 mg/100 mL[1]. The global HMO market has
grown rapidly from $200 million in 2022 and is expected to reach $780 million in 2028 [4]. As
the
main component of HMO, the application demand of 3'-SL will continue to be released with market
expansion.

In
view of its diversified and valuable applications, the demand for 3'-SL will continue to
increase. However,
the traditional extraction and chemical synthesis methods are inefficient and cannot meet the
rapidly
growing market demand. Therefore, sustainable biotechnology methods have attracted more and more
research
attention.

It is
worth noting that fermentation medium may account for 30% of the total production cost of
commercial
chemicals, which urges people to find economic alternatives[5]. soy whey is a by-product of
soybean food production process, which produces as much as 70 million tons in China every
year[6].

However, more than
80% of
carbohydrates, 20% of protein and 20% of oil in soybeans will be lost to soy whey, resulting in
soy whey
containing nearly 1.5% of carbohydrate, 0.1-0.8% of protein and about 0.4% of trace
elements[7]. Therefore, soy whey is a
nutrient-rich culture condition, which is likely to be used as a culture medium for our
biosynthesis.
However, most of the soy whey is
discharged into sewage, which not only causes serious environmental pollution, but also leads to
the waste
of its nutrients.

Our
research is based on the above background. First, using soy whey as the culture medium for our
biosynthesis
can not only save costs, but also control water pollution. Second, we synthesized 3'-SL with
high value by
microorganisms on the premise of using soy whey as culture medium, which has high economic
benefits and
market value.

![](https://static.igem.wiki/teams/5741/images/result1.webp)

Figure
1
Schematic diagram of
experimental design

# **1.Construction and verification of carbohydrate utilization module**

**1.1 Construction
of pCOLAtrc(lost O)-sacc-aga**

At
present, most of the *E.coli* can't use carbohydrates (such as Sucrose, stachyose and
raffinose) in
soy whey as carbon source, which makes it difficult to use this rich food industry by-product as
fermentation medium. In order to enable *E.coli* to make normal use of these nutrients,
we introduced
*AGA* and *SACC* into *E.coli*. AGA is α-galactosidase and SACC is
extracellular
sucrase. *AGA* and *SACC* can convert carbon sources that *E.coli* can't
use into
available carbon sources (such as glucose, fructose and galactose). After introducing them into
bacteria, we
initially obtained the strains that could be successfully constructed by antibiotic screening
and further
verified them by DNA sequencing, and the sequencing results showed that the construction of our
strains was
successful.

![](https://static.igem.wiki/teams/5741/images/result2.webp)

**Figure 2.
Constructing carbohydrate utilization module.**

**1.2
SDS-PAGE was used to verify the protein expression level**

After
confirming the success of the strain construction, we want to further verify whether these two
genes can
express the protein normally. We have carried out experimental tests in soy whey medium. The
experimental
group used IPTG to induce protein expression. The experimental results showed that the strain
could grow
normally and express Aga and Sacc proteins under the induction of IPTG in soy whey medium. After
introducing
this module, the strain can grow normally in soy whey and express protein normally under the
induction of
IPTG. Since the self-induction module of galactose has not been constructed, the protein
expression was less
when galactose is used as inducer. Therefore, the next step is to construct a self-induction
module of
galactosein bacteria.

![](https://static.igem.wiki/teams/5741/images/3result.webp)

**Figure 3
SDS-PAGE of
carbohydrate utilization module**

**1.3.
Determination of strain
growth after introducing carbohydrate utilization module**

After
confirming that the protein can be successfully induced to express, we want to know whether
these two
proteins can function normally. That is, whether *Escherichia coli* can grow normally in
soy whey
after Aga and Sacc are introduced. Therefore, We tested the growth of strains with carbohydrate
utilization
module and without carbohydrate utilization module in soy whey. The experimental results show
that the
strain can grow normally in soy whey after introducing protein, but the growth of the strain
without
introducing protein is obviously inhibited. This result shows that the strain can grow normally in
soy whey after containing carbohydrate utilization module.

![](https://static.igem.wiki/teams/5741/images/result4.webp)

**Figure 4.
Growth
ability of strains under different culture conditions.**

# **2. Construction and verification of galactose self-induction module**

**2.1.
Construction of
pCOM4-PgalP-a-EGFP, pCOM4-PgalP-b-EGFP, pCOM4-PgalP-c-EGFP**

After
constructing the carbohydrate utilization module, we have solved the normal growth problem of
bacteria in
soy whey. Next, we want bacteria to express the protein we need. At present, our bacteria need
to use
exogenous IPTG for protein induction, IPTG has potential cytotoxicity and high cost, so we
wonder if we can
build a module. After the module is introduced, bacteria can spontaneously induce protein
expression by
using galactose in soy whey. On the one hand, this can reduce the cost, on the other hand, it
can avoid
introducing chemical components that may have side effects. Galactose can not only be used as a
nutrient for
normal cell growth, but also as an inducer to induce protein expression, so how to induce
protein expression
as much as possible on the basis of maintaining bacterial growth is extremely
critical.
In order to improve the induced
yield of protein with galactose as inducer, we constructed three promoters regulated by
galactose, and
constructed EGFP fluorescent protein downstream of the promoters. By observing the expression
intensity of
fluorescent protein, we can quickly obtain the induced yield of protein. Through screening, we
can find the
promoter with the strongest inducing ability when galactose is used as a protein
inducer.
Through antibiotic screening
(Figure 5A), colony PCR
verification(Figure
5B), and DNA
sequencing(Figure
5C), we confirmed that the
construction of the strain was successful.

![](https://static.igem.wiki/teams/5741/images/result5.webp)

**Figure 5.
Construction and Verification of Promoter Screening Module.**

**A. Antibiotic
screening plate. B.** **Agarose gel electrophoresis
verification.
C.**
**The
results
of**
**DNA sequencing
verification.**

**2.2. SDS-PAGE was used to
verify the protein expression level**

After
confirming the success of the strain construction, we want to further verify whether these genes
can express
the protein normally.Three strains were cultured in LB medium, and IPTG and galactose
were used as
inducers to induce protein expression, and the protein expression level was verified by
SDS-PAGE. Through
the experimental results, we can find that EGFP protein can be expressed normally under the
culture
conditions with IPTG and galactose as inducers. Furthermore, we want to determine the expression
level of
EGFP, so as to screen the promoter with the highest induced expression intensity.

![](https://static.igem.wiki/teams/5741/images/result-6.webp)

**Figure 6
SDS-PAGE
of****galactose
self-induction module.**

**2.3. The promoter with the
strongest induction ability was screened by fluorescence intensity.**

By
measuring the fluorescence intensity, we quickly measured the induction intensity of the
promoter under the
condition of galactose induction. Through the experimental results, we can find that the
fluorescence
intensity of the blank control group and the control group has hardly changed with the increase
of time.
However, with the increase of time, the fluorescence intensity of EGFP increased with different
strains
containing different promoters. Among them, pGalp-a promoter has the strongest ability to induce
protein
expression, so we chose pGalp-a promoter for the follow-up experiment.

![](https://static.igem.wiki/teams/5741/images/result7.webp)

**Figure 7.
Fluorescence intensity determination.**

**2.4. Construction of
pCOM4-PgalP-GalP**

After
selecting the most suitable promoter, we further want to replace EGFP gene with galactose
transporter. After
adding galactose inducer, the promoter is regulated by galactose, which strongly induces the
expression of
galactose transporter downstream. Galactose transporter transports galactose to 3'-SL module and
activates
the synthesis of 3'-SL. Therefore, we first replaced EGFP gene with galactose transporter
*GALP* by
molecular biology. Through antibiotic screening (Figure 8A), colony PCR verification (Figure
8B), and DNA
sequencing(Figure 8C), we confirmed that the plasmid construction was correct.

![](https://static.igem.wiki/teams/5741/images/result8.webp)

**Figure 8.
Construction of pCOM4-PgalP-GalP. A. Antibiotic screening plate.**

**B.**
**Agarose gel electrophoresis
verification. C.**
**The
results
of**
**DNA sequencing
verification.**

**2.5. SDS-PAGE was used to
verify the protein expression level**

After
the successful construction of the strain, we also verified the protein expression level of the
strain by
SDS-PAGE. Through the experimental results **(**Figure 9), we can find that Galp protein
can be expressed normally under the induction of IPTG and galactose.

![](https://static.igem.wiki/teams/5741/images/result-9.webp)

**Figure 9. The
result of SDS-PAGE.**

# **3.Construction** **and verification of 3'-SL synthesis module**

**3.1. Construction of
pET-CSS-SaiT**

After
confirming that bacteria can survive in soy whey and can spontaneously induce protein production
by using
nutrients in soy whey, the next step is to obtain the synthesis module of 3'-SL.CMP-Neu5Ac
Synthase (CSS)
and α2,3-sialyltransferase (α2,3-SiaT) are the key enzymes for 3'-SL synthesis. CSS catalyzes
the formation
of CMP-Neu5Ac, and free N-acetylneuraminic acid (Neu5Ac) combines with CTP (cytidine
triphosphate) under the
action of CSS to form CMP-Neu5Ac. As an activated form of sialic acid, CMP-Neu5Ac is the donor
of subsequent
sialylation reaction. α2,3-SiaT is responsible for transferring Neu5Ac in CMP-Neu5Ac to
galactose residue of
acceptor lactose to form 3'-SL. We introduced these two enzymes
into *E.coli*, enabling *E.coli* to synthesize 3'-SL. First, we obtained the
strains that
could be successfully constructed by antibiotic screening(Figure 10A), and then we further
confirmed that
the construction of this strain was successful by colony PCR (Figure 10B) and DNA sequencing
(Figure
10C).

![](https://static.igem.wiki/teams/5741/images/result10.webp)

**Figure 10.
Construction and verification of 3'-SL synthesis module. A. Antibiotic screening plate.
B.** **Agarose gel
electrophoresis verification. C.** **The results
of**
**DNA sequencing
verification.**

**3.2. SDS-PAGE was used to
verify the protein expression level**

After
the successful construction of the strain, we also verified the protein expression level of the
strain by
SDS-PAGE. Through the experimental results **(**Figure 11), we can find that two proteins
can be expressed normally under the induction of IPTG and galactose.

![](https://static.igem.wiki/teams/5741/images/result-11.webp)

**Figure 11. The
result of SDS-PAGE.**

# **4. Construction and verification of three plasmid co-transformed strains**

**4.1. The Construction
of Co-transformed Plasmids**

After
all the three modules are constructed, we need to co-transform the plasmids of the three modules
into the
same bacteria to make the bacteria have complete functions. After the three plasmids were
transformed into
bacteria, we also made a preliminary screening through antibiotics to obtain the strains that
could be
successfully constructed (Figure 12A). Then we further verified it by colony PCR(Figure 12B) and
DNA
sequencing (Figure 12C), and the experimental results showed that our co-transformed strain was
constructed
correctly**.**

![](https://static.igem.wiki/teams/5741/images/result12.webp)

**Figure 12.
Construction and verification of three plasmid co-transformed strains. A. Antibiotic
screening plate.
B.** **Agarose gel
electrophoresis verification. C.** **The results
of**
**DNA sequencing
verification.**

**4.2. Determination of strain
growth curve after co-transformation**

After
transferring the plasmid of three modules, we first want to determine whether the co-transformed
bacteria
can survive normally in soy whey. Therefore, we cultured the co-transformed bacteria in LB and
soy whey
respectively and determined the growth curve. Through the determination of growth curve, we
found that
bacteria can grow normally in Soy whey after plasmid co-transformation, and the growth ability
is completely
consistent with that of bacteria cultured in LB medium. Next, we need to determine the
ability of the strain to produce 3'-SL in soy whey.

![](https://static.igem.wiki/teams/5741/images/result13.webp)

**Figure 13.****Determination
of strain growth curve after co-transformation****.**

**4.3. The synthetic amount of
3'-SL was determined by HPLC**

After
confirming that the strain can survive normally in soy whey, we need to measure the yield of
3'-SL in the
next step.
After streaking and activating
the fermentation strain, a single colony was picked and inoculated into 5 mL of LB liquid medium
(with 5 μL
Chl and Amp) and then cultured at 37°C and 200 r/min for 8-12 hours. The culture was transferred
to 100 mL
of soy whey at an initial OD600 of 0.1 and cultured at 37°C and 200 r/min for
approximately 8
hours. The culture was centrifuged at 4°C and 5000 rpm for 10 minutes. The bacterial cells were
resuspended
in 24 mL of Tris-HCl buffer (50 mmol/L, pH 7.0, containing 250 mg Neu5Ac or 500 mg Neu5AC). A
substrate
solution with a concentration of 10 g/L (1 mL of 250 g/L lactose) was added to start the
reaction.
The results of HPLC
showed that when adding 250 mg/L of Neu5Ac, *E.coli* could produce more than 80 mg/L of
3'-SL (Figure 14A) in 16 hours, and the conversion rate of 3'-SL was over 30% (Figure 14). When
adding 500
mg/L of Neu5Ac, *E.coli* can produce more than 80mg/L of 3'-SL (Figure 14C) in 24 hours,
and the
conversion rate of 3'-SL is over 40% (Figure 14D).

![](https://static.igem.wiki/teams/5741/images/111.webp)

**Figure 14.
Determination of synthetic yield of 3'-SL by HPLC.**

**A.3'-SL yield
(Neu5AC 250 mg) B. 3'-SL conversion rate (Neu5AC 250 mg)**

**C.3'-SL yield
(Neu5AC 500 mg) D. 3'-SL conversion rate (Neu5AC 500 mg)**

# **5. Future plans**

Based
on our experimental results, we constructed a strain, which can survive in soy whey and can
spontaneously
induce and express 3'-SL protein by using soy whey. First of all, our experiment broke through
the
bottleneck that *E. coli* has a defect in the utilization of carbon source in soy whey,
which enables
us to cultivate *E. coli* with cheap soy whey. Secondly, we constructed an
IPTG-independent
self-induced expression system to solve the toxicity and cost problems caused by using chemical
inducers in
protein induction. Finally, our experiment can not only synthesize high-value 3'-SL but also
control the
water pollution caused by yellow serous water, which is of great significance in protecting the
ecological
environment.

There
are also some areas that need to be optimized in our experiment. At present, the conversion rate
of 3'-SL is
about 40%. We can further optimize the promoter or other biological elements in the future, so
as to further
improve the conversion rate and increase the protein yield. Secondly, we can apply the
constructed strain to
other products. By replacing the 3'-SL synthesis module, we can realize the efficient production
of other
high value-added products. Finally, if our products need to be put into industrial production in
the future,
we need to optimize the fermentation process, and further research is needed to maintain the
stability of
strains and ensure the stable supply and quality consistency of raw materials of soy whey. And
we also need
to optimize the product separation and purification process to develop an efficient and low-cost
product
separation and purification process suitable for industrial production.

# **6. References**

---

[1] HU M, ZHANG T
2023.
Expectations for
employing *Escherichia
coli* BL21 (DE3) in the synthesis of human milk oligosaccharides. Journal of
agricultural and food
chemistry [J], 71: 6211-6212.

[2] LEE S Y,
STUCKEY D
C 2022. Separation and
biosynthesis of value-added compounds from food-processing wastewater: Towards sustainable
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[3] ZHANG J, ZHU Y,
ZHANG W, et al. 2022.
Efficient production of a functional human milk oligosaccharide 3′-sialyllactose in genetically
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[4] ZHU Y, ZHANG J,
ZHANG W, et al. 2023.
Recent progress on health effects and biosynthesis of two key sialylated human milk
oligosaccharides,
3′-sialyllactose and 6′-sialyllactose. Biotechnology advances [J], 62: 108058.

[5] WANG Z, DAI Y, AZI F, et al. 2024.
Engineering
Escherichia coli for cost-effective production of medium-chain fatty acids from soy whey using
an optimized
galactose-based autoinduction system. Bioresource technology [J], 393: 130145.

[6] LIANG J, XU N, NEDELE A-K, et al. 2023.
Upcycling
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of
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[7] LEE S Y,
STUCKEY D
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Towards
sustainable wastewater resource recovery. Journal of Cleaner Production [J], 357: 131975.

[8] WANG Z, DAI Y,
AZI
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Engineering Escherichia coli for cost-effective production of medium-chain fatty acids from soy
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130145

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