iGEM 2023 — Team hbut-china

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{% block title %}Notebook{% endblock %}
{% block pageTitle %}Notebook{% endblock %}
{% block sideBarTitle %}Notebook{% endblock %}
{% block lead %}
Document the dates you worked on your project. This should be a detailed
account of the work done each day for your project.
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# April

## Week 1 (4.24-4.30)

### Project

The team was officially established. We held the first meeting where we
met the rest of the team and some of the advisors.

# May

## Week 2 (5.1-5.7)

### Project

Members of the experimental group learned the experimental safety
guidelines and did safety training required by the laboratory. During
the first week, we completed the design of all the required primers and
began to design our experimental scheme.

## Week 3 (5.8-5.14)

### Project

We had our second meeting where we were introduced to the judging
handbook. We discussed the next series of arrangements, such as
experimental progress and promotion activities.

## Week 4 (5.15-5.21)

### Human practices

The human practices group and the experimental group members of the
HBUT-China team held a one-day booth promotion activity in the central
area of the campus, in order to promote the International Genetic
Engineering Machine Design Competition and synthetic biology.

## Week 5 (5.22-5.28)

### Project

We decided to construct expression vectors such as Cas12a, sgRNA and
donor and verify the feasibility of single and double plasmid systems.

### Human practices

Kong Xiang, Zeng Hongying and Zheng Yimeng, three members of the human
practices group, had an in-depth communication with the head of Angel
Yeast Company.

# June

## Week 6 (5.29-6.4)

### Project

Brainstorming about the molecules we want to produce continued. This
week, in addition to studying the construction of CRISPR expression
vectors, we also studied the gene knockout pathway for synthesizing
lycopene.

### Human practices

The instructor Yao Lan and the team members Kong Xiang, Zeng Hongying
and Han Yi came to the kindergarten of Hubei University of Technology.
In the process of popularizing science, there was laughter and laughter.
The children have fun and learn biology at the same time.

## Week 7 (6.5-6.11)

### Project

In this week, we started to analyze the outstanding cases and discuss
them in combination with our projects. At the same time, the members of
the experimental group read the literature related to the project to
improve the design and plan of the experiment.

### Human practices

In order to further promote the competition content and synthetic
biology of the our team to more college students, the team held a
science popularization class propaganda activity with the help of the
Yanlin Science innovation platform of the College of Bioengineering and
Food.

## Week 8 (6.12-6.18)

### Project

We decided to screen high-efficiency promoters, terminators, RBS and
other components.

## Week 9 (6.19-6.25)

### Project

#### Clonings

##### Plasmids

Plasmid with different skeleton of MAD7 was designed and synthesized,
such as PEC-MAD7,pJYS1-MAD7, PEC-cpf1 and pJYS1-MAD7. We amplified and
validated all the obtained plasmids.

##### promoter

The modified promoter fragment(PEC-PlacM-MAD7) was amplified by PCR and
the plasmid was purified.

#### Others

Members of the art group designed a lovely team LOGO and IP cultural and
creative products for the project content, and Kong Xiang, the leader of
the human practices group, was responsible for the purchasing affairs.

## Week 10(6.26-7.2)

### Project

#### Clonings

##### Promoter

The constructed plasmids were transformed into *E. coli* TOP10,
and the sequencing results of one of the clones were correct.

##### Replicon

The modified replicon fragment(PEC-PlacM-PxmB-MAD7) was amplified by PCR
and the plasmid was purified.

##### Others

Members of the art team make posters for Hubei Synthetic Biology Open
Conference.

Web group members complete the description page, wiki page validation
and release wiki tool igem-uploads.

### Collaborations and partnerships

Our team members and HUBU-SKY-China members communicated online through
the conference platform and shared their experiences in the competition.

# July

## Week 11 (7.3-7.9)

### Project

#### Clonings

##### Promoter

The plasmid was transformed into *Corynebacterium glutamicum*,
and the editing efficiency was calculated after color comparison and
verification.The results show that our optimized promoter is successful!

##### Replicon

The constructed plasmids were transformed into *E. coli* TOP10,
and the sequencing results of one of the clones were correct.

### Collaborations and partnerships

Kong Xiang, Yu Siqin and Han Yi went to Hubei University and had an
in-depth discussion with HUBU-SKY-China team on follow-up video
cooperation.

At the invitation of Hubei University, the HBUT-China team participated
in Hubei Synthetic Biology Open Conference. In the morning, the
conference officially opened, and the participating teams carried out a
preliminary display of the project. In the afternoon, the teams carried
out a comprehensive display and more in-depth communication on the
project content at the conference booth.

## Week 12 (7.10-7.16)

### Project

#### Clonings

##### Replicon

The plasmid was transferred into *Corynebacterium glutamicum*,
and the editing efficiency was calculated after color comparison and
verification. The results show that our optimized replicon is
successful!

##### PAM sites

We designed the positions of PAM sites and front and rear chains in
different skeletons, such as pEC-MAD7-TTTN, pJYS1-MAD7-TTTN,
pEC-Cpf1-TTTN, pJYS1-Cpf1-TTTN. Amplification of the fragment, then
cloning into plasmids.

### Human practices

Human group and art group members produced three funny children’s
picture books. Our team hopes that through this, children can establish
a preliminary understanding of biological knowledge and cultivate their
interest in biology.

### Collaborations and partnerships

From July 8 to 10, 2023, HBUT-China team members Jiang Yiming, Liang
Tianyi, Kong Xiang, Wang Niannian and Zeng Hongying participated in the
10th Conference of China iGEMer Community (CCiC) held at Hainan
University, bringing together teams from all over the country. Our team
members showed the team project content to the teachers and students in
the conference with confidence and fluency, and won a lot of applause
and recognition.

## Week 13 (7.17-7.23)

### Project

#### Clonings

##### PAM sites

The constructed plasmids were transformed into *E. coli* TOP10,
and the sequencing results of one of the clones were correct.

For the integrity of the experiment, we continued to design the
positions of PAM sites and front and rear chains in different skeletons,
such as pEC-MAD7-CTTN, pJYS1-MAD7-CTTN, pEC-Cpf1-CTTN, pJYS1-Cpf1-CTTN.
Amplification of the fragment, then cloning into plasmids.

#### Straw

We designed the experiment of straw processing to obtain glucose, and
prepared the materials needed for the experiment.

### Human practices

Our human practices group members, Xinqiu Hu and Yimeng Zheng, designed
a questionnaire to understand the public’s understanding of synthetic
biology, lycopene, straw treatment and utilization, further publicizing
our project.

## Week 14 (7.24-7.30)

### Project

#### Clonings

##### PAM sites

The plasmid was transferred into *Corynebacterium glutamicum*,
and the editing efficiency was calculated after color comparison and
verification.The high-quality PAM sites in the gene skeleton were
obtained by comparing the results.

#### Straw

The straw powder was dried by removing the surface lipids with anhydrous
ethanol.

### Human practices and Collaborations

in order to serve the society and contribute to the society, the human
practices group members Zeng Hongying, Zheng Yimeng, Hu Xinqiu and two
members of CUG-China team went to the nursing home, the elderly Service
center of Shizishan Street Hongshan District, to carry out volunteer
services.The activity was mainly in the form of artistic performances
and popular science lectures, bringing joy to the elderly and relevant
knowledge of popular science.

# August

## Week 15 (7.31-8.6)

### Project

#### Clonings

##### Knockout of large fragments

We used the CRISPR-MAD7(Cas12a) system to knock out large fragments, and
designed plasmids that knocked out fragments of different lengths, such
as 5kb, 10kb, 20kb, 25kb. Amplification of the fragment, then cloning
into plasmids.

#### Straw

The dried straw powder was pretreated with dilute sulfuric acid, and the
enzymatic solution was added for enzymatic hydrolysis, and the glucose
solution was obtained by solid-liquid separation.

### Collaborations and partnerships

Our team members participated in the 2nd Synthetic Biology Competition ·
Innovation Competition sponsored by the Syntheic Biology Branch of the
Chinese Society of Bioengineering at Shenzhen University of Technology.

## Week 16 (8.7-8.13)

### Project

#### Clonings

##### Knockout of large fragments

The constructed plasmids were transformed into *E. coli* TOP10.
Unfortunately, some of the clones turned out to be wrong, which means we
have to rethink our design.

#### Straw

A biosensor analyzer was used to determine the glucose content in the
glucose extract. Cryopreservation for future use.

### Human practices

Zeng Hongying, a member of the Human Practice group, returned to
Xiangyang Zhiyuan Middle School to promote the team project and
popularize synthetic biology knowledge. She encouraged the students to
actively participate in various comprehensive competitions in the
university stage, and cultivated their scientific research ability,
learning ability and teamwork ability in the competition practice.

## Week 17 (8.14-8.20)

### Project

#### Clonings

##### Knockout of large fragments

Some plasmids were transformed into *Corynebacterium glutamate*,
and the editing efficiency was calculated by color comparison and
verification. The redesigned plasmid was again transformed into
*E.coli* TOP10 and verified again.

##### Single gene knockout

We use the CRISPR/MAD7 gene editing system to knock out single genes,
such as CrtR, CrtEb, CrtYf. Amplification of the fragment, then cloning
into plasmids.

#### Others

Web group members completed software colonies-counter verification.

### Human practices

HP group members took straw treatment and utilization as the starting
point, interviewed relevant enterprise personnel, government personnel,
farmers, experts, etc, and obtained the understanding and thinking of
many people in different industries on straw treatment and utilization.

### video

We discussed the video script, style, and content, and decided on a
production date.

## Week 18 (8.21-8.27)

### Project

#### Clonings

##### Knockout of large fragments

Some plasmids were transformed into \_Corynebacterium glutam\_\_icum\_, and
the editing efficiency was calculated by color comparison and
verification. Through the analysis of the results, we found the highest
editing efficiency of the knockout fragment length in this system.

##### Single gene knockout

The constructed plasmids were transformed into *E. coli* TOP10.
Sadly, all the plasmids we’ve built have failed. So we had to go back to
the clone design.

### Human practices

Network member Han Yi went to Haikou Nongken Middle School to introduce
iGEM and our program to the students. The students showed great interest
in the competition, and Han Yi encouraged them to participate in the
competition and improve their abilities in synthetic biology in the
future.

## Week 19 (8.28-9.3)

### Project

#### Clonings

##### Single gene knockout

We redesigned the method of constructing the plasmid, amplified the
upstream and downstream two fragments, and then cloned them into the
plasmid. As time is pressing, we speed up the experiment.

The constructed plasmids were transformed into *E. coli* TOP10.
Happily, the cloned plasmid was verified by sequencing to be correct.

Plasmids were transformed into
*Corynebacterium glutamicum.* Pink colonies appeared on the
medium plate, which was amazing!

#### shake flask

Through color comparison, a recombinant strain with good growth ability
was isolated and selected from the basic culture. Glucose extracted from
straw was added to shake flask culture medium, and the growth of strain
was measured under this culture medium.

### Human practices

Our team members held charitable donation activities for cultural
creative products and picture books, transforming the project into
public charity for people in need of help.

### video

The first draft of our video is almost complete, and the animation part
is starting to be added. We were planning to complete the promotion
video within a week.

# September

## Week 20 (9.4-9.10)

### Project

#### High Performance Liquid Chromatography

After a certain period of time, a certain sample bacterial solution was
taken and the yield of lycopene in the modified strain was measured by
high performance liquid chromatography (HPLC) to evaluate the effect of
experimental manipulation on lycopene synthesis.

### Human practices

We sold the designed cultural and creative products and picture books
for charity and then contacted charity organizations to make donations.

## Week 21 (9.11-9.17)

### video

We completed the recording of the narration part of the demo video and
the editing of the animation in the video, and submitted it on time.

{% endblock %}

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{% block title %}Engineering Success{% endblock %}
{% block pageTitle %}Engineering Success{% endblock %}
{% block sideBarTitle %}Engineering Success{% endblock %}
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Demonstrate engineering success in a part of your project by going through
at least one iteration of the engineering design cycle.
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# 1. 1ST ITERATION

### Optimization of CRISPR-MAD7(Cas12a) system

## 1.1 1st ITERATION – design

For gene editing using CRISPR technology, you need to transfect the
plasmid into cells, allow it to express the CRISPR system and edit the
target gene. To improve the efficiency of gene editing, we optimized the
CRISPR system to determine the optimal CRISPR-MAD7(Cas12a) system.

The promoter is responsible for driving the transcription of genes,
which increases the localization and binding of CRISPR system on the
target genes, thus improving the editing efficiency is the basic
structure of plasmid. In order to effectively deliver CRISPR components
to the target cells, the most appropriate plasmid should be selected. In
CRISPR system, Cas protein is usually improved by identifying and
binding to the PAM site and performing DNA cutting or other
modifications to identify the recognition efficiency of Cas protein by
selecting the best PAM site.

In conclusion, we will select the optimal promoter, plasmid and PAM site
to improve the efficiency of gene editing.

## 1.2 1st ITERATION – build

A. Selection of the optimal promoter

To determine the optimal promoter, we went through literature review and
screening, the relevant promoters were Ptuf, PlacM, Ptac and Ptrc. The
insertion of these promoters into the pEC-XK99E plasmid vector resulted
in the recombinant plasmid. The constructed plasmids were transferred
into
*E. coli* and then cultured. After some time, colonies on the
medium were picked for PCR validation, an expanded culture of the
verified verified bacteria. After 12h, the plasmids were extracted and
sequenced, electrotransfer of the correctly sequenced plasmid into
*C. glutamicum* ATCC13032. Colony growth on the medium was
observed after a certain period of incubation. The optimal promoter was
identified by alignment.

B. Selection of the optimal plasmid

We found that the gene editing efficiency of the pEC-XK99E plasmid
vector that identified the best promoter was still not high enough, and
to improve the efficiency, we selected the best plasmid. We constructed
pJYS1-MAD7-TTTA, pBluescript-MAD7-TTTA and pXMJ19-MAD7-TTTA three
plasmids. The correctly sequenced plasmid was electrotransferred into
*C. glutamicum* ATCC 13032, and the colony growth on the medium
was observed, and the optimal plasmid was determined by comparison.

C. Validation of promoters on the optimal plasmid

To ensure that promoters capable of efficient expression on the
pEC-XK99E plasmid are also highly expressed on the newly determined
optimal plasmid, validations of the promoter on the optimal plasmid was
performed. The experimental manipulation of the first step was repeated
after replacing the plasmid growth of the pEC-XK99E plasmid with the
best plasmid.

D. Selection of the best PAM site

In the above experiments, we identified the optimal promoter and the
optimal plasmid, based on which we will determine the optimal PAM site.
By reviewing the literature, we selected (Table 1) PAM sites for
experiments. Changing the gRNA during plasmid construction, that is, the
PAM site was changed. The recombinant plasmid was obtained by fusion PCR
and one-step cloning technology, the sequenced plasmid was
electrotransferred to *C. glutamicum* ATCC13032, the colony
growth was observed, and the best PAM site was obtained by comparison.

Table 1. Selection of the PAM sites

| Selection of the PAM sites |
| --- |
| pJYS1-MAD7-gRNA-TTTA(+) |
| pJYS1-MAD7-gRNA-TTTC(-) |
| pJYS1-MAD7-gRNA-TTTT(-) |
| pJYS1-MAD7-gRNA-TTTG(-) |
| pJYS1-MAD7-gRNA-TTTC(+) |
| pJYS1-MAD7-gRNA-TTTA(-) |
| pJYS1-MAD7-gRNA-CTTC(+) |
| pJYS1-MAD7-gRNA-CTTA(+) |
| pJYS1-MAD7-gRNA-CTTG(-) |
| pJYS1-MAD7-gRNA-CTTT(+) |

## 1.3 1st ITERATION – test

We initially estimated the successful introduction of the plasmid into
the colonies on the plate, and verified it by colony PCR. For successful
validation, the solution after colony PCR reaction was taken for gene
sequencing to test whether successful knockout of related genes.

Selection of the optimal promoter

![](https://static.igem.wiki/teams/4687/wiki/content-pages/engineering/a-pec-ptuf-mad7.png)

(a) pEC-Ptuf-MAD7

![](https://static.igem.wiki/teams/4687/wiki/content-pages/engineering/b-pec-placm-mad7.png)

(b) pEC-PlacM-MAD7

![](https://static.igem.wiki/teams/4687/wiki/content-pages/engineering/c-pec-ptac-mad7.png)

(c) pEC-Ptac-MAD7

Figure 1. Colony growth using different promoters.(White colonies were
successful for gene knockout, and yellow colonies were unsuccessful)

![](https://static.igem.wiki/teams/4687/wiki/content-pages/engineering/qga7buhcuoagagxq1vxcvvqdn9d.png)

Figure 2. The efficiency plots of the knockdown genes using the
different promoters.

By contrast, we can obtain the optimal promoter that is PlacM.

![](https://static.igem.wiki/teams/4687/wiki/content-pages/engineering/a-pjys1-mad7-ttta.png)

(a) pJYS1-MAD7-TTTA

![](https://static.igem.wiki/teams/4687/wiki/content-pages/engineering/b-pbluescript-mad7-ttta.png)

(b) pBluescript-MAD7-TTTA

![](https://static.igem.wiki/teams/4687/wiki/content-pages/engineering/c-pxmj19-mad7-ttta.png)

(c) pXMJ19-MAD7-TTTA

Figure 3. Colony growth in the case of different plasmids.(White
colonies were successful for gene knockout, and yellow colonies were
unsuccessful)

![](https://static.igem.wiki/teams/4687/wiki/content-pages/engineering/qutgbiclwoclhmxdqxxckpehndh.png)

Figure 4. The efficiency plots of the knockdown genes using the
different plasmids.

By contrast, we can obtain the optimal plasmid that is pJYS1.

![](https://static.igem.wiki/teams/4687/wiki/content-pages/engineering/a-pjys1-ptuf-mad7.png)

(a) pJYS1-Ptuf-MAD7

![](https://static.igem.wiki/teams/4687/wiki/content-pages/engineering/b-pjys1-placm-mad7.png)

(b) pJYS1-PlacM-MAD7

![](https://static.igem.wiki/teams/4687/wiki/content-pages/engineering/c-pjys1-ptac-mad7.png)

(c) pJYS1-Ptac-MAD7

Figure 5. To verify the colony growth at the promoter.(White colonies
were successful for gene knockout, and yellow colonies were
unsuccessful)

The comparison showed that the best promoter PlacM selected when using
plasmid pEC-XK99E was also the most efficient knockout gene in plasmid
pJYS1.

1. Selection of the best PAM site

Since there are many PAM sites and many plate maps, we selected two
representative plate plots here.

![](https://static.igem.wiki/teams/4687/wiki/content-pages/engineering/a-pjys1-mad7-grna-ttta.jpg)

(a) pJYS1-MAD7-gRNA-TTTA(+)

![](https://static.igem.wiki/teams/4687/wiki/content-pages/engineering/b-pjys1-mad7-grna-tttc.jpg)

(b) pJYS1-MAD7-gRNA-TTTC(+)

Figure 6. Colony growth at the different PAM sites.(White colonies were
successful for gene knockout, and yellow colonies were unsuccessful)

![](https://static.igem.wiki/teams/4687/wiki/content-pages/engineering/chuzbcw3sorw9txbz1gccw7entc.png)

Figure 7. The efficiency plots of the knockdown genes using the
different PAM sites.

By contrast, we can obtain the optimal PAM site that is TTTC(+).

## 1.4 1st ITERATION – learn

We obtained the gene editing efficiency by comparing and calculating the
color of the colonies on the plate, and selected the best parts.

The best promoter is PlacM. The best plasmid is pJYS1. The best PAM site
is TTTC(+).

In summary, we have optimized the CRISPR system to obtain the
pJYS1-MAD7-gRNA-TTTC(+) plasmid, and we will expand the subsequent
experiments accordingly.

# 2. 2ND ITERATION

### Build strains that produce high lycopene

## 2.1 2nd ITERATION – design

In the previous research, we have optimized the CRISPR-MAD7(Cas12a)
system to improve the efficiency of gene editing of the CRISPR-MAD7
system in *C. glutamicum* ATCC13032 by selecting the plasmid,
optimal PAM site, promoter, etc.Therefore, by using the optimized
CRISPR-MAD7(Cas12a) system to edit the lycopene-producing gene of
*C. glutamicum* ATCC13032, the purpose of lycopene enrichment can
be achieved.

We first build the relevant plasmids of the CRISPR-MAD7(Cas12a) system
that have been optimized in the early stage. And designed to accurately
locate the gene *crtEb, crtR* upstream and downstream gRNA. Under
the action of this gRNA, the downstream genes *crtEb*,*crtR*
of lycopene can be accurately knocked out, so as to achieve the
accumulation of lycopene.

## 2.2 2nd ITERATION – build

* pJYS1-MAD7-*ΔcrtEb*(Knock out the *crtEb* gene)
* pJYS1-MAD7-*ΔcrtR*(Knock out the *crtR* gene)
* pJYS1-MAD7-*ΔcrtEb*:*ΔcrtR*(Knock out the
  *crtEb/crtR* dual gene)

![](https://static.igem.wiki/teams/4687/wiki/content-pages/engineering/yn6tbqgxcoqxk9xmggtckhrontc.jpg)

Figure 9. Schematic diagram of the three plasmids.

A. The plasmid was transferred into *Escherichia coli* Top10
competent cells by heat shock and cultured in LB solid medium at 37°C.

B. Verify and extract plasmid.Through resistance screening, colony PCR
verifies whether the plasmid is successfully introduced into
*Escherichia coli* Top10. For the successful verification of
bacteria, the bacteria were expanded and cultured, and the plasmid DNA
was extracted the next day and sent to the company for gene sequencing.

C. Transfer the sequenced correct plasmid into *C. glutamicum*
ATCC13032 by electroporation and culture in LB solid medium at 30°C.

![](https://static.igem.wiki/teams/4687/wiki/content-pages/engineering/m9pibe5drobniexkrxwc1ylpnye.png)

(a) *C. glutamicum* ATCC13032*-ΔcrtEb*

![](https://static.igem.wiki/teams/4687/wiki/content-pages/engineering/eolfbxtcforexjxxli9czwpnnh0.png)

(b) *C. glutamicum* ATCC13032*-ΔcrtR*

![](https://static.igem.wiki/teams/4687/wiki/content-pages/engineering/plqmbihfgorumbxyjiyclxpynsb.png)

(c) *C. glutamicum* ATCC13032*-ΔcrtEb:ΔcrtR*

Figure 11. Three modified strain medium plates.

## 2.3 2nd ITERATION – test

First, through the naked eye, we can find that the color of the colony
has changed, and the plasmid introduction is preliminarily estimated to
be successful, and the colony PCR verification is performed. For
successful verification of bacteria, the solution after colony PCR
reaction was taken for gene sequencing to verify whether the relevant
genes were successfully knocked out.

![](https://static.igem.wiki/teams/4687/wiki/content-pages/engineering/figure12-a.jpg)

(a)*C. glutamicum* ATCC13032*-ΔcrtEb*

![](https://static.igem.wiki/teams/4687/wiki/content-pages/engineering/figure12-b.jpg)

(b)*C. glutamicum* ATCC13032*-ΔcrtR*

![](https://static.igem.wiki/teams/4687/wiki/content-pages/engineering/figure12-c.jpg)

(c)*C. glutamicum* ATCC13032*-ΔcrtEb:ΔcrtR*

Figure 12. Colony PCR electrophoresis verification.

For cultures that verified successful knockout genes, we transferred
them to 300 mL flasks for scale-up. After a certain period of time, a
certain sample bacterial solution was taken and the yield of lycopene in
the modified strain was measured by high performance liquid
chromatography (HPLC) to evaluate the effect of experimental
manipulation on lycopene synthesis.

![](https://static.igem.wiki/teams/4687/wiki/content-pages/engineering/figure13.jpg)

2.4 Figure 13. Precipitation after centrifugation of strains.

## 2nd ITERATION – learn

In the previous project, we optimised the CRISPR-MAD7(Cas12a) system to
achieve the highest gene editing efficiency in
*Corynebacterium glutamicum*, and thus we wished to construct
strains with a high lycopene yield that could produce lycopene in large
quantities. We gradually constructed
*ΔcrtEb, ΔcrtR, ΔcrtEb:ΔcrtR*strains. Through the analysis we can
find that knocking out the branched genes can lead to a good increase in
lycopene yield. It helps us to better solve many problems, such as low
efficiency of lycopene in industrial production.

{% endblock %}

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

# 1. Getting carbon sources from straw

### 1.1 Aim

China itself is a large agricultural country produces a large amount of
straw waste every year, and relevant data show that 865 million tons of
straw will be produced nationwide in 2021. The purpose of this
experiment is to obtain the carbon source through straw treatment, which
can improve the nutrients for the subsequent culture of
*Corynebacterium glutamicum*, realize the overall integrity of
the experiment, and also play a role in protecting the environment. The
carbon source obtained from straw was mainly glucose obtained through
enzyme treatment after treating waste straw with diluted acid.

### 1.2 Experimental design

#### Experimental materials

* Straw
* 1% diluted sulfuric acid
* cellulase
* buffer pH4.8 (HAc-NaAc)

Before starting the experiment the straw needs to be pulverized and
screened for straw powder 48 mesh. After pretreatment with diluted
sulfuric acid, the moisture content in the solids was measured. It can
then be treated with cellulase and filtered to obtain glucose.

![](https://static.igem.wiki/teams/4687/wiki/content-pages/experiments/illustrator.jpg)

Figure 1. Extraction of glucose from the straw.

For more details on the experiments and protocols, as well as the
results of the experiments, see the link.

[Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)

[Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)

# 2. Selection of the optimal promoter

### 2.1 Aim

The purpose of this experiment is to select the promoter which are
suitable to play a good role in *Corynebacterium glutamicum*.

A promoter is a DNA sequence located in the upstream region of a gene,
which serves to initiate gene expression during transcription.
Optimizing the promoter increases the localization and binding of the
CRISPR-MAD7(Cas12a) system on the target gene, thus improving the
editing efficiency. By replacing or changing promoters, gene expression
levels can be adjusted. By making these improvements, the efficiency of
gene editing in *Corynebacterium glutamicum* by the
CRISPR-MAD7(Cas12a) system can be further enhanced.

### 2.2 Experimental design

#### Experimental materials

* pEC-XK99E plasmid vector
* Ptuf promoter
* PlacM promoter
* Ptac promoter
* Ptrc promoter

We looked for relevant promoters through the literature. First of all, a
general screening was carried out, and we constructed a recombinant
plasmid by substituting the promoter on the plasmid of pEC-XK99E. The
recombinant plasmid with correct sequencing results was transferred into
*Corynebacterium glutamicum* by electrotransformation, and the
suitable promoter were derived by observing the growth of the final
plate colony.

For more details about the experiments and protocols, as well as the
results of the experiments, please see the links.

[Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)

[Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)

![](https://static.igem.wiki/teams/4687/wiki/content-pages/experiments/w1lzbt5roomrhmxqquicfgawndf.jpg)

![](https://static.igem.wiki/teams/4687/wiki/content-pages/experiments/ma5wb29yho0drjxi7incchcvnue.jpg)

![](https://static.igem.wiki/teams/4687/wiki/content-pages/experiments/wjiub8ivpogybvxzz3dcxes4ngg.jpg)

Figure 2. The construction of pEC-Ptac-MAD7,pEC-Ptuf-MAD7 and
pEC-PlacM-MAD7.

# 3. Selection of the optimal plasmid

### 3.1 Aim

When using CRISPR-MAD7(Cas12a) for gene editing, it is often necessary
to deliver CRISPR components, such as the Cas12a protein and gRNA, into
the target cell to ensure that they work properly within the cell. To
efficiently deliver CRISPR components into the target cell, it is often
necessary to use a plasmid, due to the fact that plasmids can be used to
store, replicate and deliver exogenous DNA.

### 3.2 Experimental design

#### Experimental material

* pJYS1 plasmid
* pBluescript plasmid
* pXMJ19 plasmid
* gRNA-donor
* MAD7 system

By constructing pJYS1-MAD7-TTTA, pBluescript-MAD7-TTTA, pXMJ19-MAD7-TTTA
plasmids, which were transferred into
*Corynebacterium glutamicum* by electrotransformation, the growth
of the colonies on the culture medium was observed. The best plasmid can
only be determined by checking the situation against each other.
Considering the required situation, three BHIS media and the
corresponding antibiotics were required for the intra-experimental
process.

After repeating the experiment, the most suitable plasmid was selected
by observing the growth of the plate colonies as a basis for subsequent
experiments.

For more details about the experiments and protocols, as well as the
results of the experiments, please refer to the link.

[Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)

[Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)

![](https://static.igem.wiki/teams/4687/wiki/content-pages/experiments/a1drbx7iro3qb6xxejhckhponhb.jpg)

![](https://static.igem.wiki/teams/4687/wiki/content-pages/experiments/fgpobc4xhoinetxkwakcpqvenph.jpg)

![](https://static.igem.wiki/teams/4687/wiki/content-pages/experiments/tjeobkev6ovrqhxa6ykczsm6neb.jpg)

Figure 3. Plasmid construction process of pJYS1-MAD7,pBluescript-MAD7
and pXMJ19-MAD7.

# 4. Validation of promoters on the optimal plasmid

### 4.1 Aim

To ensure that promoters capable of efficient expression on the
pEC-XK99E plasmid are also highly expressed on the newly determined
optimal plasmid, validations of the promoter on the optimal plasmid was
performed.

### 4.2 Experimental design

#### Experimental Materials

* pJYS1 plasmid
* Ptuf promoter
* PlacM promoter
* Ptac promoter
* Ptrc promoter

The experimental manipulation of the first step was repeated after
replacing the plasmid growth of the pEC-XK99E plasmid with the best
plasmid.

For more details about the experiments and protocols, as well as the
results of the experiments, please see the link.

[Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)

[Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)

# 5. Selection of the best PAM site

### 5.1 Aim

The purpose of this experiment is to select the PAM site that can help
the CRISPR-MAD7(Cas12a) system can work better in
*Corynebacterium glutamicum*.A PAM site is a short sequence
adjacent to a target DNA sequence, which is a specific sequence that is
required by Cas proteins when they are used to recognize exogenous DNA
sequences in the CRISPR-MAD7(Cas12a) system. In the CRISPR-MAD7(Cas12a)
system, Cas proteins typically identify a target DNA sequence by
recognizing and binding to a PAM site and performing DNA cleavage or
other modifications at that location. Different types of Cas proteins
have specific PAM preferences, which limits the applicability of the
CRISPR-MAD7(Cas12a) system. On top of selecting the pJYS1 plasmid as the
basis for applying the CRISPR-MAD7(Cas12a) system in
*Corynebacterium glutamicum*, we will further select the optimal
PAM sites in order to improve the efficiency of gene editing.

### 5.2 Experimental design

#### Experimental Materials

* pJYS1-MAD7-TTTN plasmid

We firstly selected (Table 1) the PAM sites as experiments through
thesis research. By changing the difference of gRNA in the process of
plasmid construction, that is to say, changing the difference of PAM
sites, and finally linking them together by primer design. The first
step is to amplify the target bands by PCR, and then to constitute the
recombinant plasmid by one-step cloning method. The recombinant plasmid
is transformed into *E. coli* and verified by colony PCR, then
the plasmid is extracted and sent to the company for sequencing. After
the plasmid is correct, it can be transformed into
*Corynebacterium glutamicum* by electrotransformation. Finally,
the most suitable PAM site can be obtained by comparing the experimental
results.

For more details about the experiments and protocols, as well as the
results of the experiments, please see the link.

[Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)

[Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)

Table 1. Selection of the PAM sites

| Selection of the PAM sites |
| --- |
| pJYS1-MAD7-gRNA-TTTA(+) |
| pJYS1-MAD7-gRNA-TTTC(-) |
| pJYS1-MAD7-gRNA-TTTT(-) |
| pJYS1-MAD7-gRNA-TTTG(-) |
| pJYS1-MAD7-gRNA-TTTC(+) |
| pJYS1-MAD7-gRNA-TTTA(-) |
| pJYS1-MAD7-gRNA-CTTC(+) |
| pJYS1-MAD7-gRNA-CTTA(+) |
| pJYS1-MAD7-gRNA-CTTG(-) |
| pJYS1-MAD7-gRNA-CTTT(+) |

# 6. Construct strain of bacteria

### 6.1 Aim

Through a lot of experiments, we have found the plasmid, PAM site,
promoter suitable for use in *Corynebacterium glutamicum*. In the
following experiments, we need to knock out the genes affecting the
production of lycopene by using the well established CRISPR-MAD7(Cas12a)
system.

We firstly chose to knock out the *CrtEb* gene, according to the
principle that after knocking out *CrtEb*, it will reduce the
further decomposition of lycopene, so as to achieve the purpose of
enriching lycopene. And the color of
*Corynebacterium glutamicum* will change from yellow to red after
knocking out the *CrtEb* gene.

### 6.2 Experimental design

By constructing the plasmid which is the knockdown of
*CrtEb* gene as shown in the figure, the efficiency of the
CRISPR-MAD7(Cas12a) system to knockdown the *CrtEb* gene was
verified by transforming the recombinant plasmid which was sequenced
correctly into *Corynebacterium glutamicum*, and observing the
growth. And the strain with the correct knockdown gene will be saved as
the basis for subsequent experiments. At the same time, we can use the
strain that has knocked out the *CrtEb* gene as a sensory state
as a basis for further experiments in the future.

For more details about the experiments and protocols as well as the
experimental results, please refer to the link.

[Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)

[Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)

# 7. Build strains that produce high lycopene

## Optimizing chassis cells

### 7.1 Aim

This experiment aims to study the recombinant efficiency by expressing
the recE/T enzyme system in *C. glutamicum* ATCC13032, providing
a more efficient chassis for later gene manipulation. Because of the low
efficiency of gene recombination for wild
*Corynebacterium glutamicum*. Reviewing the relevant literature,
it was found that the expression of recE/T enzyme system significantly
improved the recombination efficiency of
*Corynebacterium glutamicum*, and provided a more efficient
chassis cell for later gene manipulation.

### 7.2 Experimental Design

#### Experimental Materials

* *C. glutamicum* ATCC13032
* pEC-XC99E-recET

![](https://static.igem.wiki/teams/4687/wiki/content-pages/experiments/czqcbquitogzdpxfldqcy610n4g.jpg)

Figure 5.Chassis cell construction process.

First, plasmids containing genes associated with the recE/T enzyme
system were designed and synthesized. Plasmid amplification by
transformation of TOP10 strains. The plasmid was then transferred to
*C. glutamicum*  ATCC13032 by electroconversion. Positive clones
carrying recE/T enzyme system plasmids are obtained by screening for
resistance genes (e.g. kanamycin resistance genes). Finally, the
sequencing method was used to ensure the successful introduction of
recE/T gene.

# 8. Construction of the *ΔcrtEb* strain

### 8.1 Aim

This experiment aims to explore the application of CRISPR-MAD7(Cas12a)
system in gene editing by editing the *crtEb* gene of
*Corynebacterium* *glutamicum* by using the
CRISPR-MAD7(Cas12a) system, and to construct a strain with high yield of
lycopene.

### 8.2 Experimental Design

#### Experimental Materials

* *C. glutamicum* ATCC13032-RecE/T
* pJYS1-MAD7-*ΔcrtEb*

![](https://static.igem.wiki/teams/4687/wiki/content-pages/experiments/ferrbxbgxo42rtxwvghcvoldn1e.jpg)

Figure 6. *ΔcrtEb* strain construction process.

First, plasmids containing genes and gRNAs associated with the
CRISPR-MAD7(Cas12a) system were designed and synthesized. The plasmid is
then amplified by transformation of TOP10 strains. Next, the plasmid is
transferred into *Corynebacterium glutamicum* cells by
electroconversion method, and positive clones carrying the
CRISPR-MAD7(Cas12a) system plasmid are obtained by screening for
resistance genes (such as kanamycin resistance genes). Gene knockout
verification was performed by PCR and sequencing methods to detect the
knockout of *crtEb* gene. Finally, the plasmid is cured by
subculture.

# 9. Determination of lycopene content

### 9.1 Aim

The aim of this experiment is to measure lycopene yield in
*Corynebacterium glutamicum* using high performance liquid
chromatography (HPLC) to evaluate the effect of genetic engineering on
lycopene synthesis by setting up a control group.

### 9.2 Experimental Design

#### Experimental Materials

* M solution. 60%hexane and 40%acetone
* A solution. 81%methanol,15%MTBE and 4%dd*H*2*O*
* B solution. 7%methanol,90%MTBE and 3%dd*H*2*O*

![](https://static.igem.wiki/teams/4687/wiki/content-pages/experiments/fig7-a.png)

![](https://static.igem.wiki/teams/4687/wiki/content-pages/experiments/fig7-b.png)

Figure 7. Operation steps and principles of HPLC.

1. Take 1 mL of standard product and dry weigh it at 70 degrees
   Celsius, and obtain 1ml standard with a dry weight of x mg.
2. For carotenoid extraction, centrifuge 1 mL of culture. Resuspend the
   cell pellet in 1 mL M solution with vigorous shaking of the shaker
   for 60 sec, and transfer the buffer with pigment to a glass tube.
3. Repeat the above procedure three times at 4°C until the cell pellet
   and supernatant are colorless.
4. Transfer the supernatant to a brown HPLC vial with a screw cap and
   filter the sample with a 1 mL syringe and a 0.2 μm sterile organic
   filter membrane.
5. Use [ZORBAX Eclipse, XDB-C18 (particle size 5 μm, inner diameter 4.6
   mm, length 250 mm), both of which are used in combination with
   inverting protection column and diode array detector (DAD) for
   ultraviolet/visible (Vis) spectral recording]. The α eluate was
   separated from 100% A solution to 100% B solution by binary gradient
   elution and the injection volume was 30 μL at a constant flow rate
   of 1.0 mL/min. Carotenoids were identified by comparing retention
   time and absorption spectra (410-510 nm) at the 410-510 nm peak
   λmax, resulting in lycopene yield assays.

# 10. Engineered cultivation of high-yield lycopene strains

### 10.1 Aim

The aim of this experiment was to evaluate the growth status and
lycopene yield of *Corynebacterium glutamicum* in fermenter
culture, with a view to optimizing fermentation conditions and improving
lycopene production efficiency.

### 10.2 Experimental Design

#### Experimental Materials

* Optimized media.37 g/L BHI,91 g/L sorbitol,2 g/L glucose

  ( Dissolve in deionized water, stir and autoclave)
* Controlled fermenters.The optimal temperature range is 28-32°C.
  The dissolved oxygen content is controlled at about 30%-50%. The
  optimal pH range is generally 6.0-8.0.

![](https://static.igem.wiki/teams/4687/wiki/content-pages/experiments/fig-8-a-1.png)

![](https://static.igem.wiki/teams/4687/wiki/content-pages/experiments/fig-8-b.png)

Figure 8. Operation steps and application of fermenters.

*Corynebacterium glutamicum* was cultured in flasks to detect the
effects of different media components and concentrations on bacterial
growth and lycopene yield. Based on the experimental results, the media
formula was optimized. Transfer the optimized medium to a fermenter and
inoculate with *Corynebacterium* *glutamicum* seed
fluid.Set fermentation conditions such as temperature, pH, stirring
speed, dissolved oxygen, etc. During fermentation, samples are taken
periodically to detect bacterial growth (e.g.biomass, cell density) and
lycopene yield. After the end of fermentation, the fermentation broth is
collected, and lycopene is extracted and purified by extraction,
chromatography and other methods. The yield of lycopene was detected by
HPLC and other methods.

{% endblock %}

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{% block title %}Results{% endblock %}
{% block pageTitle %}Results{% endblock %}
{% block sideBarTitle %}Results{% endblock %}
{% block lead %}
You can describe the results of your project and your future plans here.
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# 1. Incorporating the green eco component - recycling straw

Every year the world produces a lot of straw, and what to do with it is
a problem. Straw is rich in cellulose, which is a polysaccharide. With
proper treatment, we can extract glucose from straw, and we can use this
glucose to cultivate our modified *Corynebacterium glutamicum*,
which can provide a better idea for straw treatment in the future.

After treatment of the straw, we obtained the relevant solution, the
main component of which is glucose. The composition of the sugar
solution of the treated straw is as follows. 6 g of dried solid, after
treatment in 60 mL system, the concentration of the sugar solution
obtained is 27.6 g/L and 23.25 g/L.

|  |  |  |
| --- | --- | --- |
|  | Absolutely dry solid | Straw sugar concentration |
| 60ml system | 6 g | 27.6 g/L |
| 60ml system | 6 g | 23.25 g/L |

Figure 1. Straw sugar concentration obtained from treatment of waste
straws.

We configured the same concentration of glucose solution and cultured
continuously at the same concentration for 120h and measured the growth
curves every 24h. The strains we used were unmodified
*Corynebacterium glutamicum* ATCC13032,
*ΔcrtEb Corynebacterium glutamicum*  ATCC13032 and
*ΔcrtEb:crtR* *Corynebacterium glutamicum*
*ATCC13032*.We obtained the following growth curves. We get the
following growth curve.

We can find that the sugar solution produced after straw treatment is
good for cultivating *Corynebacterium glutamicum*. For group h,
after knocking out the *crtEb* gene, the culture with straw sugar
solution grows more rapidly in a short period of time compared to using
glucose solution. The genes we ultimately wanted to knock out were
*crtEb* and *crtR*, and in the growth curve below, group h
was able to reach an average level, a result that is very promising. In
future experiments, we need to explore the mechanisms that allow the
straw sugar solution to better cultivate
*Corynebacterium glutamicum*.

![]()

![](https://static.igem.wiki/teams/4687/wiki/content-pages/results/growth-curve.png)

Figure 2.(a)13032+Glu,(b)13032+Straw Sugar
Liquid,(c)*ΔcrtR*+Glu,(d)*ΔcrtR*+Straw
Sugar
Liquid,(e)*ΔcrtEb*+Glu,(f)*ΔcrtR*+Straw
Sugar
Liquid,(g)*ΔcrtEb:ΔcrtR*+Glu,(h)ΔcrtEb:*ΔcrtR*+Straw
Sugar Liquid

During the experiment we chose to use cellulase to enzymatically degrade
the straw and use the glucose produced for the culture of
*Corynebacterium glutamicum*. In order to test the effect of
using the glucose produced by the enzymatic degradation of straw to
cultivate *Corynebacterium glutamicum*, and compared with the use
of a standard glucose solution, the results are shown in the figure
below. By observing the glucose content in the solution, we can measure
whether the strain is able to utilize the glucose and whether the
glucose produced from straw has any effect on the growth of the strain.

We performed glucose backfilling after measuring the OD600
value once. The glucose produced by the enzymatic digestion of straw was
not highly utilized compared to the blank group. The glucose from the
enzymatic digestion of straw without any treatment was not well utilized
by *Corynebacterium glutamicum*, compared to the standard glucose
solution. In this regard, we need to treat the glucose obtained from
enzymatic digestion of straw.

In our experiments, we pay special attention to combining environmental
protection and sustainable development, and are committed to solving the
current problem of straw that cannot be reasonably utilized. The use of
straw sugar solution for bacterial culture is a good idea, which not
only can effectively utilize straw resources, but also can further
promote environmentally friendly research.

In the future, we will continue to explore more ways to treat straw and
integrate them more closely into our programs. In this way, we hope to
make a greater contribution to solving energy problems and environmental
governance, and to play our part in realizing the Sustainable
Development Goals. We believe that through continuous efforts and
innovation, we can make significant contributions to building a better
future.

# 2. Promoter Optimization

After identifying the PAM site with the highest editing efficiency of
MAD7, in order to further improve the editing efficiency of MAD7
nuclease, we constructed different promoters using pEC plasmid as a
vector in order to improve the transcriptional-translational efficiency
of MAD7. We constructed four promoters, namely, PlacM, Ptuf, Ptac, and
Ptrc, respectively, to enhance the transcriptional efficiency of MAD7.
Eventually, the average editing efficiency of the PlacM promoter reached
10.97%, the average editing efficiency of the Ptuf promoter was 2.98%,
and the editing efficiency of the Ptac promoter was the lowest, 0. In
this regard, we can continue to optimize the promoters on the basis of
PlacM to finally achieve the effect of a strong promoter.

![](https://static.igem.wiki/teams/4687/wiki/content-pages/results/thilbkgjnopxhuxq8xzcycfdnye.png)

Figure 3.Transformatnt number and editing efficiency of
different promoters.

# 3. Selection of the optimal plasmid

The editing efficiency of MAD7 nuclease can be maximized by using
appropriate plasmids to carry MAD7 nuclease in different situations. In
our project, we selected different plasmids to carry fragments of MAD7
nuclease into *Corynebacterium glutamicum* ATCC13032. We used
four plasmids, pJYS1, pBluescript, pEC-XK99E, and pXMJ19. as can be seen
from the graphs, the average editing efficiency and the number of
transformations did not differ much when using the pXMJ19 and
pBluescript plasmids as vectors. pEC-XK99E plasmid vector, although it
transformed the largest number of colonies, the editing efficiency was
the The pEC-XK99E plasmid vector had the highest number of transformed
colonies but the lowest editing efficiency. The highest editing
efficiency was for pJYS1, which reached 80% and had a high number of
transformations relative to pXMJ19 and pBluescript plasmids. We
transferred the plasmids into *Corynebacterium glutamicum* ATCC
13032, and continuously constructed and optimized the plasmids based on
our screened plasmids.

![](https://static.igem.wiki/teams/4687/wiki/content-pages/results/x5qsbk8kroufzdxkrybcmpkun3e.png)

Figure 4.Transformant number and editing efficiency of
different plasmids.

# 4. Selection of the best PAM site

In the course of this study, in order to explore the preference of PAM
sites of CRISPR-MAD7(Cas12a) system in
*Corynebacterium glutamicum*, to find the most suitable PAM sites
for the work of MAD7 nuclease complex, and to improve the efficiency and
accuracy of gene editing technology of CRISPR-MAD7(Cas12a) system in
*Corynebacterium glutamicum*. We tried to design a total of 11
different PAM sites,which are TTTA(+), TTTC(-), TTTT(-), TTTG(-),
TTTC(+), TTTA(-), TTTT(-), CTTC(+), CTTA(+), CTTG(-), CTTT(+). The
plasmids were constructed on this basis to explore their editing
efficiency and to find the best PAM sites.

![]()

![](https://static.igem.wiki/teams/4687/wiki/content-pages/results/inskbqm0godqptx2znycco15nxg.png)

Figure 5.Transformant number and editing efficiency of
different PAM sites.

Through the results, we can find that TTTA(+), TTTC(-), TTTT(-),
TTTG(-), TTTC(+), TTTA(-), TTTT(-), CTTG(-), CTTT(+), which are PAM
sites, are helpful for the CRISPR-MAD7(Cas12a) system to perform better
gene editing in *Corynebacterium glutamicum*. However, in terms
of colony number and transformation efficiency, TTTC(+) is superior,
which means that TTTC(+) can give the best performance compared with
other PAM sites. Of course, this also means that gene editing at this
PAM site is easier to achieve and performs well in terms of success rate
and efficiency. Based on this finding, we determined that this PAM site
would be an important basis for our subsequent experiments.

# 5. Large Segment Knockout Function Test for MAD7 System

Based on the efficiency and effectiveness of the CRISPR-MAD7(Cas12a)
system, we verified the knockdown efficiency of the CRISPR-MAD7(Cas12a)
system on *Corynebacterium glutamicum* gene fragments by randomly
knocking down genes with sizes of 5 kb, 10 kb, 15 kb, 20 kb, and 25 kb,
and obtained the following results, with specific editing efficiencies
as follows.

![](https://static.igem.wiki/teams/4687/wiki/content-pages/results/data-8.png)

Figure 6.Transformant number and editing efficiency of
large Segment Knockout

From the experimental results, it can be observed that the CRISPR-MAD7
(Cas12a) system has the highest efficiency of up to 83% for knocking out
10kb genes in *Corynebacterium glutamicum*, and more than 50% for
knocking out 5kb and 20kb genes, meanwhile, the CRISPR-MAD7 (Cas12a)
system can knock out genes of 25kb size, which is something no other
system can achieve. Of course, for the knockout of 25kb genome, we still
need to further explore and optimise the experiment. Meanwhile, these
results also indicate that the CRISPR-MAD7(Cas12a) system we found is
more suitable for gene editing in *Corynebacterium glutamicum* .

# 6. Construction of strains

After completing the selection of promoters, PAM sites and plasmids, we
started to achieve our ultimate goal of efficient lycopene production by
knocking out the relevant genes. We used pJYS1 plasmid as a vector,
PlacM as a promoter, and TTTC(+) as the final PAM site on the basis of
which we carried out the construction of plasmids for knockdown genes.
The construction of the final strain with high lycopene production was
completed. We mainly performed pJYS1-MAD7-*ΔcrtEb* (Knock out the
*crtEb* gene), pJYS1-MAD7-*ΔcrtR* (Knock out the
*crtR* gene), pJYS1-MAD7-*ΔcrtEb:ΔcrtR* (Knock out the
*crtEb/crtR* dual gene) strains were constructed. The results
were shown as follows.

## Construction of *ΔcrtEb* strains

![](https://static.igem.wiki/teams/4687/wiki/content-pages/results/fig-7-1.png)

Figure 7.Knockdown efficiency of the *crtEb* gene
by MAD7

![](https://static.igem.wiki/teams/4687/wiki/content-pages/results/miczbmjnvobvirxvyqecz45anpe.png)

Figure 8.  *C. glutamicum* ATCC13032-*ΔcrtEb*

From the results, we can see that the editing efficiency of the
constructed pJYS1-MED7-*ΔcrtEb* strain is as high as more than
90%. This is a good verification that the system we constructed is very
effective.

## Construction of *ΔcrtR* strains

![](https://static.igem.wiki/teams/4687/wiki/content-pages/results/z7lmbev9mokhcdxx0bvcdxcanld.png)

Figure 9. *C. glutamicum* ATCC13032-*ΔcrtR*

From the results, we can see that the constructed pJYS1-MED7-*ΔcrtR*
strain changed its colour significantly.

## Construction of *ΔcrtEb:ΔcrtR* strains

![](https://static.igem.wiki/teams/4687/wiki/content-pages/results/jzrjbcmyyohg3lxwl5pcf3c1nsg.png)

Figure 10. *C. glutamicum* ATCC13032-*ΔcrtEb:ΔcrtR*

From the results, we can see that the colour of the constructed
pJYS1-MAD7-*ΔcrtEb:ΔcrtR* strain changed significantly. It can be
clearly seen that the colour of the strain has turned pink indicating a
substantial increase in lycopene production.

By selecting plasmid vectors, choosing PAM sites, optimizing promoters
and performing large fragment genome knockdown experiments, we
successfully constructed a new editing system for targeting
*Corynebacterium glutamicum* for transformation. Our experiments
provide valuable references for future research teams and directions on
where to start to modify a particular bacterium.

Besides, compared with the existing system, our constructed system is
more convenient and suitable for industrialized production. And
lycopene, as an excellent antioxidant, has an important role in
biomedicine and food health. Through the modified
*Corynebacterium glutamicum*, we realized the accumulation of
lycopene, which opened a new way for the production of lycopene and
promoted the development and progress of related fields.

![](https://static.igem.wiki/teams/4687/wiki/content-pages/results/growth-curve.png)

Figure 11. (a)13032+Glu,(b)13032+Straw Sugar
Liquid,(c)*ΔcrtR*+Glu,(d)*ΔcrtR*+Straw
Sugar
Liquid,(e)*ΔcrtEb*+Glu,(f)*ΔcrtR*+Straw
Sugar
Liquid,(g)*ΔcrtEb*:*ΔcrtR*+Glu,(h)*ΔcrtEb:ΔcrtR*+Straw
Sugar Liquid

We can find that the sugar solution produced after straw treatment is
good for cultivating *Corynebacterium glutamicum*. For group h,
after knocking out the *crtEb* gene, the culture with straw sugar
solution grows more rapidly in a short period of time compared to using
glucose solution. The genes we ultimately wanted to knock out were
*crtEb* and *crtR*, and in the growth curve below, group h
was able to reach an average level, a result that is very promising. In
future experiments, we need to explore the mechanisms that allow the
straw sugar solution to better cultivate
*Corynebacterium glutamicum*.

# 7. Successful production of lycopene

![](https://static.igem.wiki/teams/4687/wiki/content-pages/results/cch8bhtiuof2x5xr8tecf6bqnyc.png)

Figure 12. *ΔcrtR* strain on the left,
*ΔcrtEb:ΔcrtR* strain on the right.

After previous strain construction, we ended up with expanded cultures
in shake flasks and ended up with the strains shown in the figure. On
the left is the *ΔcrtEb* strain and on the right is the
*ΔcrtR/crtEb* strain. Where we can see that the
*ΔcrtR*:*ΔcrtEb* strain produced significantly higher
lycopene yields.

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