iGEM 2022 — Team zhejiang-united

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{% block title %}Notebook{% endblock %}
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![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-banner.jpg)
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Notebook for experiment

2022.8.10

Team Ice-breaking
Project discussion
Determination of team’s name and logo

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-notebook01.jpg)

2022.8.11

Safety training in laboratory
LB Culture Medium cultivation
YPD Culture Medium cultivation
PCR: lac-12 & wbgl, gmd & wcaG
Culture solution configuration

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-notebook02.jpg)

2022.8.12

Gel electrophoresis
Gel extraction
Plasmid extraction
Transformation

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-notebook03.jpg)

2022.8.13

Verification of bacteria colony by PCR
Gel electrophoresis

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-notebook04.jpg)

2022.8.14

Cultivation of Yeast competent cells
Enzyme digestion

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-notebook05.jpg)

2022.8.15

Reintroduce the aim of our experiment
YPD30L2 cultivation

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-notebook06.jpg)

2022.8.16-17

Verification of S. cerevisiae colony by PCR

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-notebook07.png)

2022.8.18-23

Curve of the growth of Saccharomyces cerevisiae
Test 2’-FL content

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-notebook08.jpg)

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
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![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-banner.jpg)
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Engineering

Introduction

Breast milk oligosaccharides, also known as human milk oligosaccharides (HMOs), are the third most abundant solid
component in breast milk after lactose and fat. Over 200 sugar compounds were identified in HMOs, among which
2′-Fucosyllactose (2’-FL) has the highest proportion in HMOs (more than 30%). Studies have proved that 2'-FL not
only promotes the development of the immune system and gut microbiota but also helps to prevent allergic disease and
promotes brain function and cognitive development.
At present, there are three methods for synthesizing 2'-FL, including chemical synthesis, enzymatic synthesis, and
whole-cell biosynthesis. However, there are some limiting factors for chemical synthesis and enzymatic synthesis
including the toxic reagents and the cost of precursors. In contrast, the whole-cell biosynthesis method is
relatively low-cost and mainly performed in engineered E.coli to produce 2'-FL. But, for large-scale fermentation,
there is possible endotoxin contamination.
Thus, in this project, we designed and synthesized an engineered S. cerevisiae cellular factory producing 2'-FL from
sweet potato residues by fermentation. On one hand, producing 2'-FL in S. cerevisiae is a relatively safe cellular
factory to allow large-scale fermentation. On the other hand, it is promising that use sweet potato residues to
further extend the industrial chain, improve the utilization value of inferior biomass resources, and cut the
cost.
Our team aims to produce 2'-FL in S. cerevisiae for providing the economic strategy. In terms of raw material, there
are two pathways to produce GDP-L-fucose GDP-D-mannose (called de novo pathway) or L-fucose (called salvage
pathway). In the de novo pathway, the GDP-D-mannose converts 2'-FL and is secreted into a medium involving three
enzymes gmd, wcaG, and DDD, as well as transporter lac12. In this study, industrial diploid Saccharomyces cerevisiae
CCTCC M94055 was used as the host to express 2 ' -FL pathway genes by chromosome integration and complete molecular
construction(Figure 1).

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-engineering01.jpg)

Figure 1. The metabolic pathway of biosynthesis 2’-FL in S. cerevisiae.
The purple part represents the heterologous pathway gene

Design

Firstly, we design the integration backbone plasmids XI-2 and X-3, which contain the up and down homology arms of
XI-2 and X-3 genome site in yeast, respectively. Then, we designed the two genes WbgL and lac12 inserted into the
integration backbone plasmids XI-2, and the other two genes gmd and wacG inserted into the integration backbone
plasmids X-3. Those four genes are key to producing 2'-Fucosyllactose(2'-FL) in a yeast cellular factory. In
addition, we added the promoter and terminator to flanking regions of these exogenous genes in order to facilitate
expression in the engineered yeast. The map of these four plasmids are as figure 2.

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-engineering02.jpg)

Figure 2. The map of recombinant plasmids.
A. The map of XI-2 site integration backbone plasmid
B. The map of X-3 site integration backbone plasmid
C. The map of XI-2-WbgL-lac12 integration plasmid
D. The map of X-3-gmd-wacG integration plasmid

Build

1. Construction of XI-2 site integration backbone plasmid

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-engineering03.jpg)

Figure 3. XI-2 site integration backbone plasmid by colony PCR verification

XI-2 site target gene integration backbone plasmid, containing 200 bp upstream and downstream homology arms of
chromosome XI-2 site. Randomly pick 10 transformants from the LB-Amp plate, and use the upstream and downstream
primer pairs of the homology arm to verify whether the fragment inserted. The target band is about 450 bp. It’s
possible that 2, 3, 4, 5, 9 and 10 transformants has inserted the DNA fragments as shown in Figure 3.

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-engineering04.jpg)
Figure 4. Validation of XI-2 site target gene integration backbone plasmid by digestion

The plasmids of transformants 2, 3, 4, 5, 9, and 10 were extracted and further verified by NotI digestion (Figure
4). If the cut band size is correct as shown in Figure 4, we picked No. 10 to perform Sanger sequencing.

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-engineering05.jpg)
Figure 5. The details of sequence alignment of No. 10

The sequence of the transformant plasmid No. 10 is no mutation and mismatch as shown in Figure 5, indicating we
successfully constructed XI-2 site integration backbone plasmid.

2. Construction of X-3 site integration backbone plasmid

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-engineering06.jpg)
Figure 6. X-3 site target gene integration backbone plasmid by colony PCR verification

The X-3 site contains 200 bp of homology arms, which on the upper and lower sides of the chromosome X-3 site. 10
transformants were randomly picked from the LB-Amp plate, and the upstream and downstream primer pairs of the
homology arms were used to verify whether the fragment inserted. The target band was about 450 bp. As shown in
Figure 6, except for 6 and 7, the rest of the transformants might insert fragments.

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-engineering07.jpg)

Figure 7. X-3 site target gene integration backbone plasmid by digestion verification

The plasmid of transformants 2, 3, 5, and 8 were extracted and further verified by by Not1 digestion (Figure 7). The
target band was 3762+430 bp. Thus, we picked No. 5 for sequencing, and the results are shown in Figure 8.

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-engineering08.jpg)

Figure 8. Sequencing of the transformant No. 5

The sequence alignment well matched, indicating that the X-3 site integrated backbone plasmid was constructed
successfully.

Secondly, the wbgL-lac12 and gmd-wcaG incorporated into the integration plasmids XI-2 and X-3, respectively. The
verification results were shown as follows.

3. Construction of XI-2-WbgL-lac12 integration plasmid

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-engineering09.jpg)
Figure 9. Validation of XI-2-wbgL-lac12 plasmid BamH1+Xho1 digestion

In the XI-2-wbgL-lac12 plasmid, the wbgL and lac12 gene expression cassettes are inserted between the XI-2 homology
arm, in different orientations. The wbgL+lac12 gene expression plasmid for XI-2 site integration was constructed by
a two-step digestion cloning method. After wbgL gene expression cassette was integrated, lac12 inserted though Xho1
and BamH1 digestion sites.
Plasmids of 10 transformants were extracted and verified by Xho1+BamH1 double-enzyme digestion (Fig. 9). The
positive transformant band was 6007+2635 bp, and the correct NO.11 was selected for sequencing. The results were
shown as Figure 10.

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-engineering10.jpg)
Figure 10. Sequencing of plasmid 11 correctly digested

The sequence alignment results showed well matched, indicating that the XI-2-wbgL-lac12 plasmid was constructed
successfully.

4. Construction of X-3-gmd-wacG integration plasmid

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-engineering11.jpg)
Figure 11. X-3-gmd-wcaG plasmid Xho1+Mss1 digestion verification

In the X-3-gmd-wcaG plasmid, the gmd and wcaG gene expression cassettes are inserted between the upstream and
downstream of the X-3 homology arm, in the same direction. The gmd+wcaG gene for X-3 site integration was
constructed by a two-step digestion cloning method. After the gmd gene expression cassette was integrated, the wcaG
was introduced using Xho1 digestion site.
Plasmids of 12 transformants were extracted and verified by Xho1+Mss1 double-enzyme digestion (Fig.11). The positive
transformant band was 6220+1896 bp, and the No.11 plasmid with correct digestion was randomly selected and sent for
sequencing.

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-engineering12.jpg)
Figure12. Sequencing of the correct transformant plasmid of No. 11

The details of sequence alignment showed there is no mutation, indicated that the X-3-gmd-wcaG plasmid was
constructed successfully.

5. Using CRISPR-Cas9 technology to integrate the target genes into the genome of S. cerevisiae

Then, we used CRISPR-Cas9 technology to integrate the target four genes WbgL, lac12,
gmd, and wacG into the genome of S. cerevisiae. The constructed X-3-gmd-wcaG integration plasmid and XI-2-wbgL-lac12
integration plasmid were digested with NotI, respectively. The large fragments were transformed into the yeast
competent cell that already contains the Cas9 expression plasmid by the lithium acetate transformation method,
together with the gRNA. Finally, colony PCR was used to verify the integration of exogenous genes.

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-engineering13.jpg)

Figure 13. Validation of exogenous gene integration by colony PCR
From left to right, No.1, 2, 3, 4 transformants.

Colony PCR was used to verify whether the X-3 and XI-2 loci gene fragments were integrated into S. cerevisiae
strains. The results are shown in Figure 13. The integrated copy number of the four transformants was verified using
four different primer pairs. If the internal primers (X-3 inner-primer pairs and XI-2 inner-primer pairs) can
amplify the target band, and the outer primers (X-3 outer-primer pairs and XI-2 outer-primer pairs) cannot amplify
the target band with the size of the integration homology arm (4346bp and 4880bp), it means that 2 copies have been
integrated. If the primers outside the site amplify the target band of the size of the integration homology arm, it
means that 1 copy of the target gene has been integrated. Based on the above analysis, we judged that the middle
transformants 2 and 3 have clearly integrated one copy of the target gene, and can be tested for subsequent
experiments.

Test

1. The engineered strain utilizes synthetic medium to produce 2'-FL

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-engineering14.jpg)

Figure 14. Fermentation of recombinant yeast in YPD30L2 medium to produce 2'-FL

The productivity of 2'-FL by recombinant yeast in the synthetic medium YPD30L2 was tested. As shown in Figure 14, 30
g/L glucose was completely consumed within the initial 4 h, and then the strain began to use the produced ethanol as
a carbon source, showing a secondary growth state (Figure 14A). The initial addition of 2 g/L of lactose was
undetectable after 48 h, resulting in about 0.7 g/L of 2'-FL (Figure 14B). The theoretical conversion rate of
lactose to 2'-FL was 100%. Part of 2'-FL accumulated intracellularly and failed to be effluxed into the medium.

2. Production of 2'-FL from sweet potato residues by engineered strains

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-engineering15.jpg)

Figure 15. Production of 2'-FL from sweet potato residues in recombinant yeast

The productivety of 2'-FL by recombinant yeast from sweet potato residues was tested, as shown in Figure 15.
Generally, the growth of the strain was slightly worse than that of the synthetic medium (Figure 15A), which may be
due to the presence of some inhibitor for yeast growth in the sweet potato residues. The presence of lactose was not
detected at 48 h, and the final yield was about 0.6 g/L 2'-FL (Figure 15B).

Learn

By expressing exogenous WbgL, lac12, gmd, and wacG genes, we constructed the metabolic pathway of 2'-FL and obtained
a yeast cell factory with high production of 2'-FL. Our fermentation experimental data show that the gene-edited
yeast cells can produce 2'-FL at a high level of 0.6-0.7g/L. At the same time, we integrated the exogenous gene
lac12 into yeast, so that yeast cells have the ability to efflux 2'-FL into the medium. This is in line with the
fact that the fermentation of high-yield 2'-FL by Saccharomyces cerevisiae, which is mainly based on food safety,
has become a research hotspot and difficulty, and it is also the key to solving practical social problems. Last but
not least, the CRISPR-Cas9 gene editing technology we used in this project is a good case. In the future, we can use
this technology to continue editing yeast genomes to create more diverse yeast cell factories and produce more
meaningful metabolites.

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

Content
1、 Medium preparation:
2、 Construct design
3、 Transformation of yeast

Medium preparation

**LB medium preparation：**Yeast extract 5 g/L，Tryptone 10 g/L，NaCl 10 g/L. For solid, add 20 g/L agar.
**Yeast medium preparation：**Yeast extract 10 g/L，Tryptone 20 g/L，D-glucose 30 g/L(separate)，Lactose 2 g/L. For
solid, add 20g/L agar.
**YPD30L2 preparation：**Yeast extract 10 g/L, Tryptone 20 g/L, D-glucose 30 g/L, Lactose 2 g/L.

The medium, as mentioned above, are put into autoclave at 120 °C for 20 mins.

Construct design

PCR

Primer pairs h-bb-x-3d-f1 and h-bb-x-3u-r1 amplify vector using pHCas9-Nours plasmid as DNA template to obtain the
integration backbone plasmid. Size: 3776bp.
Primer pairs h-x-3u-bb-f1 and h-x-3d-bb-r1 amplify the flanking region of homologous arms using genomic CCTCC M94055
as DNA template. Size 444bp.

Primer pairs h-bb-xi-2d-f1 and H-bb-xi-2u-r1 amplify vector using pHCas9-Nours plasmid as DNA template to obtain the
integration backbone plasmid. Size 3776bp.
Primer pairs h-xi-2u-bb-f1 and h-xi-2d-bb-r1 amplify the flanking region of homologous arms using genomic CCTCC
M94055 as DNA template. Size 444 bp.

**PCR reaction setup**

| Component | Volume (μL) |
| --- | --- |
| 2× Mix | 25 |
| Primer F (10 μM) | 2.5 |
| Primer R (10 μM) | 2.5 |
| DNA template | 2 |
| ddH2O | 18 |
| Total | 50 |

**PCR instrument set**

| Steps | Temperature(℃) | Time |
| --- | --- | --- |
| 1 | 98 | 3min |
| 2 | 98 | 30sec |
| 3 | 55 | 30sec |
| 4 | 72 | 2min |
| 5 | 72 | 10min |
| 6 | 4 | Forever |

\*Repeat step2 to 4 for 30 times.

Agarose gel electrophoresis

1. Agarose gel preparation: weight out 0.5 g Agarose in a conical flask. Add 50 mL 1×TAE buffer to the conical
flask. Melt the conical flask using a microwave oven until the solution become clear. While heating, swirl the flask
occasionally. Add 5 μL nucleic acid staining dye (Gel Red) to Agarose gel when this solution cooled down to ~55℃.
Pour the molten gel to into casting tray. And, insert the comb at the end of the casting tray. Avoid air bubbles.
When it completely solidified, remove the comb to form the wells. Put the gel into the electrophoresis bath, add
1×TAE until it covered the gel.
2. Each sample was mixed the loading dye. And then the samples were added sequentially to the wells of the gel
(avoid to break the gel).
3. Electrophoresis: cover the electrophoresis bath, samples had a net negative charge, and an electrical current was
applied in 180 V for 15 min.
4. Turn off the power, and observe gel when it exposed to a short wave ultraviolet light source. The size of DNA
fragments was determined according to the position of DNA ladder.

Gel extraction (according to commercial kit)

1. Cut the band that contains the DNA fragment.
2. Weight the gel, then add appropriate amount of Buffer B2.
3. Heat in a water bath at about 50℃ until the gel is completely dissolved.
4. DNA binding on membrane on adsorption column and centrifuge at 8000 xg for 30sec. Discard the filtrate.
5. Add 500 l wash buffer for cleaning to column and centrifuge at 9000 xg. Discard the filtrate. Repeat this
step.
6. Adding DNA eluent for elution. And purified DNA (30μL) was obtained.
7. inoculate X-3 XI-2 gRNA/DH5α（Amp）, X-3（Amp）, XI-2（Amp）, pHCas9-Nourse-1（Amp）, AQ yeast (no antibiotic）add 5 μL
ampicillin (working concentration: 100 μg/mL）and 100-200μL inoculant to 5mL LB medium, Incubator shaker foster at
220 rpm (30℃) overnight.

Plasmid extraction (according to commercial kit)

1. Harvest E. coli by centrifugation at 12000 rpm for 1 minute, and then remove the supernatant.
2. Pipette 250 μL Buffer S1 into the tubes (cool down at 4 degrees) to suspend the precipitation.
3. Pipette 250 μL Buffer S2 into the tubes with gently mixing to break the bacteria (this step must be done in 5
min)
4. Pipette 350μL Buffer S3 into the tubes with gently mixing and centrifuge at 12000rpm for 10 min, and then the
supernatant can be purified using spin filter (neutralization reaction, remove impurity like protein) .
5. Extract plasmids DNA using the column extraction method.

5.1. Centrifuge at 12000rpm for 1 min and discard the flow-through in the column.
5.2. Add 500 μL Buffer W1 to the column and centrifuge at 12,000 for 1 min.
5.3. Repeat the washing procedure twice with 700 μL Buffer W2
5.4. Discard the flow-through in the tube and centrifuge at 12,000rpm for 1 min again
5.5. Wait for 1 to 2 minutes until the alcohol in Buffer W2 finishes evaporate
5.6. Add 50μL ddH20 at 60-65 degrees and incubate the cells into a water bath for 1-2 minutes at 60-65 degrees,
and centrifuge for 1 minute.

Restriction Enzyme Double Digestion

All the PCR products were treated with the restriction enzymes NotI and XhoI, including the
cassettes of gmd-wcaG
and wbgL-lac12, as well as X-3 and XI-2 plasmid.
**DNA fragment digestion**

| Component | Volume (μL) |
| --- | --- |
| PCR fragments (Homologous recombination templates) | 25 |
| Not1 | 1 |
| Xho1 | 1 |
| CutSmart | 5 |
| ddH2O | 18 |
| Total | 50 |

**Plasmid digestion for vector preparation**

| Component | Volume (μL) |
| --- | --- |
| Plasmid vector | 30 |
| Not1 | 1 |
| Xho1 | 1 |
| CutSmart | 5 |
| ddH2O | 13 |
| Total | 50 |

\*Gel extraction (same as before) for DNA fragment
\*Gel electrophoresis for vector

DNA ligation

The system: Room temperature/30 minutes

| Component | Volume (μL) |
| --- | --- |
| Cut Vector | 10 |
| PCR fragments after digestion (Homologous recombination templates) | 7 |
| T4 ligase | 1 |
| T4 ligase buffer | 2 |
| Total | 20 |

Transformation

1. Add 10 μL recombination products to 2 tubes of 100 μL E. coli DH5α competent cells and incubate cells on ice for
30 minutes;
2. Heat-shock cells for 90 seconds in a 42 degrees water bath and cool down on ice for 3 minutes immediately;
3. Add 900 μL LB medium to the tubes and put it on the shaker at 37 degrees for 1 hour;
4. Centrifuge at 5,000 rpm for 5 minutes and remove 900 μL supernatant;
5. Suspend the precipitation and pipette the solution into the LB culture plate with ampicillin. For cultivation of
stains, the plate was put on the incubator at 37 degrees overnight.

Transformation of yeast

Preparing competent yeast cells

1. Culture yeast Y2H in YPDA medium at 30 degrees;
2. Pick single yeast into 2 ml tubes and add 1 ml YPD medium, and shake to grow the cells;
3. Pipette the solution into YPD medium at the ratio of 1:100 and incubate at 30 degrees for 18-24 hours until the
OD600 reaches 1.5-2；
4. Pipette the culture at the ratio of 1:4 into YPD medium at 30 degrees for 3 hours until the OD600 reaches
0.2;
5. Pipette the yeast culture into 1.5 ml tube and centrifuge at 12,000 rpm for 1 minute;
6. Remove the supernatant and add 1ml TE buffer and suspend the precipitation;
7. Remove the supernatant and add 0.1ml TE-LiAc solution, and suspend the precipitation.

The heat shock transformation of the competent yeast cells

1 boil the salmon sperm DNA (10μL/μg) for 10-20 minutes and put it in an ice bath for 10 minutes;
2 put 10μL salmon sperm DNA in 100μL yeast competent cells and 5μL plasmids and shake it;
3 add 600μL TE-LiAc-PEG solution and mix it through vortex;
4 incubate on the shaker at 200rpm and 20 degrees for 30 minutes;
5 add 70μL DMSO and mix;
6 heat shock for 15 minutes at 15 minutes;
7 put it in an ice bath for 2-10 minutes;
8 centrifuges at 12000rpm for 1 minutes and remove the supernatant;
9 add 100μL 1\*TE and suspend the precipitation.

Select the Transformant colonies for PCR verification.

place the validated strains in glycerol tubes to obtain E. coli with the target plasmid and stored at -80 °C until
use. The samples were identified by colony PCR, NotI and XhoI double enzyme digestion, and then sent for Sanger
sequencing. Positive colonies which were as X-3-gmd wcaG and XI-2-wbgL-lac12 were picked and inoculated in 3 mL LB
supplemented 3 μL Amp overnight.

S. cerevisiae cultureS. cerevisiae culture

0.4 ml of S. cerevisiae culture were inoculated 40 ml YPD20 medium for 8h. Collect bacteria after several period of
growth. Then, the culture transferred to 20 ml YPD30L2 medium to produce 2'-FL. After initial growth, OD600 reached
10 and regularly taking samples to measure.

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{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block header\_content %}
![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-banner.jpg)
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Results

1. Gene integration plasmid construction: Wbgl and lac12; gmd and wacG

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-results01.jpg)

We constructed two plasmids including four genes WbgL, lac12, gmd, and wacG which are key genes to produce
2'-Fucosyllactose(2'-FL) in a yeast cellular factory. In addition, we need to added the promoter and terminator to
flanking regions of these exogenous genes in order to facilitate expression in the engineered yeast. The components
were incorporated into the integration backbone plasmid with NotI and XhoI sites. Firstly, we constructed the
integration backbone plasmids XI-2 and X-3. These colonies were verified by colony PCR and Sanger sequencing and the
results were shown as follows.

A.
Validation of the construction of XI-2 site integration backbone plasmid

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-results02.png)

Figure1. XI-2 site integration backbone plasmid by colony PCR verification

XI-2 site target gene integration backbone plasmid, containing 200 bp upstream and downstream homology arms of
chromosome XI-2 site. Randomly pick 10 transformants from the LB-Amp plate, and use the upstream and downstream
primer pairs of the homology arm to verify whether the fragment inserted. The target band is about 450 bp. It’s
possible that 2, 3, 4, 5, 9 and 10 transformants has inserted the DNA fragments as shown in Figure 1.

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-results03.png)

Figure 2 Validation of XI-2 site target gene integration backbone plasmid by digestion

The plasmids of transformants 2, 3, 4, 5, 9, and 10 were extracted and further verified by NotI digestion (Figure
2). If the cut band size is correct as shown in Figure 2, we picked No. 10 to perform Sanger sequencing.

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-results04.jpg)

Figure 3 The details of sequence alignment of No. 10

The sequence of the transformant plasmid No. 10 is no mutation and mismatch as shown in
Figure 3, indicating we successfully constructed XI-2 site integration backbone plasmid.

B
Validation of the construction of X-3 site integration backbone plasmid

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-results05.png)

Figure 4. X-3 site target gene integration backbone plasmid by colony PCR verification

The X-3 site contains 200 bp of homology arms, which on the upper and lower sides of the chromosome X-3 site. 10
transformants were randomly picked from the LB-Amp plate, and the upstream and downstream primer pairs of the
homology arms were used to verify whether the fragment inserted. The target band was about 450 bp. As shown in
Figure 4, except for 6 and 7, the rest of the transformants might insert fragments.

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-results06.png)

Figure 5. X-3 site target gene integration backbone plasmid by digestion verification

The plasmid of transformants 2, 3, 5, and 8 were extracted and further verified by by Not1 digestion (Figure 5).
The target band was 3762+430 bp. Thus, we picked No. 5 for sequencing, and the results are shown in Figure 6.

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-results07.jpg)

Figure6. Sequencing of the transformant No. 5

The sequence alignment well matched, indicating that the X-3 site integrated backbone plasmid was constructed
successfully.

Secondly, the wbgL-lac12 and gmd-wcaG incorporated into the integration plasmids XI-2 and X-3, respectively. The
verification results were shown as follows.

C
Validation of the construction of XI-2-wbgL-lac12 plasmid

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-results08.png)

Figure 7 Validation of XI-2-wbgL-lac12 plasmid BamH1+Xho1 digestion

In the XI-2-wbgL-lac12 plasmid, the wbgL and lac12 gene expression cassettes are inserted between the XI-2
homology arm, in different orientations. The wbgL+lac12 gene expression plasmid for XI-2 site integration was
constructed by a two-step digestion cloning method. After wbgL gene expression cassette was integrated, lac12
inserted though Xho1 and BamH1 digestion sites.

Plasmids of 10 transformants were extracted and verified by Xho1+BamH1 double-enzyme digestion (Fig. 7). The
positive transformant band was 6007+2635 bp, and the correct NO.11 was selected for sequencing. The results were
shown as Figure 8.

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-results09.jpg)

Figure 8 Sequencing of plasmid 11 correctly digested

The sequence alignment results showed well matched, indicating that the XI-2-wbgL-lac12 plasmid was constructed
successfully.

D
Validation of the construction of X-3-gmd-wcaG plasmid

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-results10.png)

Figure 9 X-3-gmd-wcaG plasmid Xho1+Mss1 digestion verification

In the X-3-gmd-wcaG plasmid, the gmd and wcaG gene expression cassettes are inserted between the upstream and
downstream of the X-3 homology arm, in the same direction. The gmd+wcaG gene for X-3 site integration was
constructed by a two-step digestion cloning method. After the gmd gene expression cassette was integrated, the
wcaG was introduced using Xho1 digestion site.

plasmids of 12 transformants were extracted and verified by Xho1+Mss1 double-enzyme digestion (Fig. 9). The
positive transformant band was 6220+1896 bp, and the No.11 plasmid with correct digestion was randomly selected
and sent for sequencing.

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-results11.jpg)

Figure10. Sequencing of the correct transformant plasmid of No. 11

The details of sequence alignment showed there is no mutation, indicated that the X-3-gmd-wcaG plasmid was
constructed successfully.

2. Using CRISPR Cas-9 technology to integrate the target genes into the genome of S. cerevisiae

The constructed X-3-gmd-wcaG and XI-2-wbgL-lac12 plasmids were digested with Not1, respectively, and the large
fragments were extracted from the gel. Using the lithium acetate transformation method, together with the gRNA, they
were introduced into the yeast competent cell that already contains the Cas9 expression plasmid. In yeast CCTCC
M94055, colony PCR was used to verify the integration of exogenous genes.

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-results12.jpg)

Figure11. Validation of exogenous gene integration by colony PCR
From left to right, No.1, 6, 7, 10 transformants.
Primer pairs we used were shown as follows.
X-3 outer-primer: h-x-3u-bb-f1/h-x-3d-bb-r1.
XI-2 outer primer: h-xi-2u-bb-f1/h-xi-2d-bb-r1.
X-3 inner-primer pairs: h-x-3u-bb-f1/h-gap-gmd-r1.
XI-2 inner-primer pairs: h-xi-2u-bb-f1/h-wbgL-cyc1t-r1.

Colony PCR was used to verify whether the X-3 and XI-2 loci gene fragments were integrated into S. cerevisiae
strains. The results are shown in Figure 11. The integrated copy number of the four transformants was verified using
different primer pairs. If the internal primers (inner-primer pairs) can amplify the target band, and the outer
primers (outer-primer pairs) cannot amplify the target band with the size of the integration homology arm, it means
that 2 copies have been integrated. If the primers outside the site amplify the target band of the size of the
integration homology arm, it means that 1 copy of the target gene has been integrated. Based on the above analysis,
we judged that the middle transformants 6 and 7 have clearly integrated one copy of the target gene, and can be
tested for subsequent experiments.

3. The engineered strain utilizes synthetic medium to produce 2'-FL

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-results13.png)

Figure 12 Fermentation of recombinant yeast in YPD30L2 medium to produce 2'-FL

The productivity of 2'-FL by recombinant yeast in the synthetic medium YPD30L2 was tested. As shown in Figure 12, 30
g/L glucose was completely consumed within the initial 4 h, and then the strain began to use the produced ethanol as
a carbon source, showing a secondary growth state (Fig. 12A). The initial addition of 2 g/L of lactose was
undetectable after 48 h, resulting in about 0.7 g/L of 2'-FL (Fig. 12B). The theoretical conversion rate of lactose
to 2'-FL was 100%. Part of 2'-FL accumulated intracellularly and failed to be effluxed into the medium.

4. Production of 2'-FL from sweet potato residues by engineered strains

![](https://static.igem.wiki/teams/4292/wiki/t-zhejiang-united-results14.png)

Figure 13 Production of 2'-FL from sweet potato residues in recombinant yeast

The productivety of 2'-FL by recombinant yeast from sweet potato residues was tested, as shown in Figure 18.
Generally, the growth of the strain was slightly worse than that of the synthetic medium (Figure 13A), which may be
due to the presence of some inhibitor for yeast growth in the sweet potato residues. The presence of lactose was not
detected at 48 h, and the final yield was about 0.6 g/L 2'-FL (Fig. 13B).

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