iGEM 2022 — Team worldshaper-hzbiox

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Worldshaper-HZBIOX

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# About

February to March, 2022

Recruitment of team members

Team registration

April, 2022

Project brainstorming

iGEM medal list studying
Biology theory studying

May, 2022

Project background survey
Lab safety and security training
HP work startup
Team logo and jersey design
Experimental principles learning
Web design learning

June, 2022

Team uniform and logo production
Distribution and collection of questionnaires
Project design and expert interview
Promotion video design
Completion of crowdfunding
Hardware design learning
Experiment preparation
Developing an online platform, including ins, WeChat, Bilibili, weblog
Face to face talk held in Hangzhou foreign language school
Shotting the publicity video
Start to construct a wiki framework

July, 2022

Experimental skills training

Effects exploring of arginine on colon cancer cell line CT26
Knocking out of argR gene of ECN 1917
Construction of △argR-argJ ECN 1917 and function test
Interview planning & executing & summarizing, round 1
interviewing planning & executing & summarizing, round 2
Planning on publicity events (School, Speech…) online publication& announcement
Start PR and raise attention to the project
Self-media update (reality show born)
Wiki design and making
Picture book design
Project modeling
Sponsoring executing
finish project background research &description
online announcement twice

August, 2022

Arginine production analysis

Effects verification of △argR-argJ ECN 1917 on CT26 cells
Construction of pGLO-Lysin plasmid
Cell lysis test of pGLO-Lysin plasmid after transforming into E. Coli DH 5alpha, E. Coli MG1655, ECN 1917
Offline public engagement activity in Hangzhou low carbon science and technology museum
Publicity events with partner
Self-media update
Applied design
Social research and interviews with experts in the immunotherapy field
Social research and interviews with experts in Gut Microbiome
Field trip for pharmacy factory in Changsheng
Web Design and Optimization
Cohost an online meetup with Worldshaper-Shanghai
Promotional video production

September, 2022

Improvement of pGLO-Lysin plasmid (with LZU-China)

Applied design optimization (Collaborated with LZU-China 2022iGEM team)
Wiki page design and making
Manuscripts writing
Preparation of PPT
Collaborating on a three-line poem activity with a synthetic biology theme with NEFU\_China
Cooperation with worldshaper-SH for academic exchange activities

October, 2022

Optimization of Wiki

Preparation of presentation video
Submission of final judging form
Parts registry
Participation of 2022 giant jamboree

detailed information

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© 2022-Content on this site is licensed under a [Creative Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).

The repository used to create this website is available at [gitlab.igem.org/2022/worldshaper-hzbiox](https://gitlab.igem.org/2022/worldshaper-hzbiox).

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Worldshaper-HZBIOX

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## **Summary**

Cancer is currently the trickiest enemy for human beings.
it is said that colorectal cancer ranks second among the most common types of
cancer in terms of mortality
worldwide.[(https://www.spandidos-publications.com/10.3892/mco.2021.2433)](https://www.spandidos-publications.com/10.3892/mco.2021.2433)Our
project aims to develop a modified E. coli Nissle 1917 (EcN1917) that can
persistently produce arginine in the tumor microenvironment (TME). There are
two reasons to increase the arginine concentration in the TME: First, arginine
can kill cancer cells. Plus, supplement of arginine activates the immune
system that can attack cancer
cells.[(https://www.x-mol.com/paper/1262894917545467904/t?recommendPaper=1311354128826601472)](https://www.x-mol.com/paper/1262894917545467904/t?recommendPaper=1311354128826601472) Native EcN1917 has pathways
for arginine synthesis but they are inhibited by the feedback of arginine in
the environment. Therefore, to reach our goals, we constructed EcN1917 to
maximize arginine production by avoiding these feedback inhibitions.

## **Here are the project steps:**

(1) We knocked out the argR gene which encodes ArgR
protein. ArgR represses the expression of enzyme genes important for arginine
biosynthesis in response to the arginine level in the cell.

(2) We added the argJ gene derived from Corynebacterium
to remove the arginine inhibition on NAGS enzyme encoded by argA gene. NAGS is
responsible for the acetylation of glutamate, an important step in arginine
biosynthesis in EcN1917, but is inactivated by arginine. argJ encodes
bifunctional glutamate N-acetyltransferase/amino-acid acetyltransferase, which
can also acetylate glutamate and can function well at high concentration of
arginine.

(3) To ensure biosafety, we inserted the lysin gene into
our designed EcN1917 so that it would be lysed in response to
arabinose after the bacteria
have done their work.

In our project, we successfully constructed nine parts to
fulfill the above goals (Table 1).

Table 1 Part list

| No. | Name | Type | Description | Designer | Length |
| --- | --- | --- | --- | --- | --- |
| 1 | [BBa\_K4410000](http://parts.igem.org/Part%3ABBa_K4410000) | Basic | Fragment H1 from E. coli Nissle 1917 | Ruyi Shi | 1000bp |
| 2 | [BBa\_K4410001](http://parts.igem.org/Part%3ABBa_K4410001) | Basic | Fragment H2 from E. coli Nissle 1917 | Ruyi Shi | 1000bp |
| 3 | [BBa\_K4410002](http://parts.igem.org/Part%3ABBa_K4410002) | Basic | argR | Ruyi Shi | 472bp |
| 4 | [BBa\_K4410003](http://parts.igem.org/Part%3ABBa_K4410003) | Basic | KanR | Ruyi Shi | 795bp |
| 5 | [BBa\_K4410004](http://parts.igem.org/Part%3ABBa_K4410004) | Basic | argJ | Ruyi Shi | 1167bp |
| 6 | [BBa\_K4410005](http://parts.igem.org/Part%3ABBa_K4410005) | Composite | H1-argR-H2 | Ruyi Shi | 2472bp |
| 7 | [BBa\_K4410006](http://parts.igem.org/Part%3ABBa_K4410006) | Composite | H1-KanR-H2 | Ruyi Shi | 2795bp |
| 8 | [BBa\_K4410007](http://parts.igem.org/Part%3ABBa_K4410007) | Device | J23100-argJ | Ruyi Shi | 1334bp |
| 9 | [BBa\_K4410008](http://parts.igem.org/Part%3ABBa_K4410008) | Basic | Terminator | Ruyi Shi | 87bp |

## **1. Natural EcN1917 survivability under arginine**

Before engineering EcN1917, we first used MTT assay to test
whether high arginine concentration might inhibit the growth of native
EcN1917. The results showed that EcN1917 survived
and grew well at arginine concentration ranging from 0
µg/mL to 10 mg/mL (Figure 1). ≥20
mg/mL arginine suppressed the survival of EcN1917. This
tolerance range of arginine concentration indicated that EcN1917 would be
viable when it was engineered to be an arginine synthesizing factory in
vivo.

![](https://static.igem.wiki/teams/4410/wiki/engineering-files/engineering2972.png)

Figure 1: The
effect of arginine on the growth of native
EcN1917

## **2. Engineering EcN1917**

**Step 1: Construction of
EcN1917****△****argR****strain:
Knock out argR**

ArgR protein acts as an arginine repressor of the
arginine synthetic genes in EcN1917, we successfully knocked down the argR
gene in EcN1917 to increase the production of arginine in the strain.

We first linearized the H1-argR-H2 fragment with upper
and lower homologous arms by PCR from EcN1917
WT. The pMD19T vector
and H1-argR-H2 were connected to form Recombinant pMD19T by
TA cloning. H1-pMD19T-H2 was linearized by PCR. The
KanR gene was linearized from pKD4
vector by PCR. KanR gene and
H1-pMD19T-H2 gene were combined into recombinant pMD19T by one-step cloning.
The recombinant pMD19T was heat transformed into a DH5a colony (Figure
4-E). All above steps were
validated by electrophoresis
(Figure 4 A-D).

Donor DNA
(H1-KanR-H2) was linearly extracted from
successfully transformed DH5a by PCR.
Donor DNA was precipitated by alcohol and then electrotransformed
into EcN1917 WT. All above steps were
validated by electrophoresis
(Figure 5 A-B). We
used kanamycin to verify that the argR gene was knocked out
(Figure 5-C). Hence,
 EcN1917△argR strain,
EcN1917 with argR gene knocked out, was
constructed
successfully.

![](https://static.igem.wiki/teams/4410/wiki/engineering-files/engineering4255.png)

Figure 3:Schematic
illustration of the λ red recombination system

![](https://static.igem.wiki/teams/4410/wiki/engineering-files/engineering4324.png)

Figure 4: (A) The electrophoretogram of
H1-argR-H2; (B) The electrophoretogram of
recombinant pMD19T(2680bp); (C) The electrophoretogram of the
H1-pMD19T-H2(4757bp); (D) Electrophoretogram of the
donor DNA (3589bp); (E) DH5α bacteria colony
transformed with the recombinant pMD19T

![](https://static.igem.wiki/teams/4410/wiki/engineering-files/engineering4609.png)

Figure 5: (A) The
electrophoretogram of donor DNA
(H1-KanR-H2) linearly extracted from DH5a
by PCR (3589bp); (B) Electrophoretogram verification of the
successful transformation of EcN1917 with the
donor DNA (2021bp); (C) EcN1917 colonies
electrotransformed with the donor
DNA.

**Step 2: Construction of****E****cN1917****∆****argR****(****arg****J****):
Insert argJ gene into
EcN1917****△****argR**

We first constructed a pGLO-J23100-argJ vector containing
the argJ gene. To construct this vector, we used PCR to clone out a linearized
pGLO-J23100 vector abandoning the GFP gene sequence. Gel electrophoresis
showed that the length of the linearized pGLO-J23100 was about 4524bp, which
was within expectation (Figure 6-A). We then used BW25113 as template to
extract argJ gene fragment by PCR. Gel electrophoresis indicated that the
length of the argJ fragment was about 1223bp, as expected (Figure 6-B).
Finally, we constructed the pGLO-J23100-argJ vector by directly ligating the
linearized pGLO-J23100 and the argJ fragment via one-step cloning. The
pGLO-J23100-argJ vector was then electrotransformed into
EcN1917△argR. The result of gel
electrophoresis for pGLO-J23100-argJ bacterial colony showed that the whole
gene sequence length is about 2025bp, which corresponded to our expectation
(Figure 6-C).

**Functional Verification of****E****cN1917****∆****argR****(****arg****J****)**

The production of L-arginine of the engineered bacteria
was detected by Hitachi amino acid analyzer. Wild type EcN1917 was used as
control. The 24- and 48-hour fermentation broth of the bacteria were collected
and broken by ultrasonic crusher, and detected by automatic amino acid
analyzer (Figure 7). The results showed that
EcN1917∆argR (argJ)
had a yield of 3.6 mM L-arginine.

![](https://static.igem.wiki/teams/4410/wiki/engineering-files/engineering6310.png)

Figure 6: (A) Nucleic acid
gel electrophoresis result of pGLO-J23100. (B)
Nucleic acid gel electrophoresis result of argJ fragment. (C) Nucleic acid gel
electrophoresis result of pGLO-J23100-argJ bacterial colony.

![](https://static.igem.wiki/teams/4410/wiki/engineering-files/engineering6527.png)

Figure 7
L-Arginine production of ECN1917
WT (upper) and
ECN1917∆argR (argJ) (bottom)
after incubation for 48h.

**Step 3: Insert lysin gene into
EcN1917**

We constructed a pGLO-Lysin plasmid by
adding the lysin gene into a pGLO
vector. Gel electrophoresis showed
that the length was as we expected
(Figure 8-A). The pGLO-Lysin plasmid
was successfully electrotransformed into EcN1917
(Figure 8
B-D). EcN1917 transformed
with pGLO-Lysin were lysed significantly by
10-hour treatment of 30mM arabinose (Figure 9).

![](https://static.igem.wiki/teams/4410/wiki/engineering-files/engineering7042.png)

Figure 8 (A) Nucleic acid gel electrophoresis result of
pGLO-lysin plasmid
(5530bp); (B-D)
EcN1917 colonies after transformation (Experimental
group: EcN1917 transformed with
pGLO-Lysin plasmid, Positive control:
EcN1917 transformed with
pSU plasmid, Negative control:
competent EcN1917)

![](https://static.igem.wiki/teams/4410/wiki/engineering-files/engineering7334.png)

Figure 9 Effect of
arabinose on EcN1917 transformed with
pGLO-Lysin plasmid (Left: bacteria solution
without arabinose, right: bacteria solution treated
with 30mM
arabinose for 10h)

## **3. Real sample test**

EcN1917 is one of the kinds of probiotics that is
extremely proper to be used in medicine. This kind of bacteria will not
trigger some inflammations in human bodies. Thus, we choose it as our
engineering-based probiotics. The final bacterial product is equipped to
produce large amount of arginine without feedback inhibition, and can lyse in
response to arabinose. Our product can be used as a treatment approach for
cancer patients by increasing arginine concentration in the tumors and help
the immune system to kill cancer cells. When the treatment is done, arabinose
can be taken to get rid of the bacteria in the body.

To verify the anti-cancer effect of arginine. We had
several cellular experiments on colon cancer CT 26
cells. MTT assay showed that arginine
affected the viability of colon cancer cells
through concentrations dependent manner (Figure 10-A).
As a substrate for endogenous NO synthase, arginine can regulate
the tumor microenvironment through the NO pathway. It is generally believed
that increasing NO level can increase the blood flow to tumors and alleviate
hypoxia in tumors. In order to further explore the effect of L-arginine on the
anaerobic metabolism of tumor cells, different concentrations of L-arginine
were incubated with CT26 cells to detect the level of lactic
acid in the cell supernatant. The experimental results
showed that arginine at low concentrations (10 and 20 mM)
down-regulated the level of lactate metabolism
in tumor cells without affecting cell viability (Figure
10-B). When the concentration of arginine
reached 50 mM, it
had a certain inhibitory effect on cell
viability, and the
cells’ regulation
of lactate level was limited at this time. Since
tumor metabolism is closely related to the tumor immunosuppressive
microenvironment, tumor cells can promote T
lymphocyte apoptosis by upregulating PD-L1 expression. To verify that arginine
can regulate the tumor immunosuppressive
microenvironment, we detected the mRNA expression level of PD-L1 in CT26 cells
after arginine exposure by RT-qPCR. The results showed that arginine at a
concentration of 50 mM could significantly down-regulate the mRNA expression
level of PD-L1 in CT26 cells (Figure
10-C). These experiments indicated that
arginine at high concentrations (100 and 200 mM) could exert a direct
tumor-killing effect. At low concentrations, arginine
could regulate
tumor microenvironment by
down-regulating the expression level of lactic
acid and the mRNA expression level
of PD-L1 in tumor cells,
which was beneficial for tumor
immunotherapy.

![](https://static.igem.wiki/teams/4410/wiki/engineering-files/engineering10099.png)

Figure 10: (A)
Effect of arginine on the viability of CT26 cells (colon cancer cells): the
higher concentration of arginine, the lower survival rate in percent of CT26
cells ; (B) Effect of arginine on
the lactic acid level of CT26 cells ; (C)
Effect of arginine on the PD-L1 mRNA expression of CT26
cells

We also co-cultured
CT26 cells with
the
EcN1917△argRstrain
at MOIs of 25, 50,100, and 200 (number of bacteria cells: number of CT26
cells) for 3 hours, and
evaluated the viability of the CT26
cells by MTT assay, with wild
type EcN1917 (EcN1917)
used as
control.  As
shown in Figure 11,
EcN1917△argR-argJ could
significantly decrease the viability of CT26, as compared to
 EcN1917, which indicating that arginine
producing EcN1917 was capable to exert anti-cancer
effect against colon cancer cells.

![](https://static.igem.wiki/teams/4410/wiki/engineering-files/engineering10907.png)

Figure 11: Effect of
EcN1917△argR on
the viability of CT26
cells (MOI: multiplicity of infection, the ratio of the
number of bacteria to the number of target cells)

## 4. Future work

During the whole progress of the experiments, the
function-testing part is not that efficient to examine how our engineered
probiotics works. We may further do some trials on laboratory mice and deduce
the function on human bodies, which can be better for us to ensure that
arginine will work successfully and the bacteria will not cause any other
damage to human.

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© 2022-Content on this site is licensed under a [Creative Commons Attribution 4.0
International license](https://creativecommons.org/licenses/by/4.0/).

The repository used to create this website is available at [gitlab.igem.org/2022/worldshaper-hzbiox](https://gitlab.igem.org/2022/worldshaper-hzbiox).