iGEM 2021 — Team SZPT-CHINA

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# Team:SZPT-CHINA/Notebook

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* [HOME](https://2021.igem.org/Team%3ASZPT-CHINA)
* TEAM
  + [MEMBERS](https://2021.igem.org/Team%3ASZPT-CHINA/Team)
  + [ATTRIBUTIONS](https://2021.igem.org/Team%3ASZPT-CHINA/Attributions)
  + [COLLABORATIONS](https://2021.igem.org/Team%3ASZPT-CHINA/Collaborations)
  + [PARTNERSHIP](https://2021.igem.org/Team%3ASZPT-CHINA/Partnership)
* PROJECT
  + [DESCRIPTION](https://2021.igem.org/Team%3ASZPT-CHINA/Description)
  + [DESIGN](https://2021.igem.org/Team%3ASZPT-CHINA/Design)
  + [SAFETY](https://2021.igem.org/Team%3ASZPT-CHINA/Safety)
  + [NOTEBOOK](https://2021.igem.org/Team%3ASZPT-CHINA/Notebook)
* EXPERIMENTS
  + [MODEL](https://2021.igem.org/Team%3ASZPT-CHINA/Model)
  + [EXPERIMENTS](https://2021.igem.org/Team%3ASZPT-CHINA/Experiments)
  + [RESULTS](https://2021.igem.org/Team%3ASZPT-CHINA/Results)
  + [FUTURE WORK](https://2021.igem.org/Team%3ASZPT-CHINA/Future_Work)
* PARTS
  + [OVERVIEW](https://2021.igem.org/Team%3ASZPT-CHINA/Parts)
  + [CONTRIBUTION](https://2021.igem.org/Team%3ASZPT-CHINA/Contribution)
  + [ENGINEERING](https://2021.igem.org/Team%3ASZPT-CHINA/Engineering)
  + [IMPROVEMENT](https://2021.igem.org/Team%3ASZPT-CHINA/Improvement)
* IMPLEMENTATION
  + [HARDWARE](https://2021.igem.org/Team%3ASZPT-CHINA/Hardware)
  + [IMPLEMENTATION](https://2021.igem.org/Team%3ASZPT-CHINA/Implementation)
  + [PROOF OF CONCEPT](https://2021.igem.org/Team%3ASZPT-CHINA/Proof_Of_Concept)
* HUMAN PRACTICES
  + [INTEGRATED HUMAN
    PRACTICES](https://2021.igem.org/Team%3ASZPT-CHINA/Human_Practices)
  + [COMMUNICATION](https://2021.igem.org/Team%3ASZPT-CHINA/Communication)
  + [EDUCATION](https://2021.igem.org/Team%3ASZPT-CHINA/Education)
  + [INCLUSIVITY](https://2021.igem.org/Team%3ASZPT-CHINA/Inclusivity)
* [AWARDS](https://2021.igem.org/Team%3ASZPT-CHINA/Awards)

![](https://static.igem.org/mediawiki/2021/6/6f/T--SZPT-CHINA--iconGIF.png)

● week 4(1.17-1.23)

Project:

1. Built a team.
2. Meeting to communicate the future work direction of our team.

● week 5(1.24-1.30)

Project:

1. The principal investigators taught the basic knowledge of synthetic biology for the team members,
and
set
up courses on the learning app to upload relevant materials for everyone to learn at any time.

● week 6(1.31-2.6)

Project:

1. The principal investigators continued to teach the members the basic of synthetic biology.
2. The advisers guided us to learn about art skills, such as graphic design, video edit.
3. Brainstormed and collected everyone's ideas.

● week 7(2.7-2.13)

Project:

1. The team members browsed and studied a lot of outstanding projects in previous years
during the winter
vacation.

2. The principal investigators helped the team members learn the knowledge of synthetic biology
through
online courses.
3. Have the first online meeting to brainstorm, exchange ideas and clash of viewpoints.

● week 8(2.14-2.20)

Project:

1. We witnessed a marked growth of fire accidents in the news and paid attention to the problems
with
burn treatment.

2. We extensively consulted the literature to search for global burn data and clinical burn
treatment
methods.

● week 9(2.21-2.27)

Project:

1. Having the second online meeting to analyze the results of the brainstorming, screen out feasible
ideas,
and conduct preliminary research on them.

● week 10(2.28-3.6)

Project:

1. Reported the results of skills learning and presented the analysis of previous projects in the
form
of
groups.

2. We continued to learn the knowledge of synthetic biology.

● week 11(3.7-3.13)

Project:

1. The team was divided into three groups: experimental group, human practice group and technical
group.

Lab:

1. The experimental group learned the laboratory safety regulations and began experimental skill
training.

● week 12(3.14-3.20)

Project:

1. Learned genetic engineering design under the guidance of instructor.
2. Participated in Ambassador Calls: iGEM 2021 Asian Community Meeting.

Lab:

1. The experimental group continued to learn experimental skills.

● week 13(3.21-3.27)

Project:

1. Each groups planed future work.

● week 14(3.28-4.3)

Project:

1. Conducted literature review and data research.

● week 15(4.4-4.10)

Project:

1. Conducted literature review with regard to *Pseudomonas aeruginosa.*

Human practice:

1. Interviewed firefighters to learn the cause of the fire.

2. Interviewed with the burn doctor of Shenzhen Longgang Central Hospital to consult about burn
treatment
methods, clinical drugs and other issues. And learned that the most common causative agente of burn
infection is bacterial infection, among which *Pseudomonas aeruginosa* has a strong intrinsic
drug
resistance
and the ability to produce drug resistance.

● week 16(4.11-4.17)

Project:

1. Confirmation that the anti-infective target bacteria group is *Pseudomonas aeruginosa.*

Human practice:

1. We consulted with Dr. Jun Li, Department of Burn and Skin Surgery of Xijing Hospital in an
interview
online.

● week 17(4.18-4.24)

Project:

1. It was found in the literature that *Pseudomonas aeruginosa* infection may also occur in
chronic
wounds.

● week 18(4.25-5.1)

Human practice:

1. Offline interviewed with Dr. Taofang He, Department of Chronic Wounds of the Affiliated Hospital
of
Shenzhen Southern University of Science and Technology to learn about the infection of
*Pseudomonas
aeruginosa* in chronic wounds.

2. We consulted with Dr. Jinyu Zhu, Department of Orthopedics and Joint Surgery, Affiliated Hospital
of
Southern University of Science and Technology in an interview online.

● week 19(5.2-5.8)

Project:

1. After careful consideration, we decided to carry out the project in the treatment of burn wounds.

Human practice:

1. In collaboration with associations in Shenzhen Polytechnic, we held the first of the
fluorescent
protein
DIY activity in a school laboratory, introducing synthetic biology.

● week 20(5.9-5.15)

Human practice:

1. Held the second and third of the fluorescent protein DIY activity.

2.Popularized burn knowledge in Buji community, Longgang District, Shenzhen, Guangdong Province.

● week 21(5.16-5.22)

Human practice:

1. Offline interviewed with Dr. Dongyuan Sun, Department of Burn of Shekou People's Hospital in
Shenzhen.

2. We consulted with doctors who from the Department of Burn in northern China in an interview
online.

● week 22(5.23-5.29)

Project:

1. According to relevant literature, we started to design the module of Antipseudomonal Drug
Synthesis
under
the guidance of our instructor.

Human practice:

1. Conducted a lecture on synthetic biology in Shenzhen Guangming District High School.

● week 24(6.6-6.12)

Project:

1. We decided to use bacterial cellulose as the material of wound dressings. This is because
biological
dressings can reduce infection, improve wound healing quality, alleviate patient pain and suffering
compared
with traditional wound dressings.

2. It was found in the literature that visible light is widely used in the medical field, so we were
considering using the new-fangled methods of optogenetics as a gene circuit design method. we
started to
design the module of C-di-GMP Signaling and BC Film Production under the guidance of our instructor.

Human practice:

1. Communicated with Dr. Mingxing Tang in a school lab.

● week 25(6.13-6.19)

Project:

1. Participated in the round-table meeting held by CCIC.
2. Production of BC film was performed using *Gluconacetobacter hansenii* ATCC 53582 and make
it as the
host
chassis.
3. We had finished all the design of the module of Antipseudomonal Drug Synthesis.

Human practice:

1. Paid a return visit to Dr. Weijia Ding from the Burns Department of Longgang Central Hospital in
Shenzhen, asking the doctor about the feasibility of bacterial cellulose and the use of existing
wound
dressings.
2. Communicated with the Professor Jin Fan, he introduced applications of optogenetic to us.

● week 26(6.20-6.26)

Project:

1. Worked with our instructor to improve the gene circuit design of the module of c-di-GMP signaling
and
BC
film production.

Human practice:

1. Opened a new section of synthetic biology on the WeChat official account.

Lab：

1. Planed experimental details and started experiments of the module of C-di-GMP Signaling and BC
Film
Production.

2. Constructed a series of parts for c-di-GMP synthesis enzyme and hydrolysis enzyme in *E.
coli* DH5α.

● week 27(6.27-7.3)

Project:

1. Here we engineered *Gluconacetobacter hansenii* ATCC 53582 with a light-activated
transcriptional
promoter
pDawn to optically regulate expressions of a lysis protein, taking into account of the
actingmeachanism
of
antipseudomonal drugs and environmental and safety issues.

2. After finishing all the gene circuit design of 3 modules, we determined to name them after
Antipseudomonal Drug Synthesis module, C-di-GMP Signaling and BC Film Production module, Safety and
Drug
Release module.

Human practice:

1. Made a synthetic biology summer camp plan for students on campus.

Lab：

1. A series parts of c-di-GMP synthesis enzyme and c-di-GMP hydrolysis enzyme was transferred into
*Gluconacetobacter hansenii* ATCC 53582 with electrotransformation method.
2. PCR products were analyzed using 1% agarose gel electrophoresis to identify bands of expected
sizes.
3. Minimum inhibitory concentration (MIC) was determined to use different antibiotics in
*Gluconacetobacter
hansenii* ATCC 53582.

● week 28(7.4-7.10)

Project:

1. We were designing the main wiki page.

Lab：

1. We did an experiment to screen out the best medium for *Gluconacetobacter hansenii* ATCC
53582 growth.

2. We did an experiment to explore the BC film production of *Gluconacetobacter hansenii* ATCC
53582 in
SOC
and HS medium, using J23100-mcherry-pSEVA331-*G.hansenii* ATCC 53582 and *Gluconacetobacter
hansenii* ATCC 53582.

● week 29(7.11-7.17)

Project:

1. Completed the promotion video.

Human practice:

1. The members of the team carried out the project of bringing home synthetic biology and fire
prevention
knowledge, to preach our project and knowledge of synthetic biology in urban villages in Guangzhou,
Xiamen,
and Chaoshan respectively.
2. More than 70 college students were convened to give lectures on synthetic biology in the
classroom
offline.
3. Started a one-week synthetic biology summer camp, which would provide basic knowledge and
laboratory
skills in popular science microbiology and genetic engineering for college students.

Lab：

1. In order to improve the efficiency of electrotransform plasmid into *Gluconacetobacter
hansenii* ATCC
53582, we did an electrotransformation condition exploratory experiment. Finally, 3kV was selected
as
the
condition of our following experiments.
2. Screen the parts of c-di-GMP synthesis enzyme and c-di-GMP hydrolysis enzyme through exploring
the BC
film production of *Gluconacetobacter hansenii* ATCC 53582 in 12-well plates.

● week 30(7.17-7.24)

Human practice:

1. Connected with Professor Haifeng Ye from East China Normal University, and asked about the
optimization
of near-infrared light parameters.
2. Communicated with Xili Fire Squadron and discovered their learning needs of hazardous
chemicals.
3. We conducted three courses at Yang Qi Culture and Arts Training Institution.

Lab：

1. Here we engineered *E. coli* DH5α with a light-activated transcriptional promoter pDawn to
optically
regulate expressions of S105（BBa\_K3740024）, φX174 E (BBa\_K2656015) and Lysis Cassette LKD16
(BBa\_K3740032),
which uses a random primer labeling method.
2. The lysis strains were preliminarily screened out with 470nm blue light.

● week 31(7.25-7.31)

Project:

1. We chose BphS as the c-di-GMP synthetase, FcsR as the c-di-GMP hydrolase in our system.

2. The light-activated transcriptional promoter pDawn could response to blue light and induce
expression
in
*Gluconacetobacter hansenii* ATCC 53582.

Human practice:

1. We invited a professional with experienced in information dissemination and social organizations
to
guide
us in communication and public outreach of synthetic biology.
2. Participated in the second round-table meeting held by CCIC.
3. Listed chemicals that should not be watered in case of fire for firefighters on the WeChat
official
account.
4. We invited firefighters to conduct fire drills in the community's plaza, while our team members
promote
knowledge of burns.

Lab：

1. Verified the parts of c-di-GMP synthesis enzyme and c-di-GMP hydrolysis enzyme through exploring
the
BC
film production of *Gluconacetobacter hansenii* ATCC 53582 in 12-well plate repeatedly.
2. The vector with the target lysis gene was transferred into *G. hansenii* ATCC 53582 with
electrotransformation method.
3. Verified the function of pDawn promoter in *G.hansenii* ATCC 53582 in response to blue
light.

● week 32(8.1-8.7)

Project:

1. Participated in the Southern China Regional Meeting.
2. Considered that the product is realized in the form of live bacteria.
3. The lysis protein that proves the lysis effect in *Gluconacetobacter hansenii* ATCC 53582 is
X174 E
and
Lysis Cassette LKD16.

Human practice:

1. It has been found in the literature that Bacillus subtilis spray is suitable for the
prevention and
treatment of medium and small area second-degree burns and scald wound infections.
2. According to the game mode of 2048MINI game, we made the "Synthetic BC Film" mini game.

Lab：

1. We constructed a series of light-controlled complex parts in *E. coli* DH5α, using the
c-di-GMP
synthetase BphS and hydrolase FcsR.

2. Verified the ability of different lysis proteins to play a role in *Gluconacetobacter
hansenii* ATCC
53582.

● week 33(8.8-8.14)

Project:

1. The prototype of the planned product is the live bacteria wound dressings.
2. It was proved that the protein that can stably perform the function of lysis in
*Gluconacetobacter
hansenii* ATCC 53582 is X174 E.

Human practice:

1. We visited Shenzhen Lando Biomaterials Co.,Ltd, to understand the application and production
process
of
artificial skin.

2. We went to Dawang Xintian Village, an urban village in the Luohu District of Shenzhen, Guangdong
Province, where we taught the children a picture book on synthetic biology of germ warfare and had
English lessons. We also played a synthetic biology poker game and made drawings on paper plates
together.

Lab：

1. The target gene expression vector of c-di-GMP light-controlled complex parts was transferred into
*G.hansenii* ATCC 53582 with electrotransformation method.

2. PCR products were analyzed using 1% agarose gel electrophoresis to identify bands of expected
sizes.

3. Verified the lysis function of X174 E and Lysis Cassette LKD16, which can play a role in
*Gluconacetobacter hansenii* ATCC 53582 repeatedly.

● week 34(8.15-8.21)

Project:

1. There was a significant difference of the yield of BC under the condition of NIR light and dark
for
J23100-bphS-J23109-fcsR-pSEVA331-*G.hansenii* ATCC 53582 and
J23100-bphS-J23110-fcsR-pSEVA331-*G.hansenii*
ATCC
53582, but we can’t make *G.hansenii* ATCC 53582 completely do no BC film production under the
dark
condition.

Human practice:

1. We visited pharmacys near the school and ask about the existing burn treatment products and the
forms
of
living bacteria products.
2. In order to determine the product format, we consulted Dr. Weijia Ding from the burn department
about
his
views on live bacterial dressings once again.
3. We told stories for children in urban villages, using cartoon paper-cuttings.

Lab：

1. Verified a series of c-di-GMP light-controlled complex parts in *Gluconacetobacter hansenii*
ATCC 53582
through exploring the BC film production in 12-well plate.

● week 35(8.22-8.28)

Project:

1. Determined the product form as a live bacteria by-product and preliminarily determine it be made
into
the
emulsion.

Human practice:

1. We consulted with Xi'an Donghai Viable Mushroom Brand Promotion Co., Ltd, in an interview
online.
2. Offline interviewed with Aegeli Eco-Technology Co., Ltd.
3. Issued a questionnaire to survey some questions related to living bacteria products.
4. We went to the urban villages, teaching children to fold DNA models and playing jigsaw puzzles of
synthetic
biology.

Lab：

1. Planed experimental details and started experiments on the Antipseudomonal Drug Synthesis module,
then
constructed a series of parts in *E. coli* DH5α.
2. Two plasmids S2-PET28A and SE-PET28A that were transferred into *E. coli* BL21 with
electrotransformation
method.

● week 36(8.29-9.4)

Project:

1. The wiki page design was finished.

Human practice:

1. We launched a charity sale activity with the theme of "The love of 'Kissed by light'".

2. Interviewed with Dr. Yan Xiong, a biological security expert, assessed whether the bacteria
we
used
are
dangerous.

Lab:

1. Verified the expression of S2 and SE protein by SDS-PAGE.

● week 37(9.5-9.11)

Project:

1. Proved that the SE-fusion protein can target *Pseudomonas aeruginosa.*

Human practice:

1. We launched a live broadcast for non-biological college students on the Bilibili platform. We
mainly
introduced the project and talked about synthetic biology relevant to our lives.

Lab:

1. Verified the SE-fusion protein whether could target *Pseudomonas aeruginosa.*

●
week 38(9.12-9.18)

Project:

1. Determined to use pR-RBS300-SE-B1006-J23118-RBSII-IMM-rrB T1 (BBa\_K3740050) as the final gene
circuit
of
Antipseudomonal Drug Synthesis module.

Lab:

1. Carry out batch screening and verification experiments of SE-fusion protein for antipseudomonal
performance.

●
week39 (9.19-9.25)

Project:

1. Carried out data sorting work.

Human practice:

1. Organized product experience activities.

●
week 40(9.26-10.2)

Project:

1. Started writing the wiki page.

●
week 41(10.3-10.9)

Human practice:

1. Organized the fluorescent protein DIY activity again.

●
week 42(10.10-10.16)

Project:

1. Wiki writing finished.

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:SZPT-CHINA/Engineering

[![logo](https://static.igem.org/mediawiki/2021/7/7c/T--SZPT-CHINA--icon.png)](https://2021.igem.org/Team%3ASZPT-CHINA)

* [HOME](https://2021.igem.org/Team%3ASZPT-CHINA)
* TEAM
  + [MEMBERS](https://2021.igem.org/Team%3ASZPT-CHINA/Team)
  + [ATTRIBUTIONS](https://2021.igem.org/Team%3ASZPT-CHINA/Attributions)
  + [COLLABORATIONS](https://2021.igem.org/Team%3ASZPT-CHINA/Collaborations)
  + [PARTNERSHIP](https://2021.igem.org/Team%3ASZPT-CHINA/Partnership)
* PROJECT
  + [DESCRIPTION](https://2021.igem.org/Team%3ASZPT-CHINA/Description)
  + [DESIGN](https://2021.igem.org/Team%3ASZPT-CHINA/Design)
  + [SAFETY](https://2021.igem.org/Team%3ASZPT-CHINA/Safety)
  + [NOTEBOOK](https://2021.igem.org/Team%3ASZPT-CHINA/Notebook)
* EXPERIMENTS
  + [MODEL](https://2021.igem.org/Team%3ASZPT-CHINA/Model)
  + [EXPERIMENTS](https://2021.igem.org/Team%3ASZPT-CHINA/Experiments)
  + [RESULTS](https://2021.igem.org/Team%3ASZPT-CHINA/Results)
  + [FUTURE WORK](https://2021.igem.org/Team%3ASZPT-CHINA/Future_Work)
* PARTS
  + [OVERVIEW](https://2021.igem.org/Team%3ASZPT-CHINA/Parts)
  + [CONTRIBUTION](https://2021.igem.org/Team%3ASZPT-CHINA/Contribution)
  + [ENGINEERING](https://2021.igem.org/Team%3ASZPT-CHINA/Engineering)
  + [IMPROVEMENT](https://2021.igem.org/Team%3ASZPT-CHINA/Improvement)
* IMPLEMENTATION
  + [HARDWARE](https://2021.igem.org/Team%3ASZPT-CHINA/Hardware)
  + [IMPLEMENTATION](https://2021.igem.org/Team%3ASZPT-CHINA/Implementation)
  + [PROOF OF CONCEPT](https://2021.igem.org/Team%3ASZPT-CHINA/Proof_Of_Concept)
* HUMAN PRACTICES
  + [INTEGRATED HUMAN
    PRACTICES](https://2021.igem.org/Team%3ASZPT-CHINA/Human_Practices)
  + [COMMUNICATION](https://2021.igem.org/Team%3ASZPT-CHINA/Communication)
  + [EDUCATION](https://2021.igem.org/Team%3ASZPT-CHINA/Education)
  + [INCLUSIVITY](https://2021.igem.org/Team%3ASZPT-CHINA/Inclusivity)
* [AWARDS](https://2021.igem.org/Team%3ASZPT-CHINA/Awards)

![](https://static.igem.org/mediawiki/2021/6/6f/T--SZPT-CHINA--iconGIF.png)

### Engineering

Overview

Engineering Success

BBa\_K3740050

Design

Strategy

Experimental Results

BBa\_K3740030

Design

Strategy

Experimental Results

Learning

BBa\_K3740044

Design

Strategy

Experimental Results

Reference

● Overview：

The goal of project is to use optogenetically engineered *Gluconacetobacter
hansenii* to
fight
with *Pseudomonas aeruginosa* infection and produce a bacterial cellulose dressing
simultaneously. The
genetic engineering consists of three modules, antipseudomonal drug production module, c-di-GMP
signaling and BC film production module, safety and drug release module.
We have so far made a lot of progress in this project. Hereby, we presented a summary of our
engineering
data. Although this project is still ongoing and improvements are needed to make in some sections,
we
believe that our work can contribute to the iGEM community.

● Engineering Success：

I. Antipseudomonal drug production module（[BBa\_K3740050](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3740050)）

1. Design

We designed an antipseudomonal drug production module to specifically combat *P.
aeruginosa*
infection. The receptor domain, the translocase domain of pyocin S2 and the nuclease domain of
colicin
E3
are fused together to get a new chimeric bacteriocin termed SE. The antipseudomonal SE protein can
target and inhibit *P. aeruginosa*, even including those harbouring a S2 immunity protein
IMMS2. IMME3
is
an immunity protein derived from *Escherichia coli*. It can bind to the nuclease domain of SE
specifically
and inhibit its activity, thus rendering the host cell immune to SE. A similar expression level of
the
antipseudomonal protein SE and the immunity protein IMM is designed to be expressed in the
engineered
bacteria, thus preventing the bactericidal effect of SE protein on the bacterial chassis itself.

2. Strategy

Two plasmids S2-PET28A and SE-PET28A were initially constructed and introduced into
*E.
coli*
BL21. Following the induction of lysis protein by IPTG, bacterial cells were in turn disrupted and
the
supernatant containing the soluble SE proteins was collected. The molecular weight of SE protein was
checked by SDS-PAGE while its specificity to *P. aeruginosa* was also verified using the drop
plate
assay;
In order to improve the antipseudomonal effect, we engineered the RBS to modulate its strength in
controlling SE protein expression. Finally, we identified pR-RBS300-SE-B1006-J23118-RBSII-IMM-rrnB
T1([BBa\_K3740050](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3740050)) as our
optimal genetic circuit.

Figure 1. The gene circuit of pR-RBS300-SE-B1006-J23118-RBSII-IMM- rrnB T1
([BBa\_K3740050](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3740050))

3. Experimental results:

3.1 Sequencing

Plasmids from *E. coli* cells were extracted and sequenced. Analysis based on
sequencing
data indicated that the SE-encoded sequence and pR-RBS300-SE-B1006-J23118-RBSII-IMM-rrnB T1
([BBa\_K3740050](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3740050)) have been
successfully integrated into the plasmid vectors.

3.2 Characterization of fusion protein SE

3.2.1 SDS-PAGE

Method: IPTG was added to a final concentration of 0.1 mmol/mL to induce SE protein
expression in *E. coli* BL21 and subsequently incubated at 25℃ for 12 hours. Supernatant
containing SE
or
S2 proteins was obtained by sonication and centrifugation respectively. The crude protein extract
was
loaded into SDS-PAGE gel and stained by Coomassie Blue to verify the presence of target proteins
(For
more detailed steps, please click [Experiments](https://2021.igem.org/Team%3ASZPT-CHINA/Experiments)).

After induction by IPTG, S2 and SE protein expression in S2-PET28A-BL21 (lane 2) and SE-PET28A-BL21
(lane 3) has been identified. By contrast, target proteins were not expressed in the strain with
S2-PET28A-BL21 (lane 1) and SE -PET28A-BL21 (lane 4) in the absence of IPTG,  **indicating
that
expression
of S2 and SE proteins can be successfully induced in *E. coli* transformed with S2-PET28A
and
SE-PET28A,
respectively.**

Figure 2. SDS-PAGE analysis of protein expression in engineered *E. coli* BL21.
Lane 1,
S2-PET28A-BL21 without IPTG induction; Lane 2, S2-PET28A-BL21 with induction by 0.1mmol/mL IPTG;
Lane 3,
SE-PET28A-BL21 with induction by 0.1mmol/mL IPTG; Lane 4, SE-PET28A-BL21 without IPTG induction.

3.2.2 SE protein targeting verification

Method: Supernatant containing SE or S2 protein was respectively mixed with the culture
of
*Pseudomonas aeruginosa* PAO1, *P. aeruginosa* PAO1Δ1150-1151(PAO1 Δ1150-1151, PAO1
knocked out
of
S2+IMMS2)
and *E. coli* MG1655. PBS was used as a negative control. Inhibitory effect of supernatant on
the *P.
aeruginosa* growth was determined by measuring OD600 using SYNERGY H1 micro-plate
reader. (For more
detailed steps, please click [Experiments](https://2021.igem.org/Team%3ASZPT-CHINA/Experiments))

We found that the OD600 values of both *P. aeruginosa* strains cultured with SE
protein, were
much
lower
than that of the control group PBS. This proves that SE protein has antipseudomonal properties. In
contrast, pyocin S2 could only act on the immune-deficiency strain PAO1∆1150-1151. This proves that
SE
protein has a broader antipseudomonal spectrum than S2. Meanwhile, the OD600 value of
*E. coli*
MG1655
cultured with SE protein was a little higher than that of the control group PBS. This indicates that
SE
protein does not work on *E. coli*.  **This result indicated that the fusion protein SE
can
specifically
kill
 *P. aeruginosa***.

Figure 3. Optical density of bacterial culture of PAO1, PAO1 Δ1150-1151 and *E.
coli*
MG1655
treated by the supernatant of S2-PET28A-BL21 or SE-PET28A-BL21 for 790 minutes. PBS was used as the
negative control.

3.3 Batch screening and verification of SE protein antibacterial performance of
composite
parts

Method: PAO1 growth inhibition verification. *G. hansenii* ATCC 53582 culture
supernatant
containing SE proteins that were expressed under control of different RBS were obtained by
sonication
and centrifugation and further purified by infiltration with micro-pored membrane. Then, supernatant
in
different groups was individually mixed with PAO1 culture, and incubated at 30℃ for 12 hours.
SYNERGY H1
micro-plate reader was used to measure the OD600 to determine the effect on PAO1 growth.
Inhibition zone experiment. 3*μL* of the supernatant containing SE protein was dropped on FAB
plate
with
PAO1（For more detailed steps, please click [Experiments](https://2021.igem.org/Team%3ASZPT-CHINA/Experiments)).

As shown in Figure 4 (a), we found that the OD600 values of PAO1 strains cultured with
supernatant
of
the 3rd, 4th, 7th, and 8th colonies bacterial lysate,
were much lower than that of PBS in the control
group. This proves that culture supernatant of the 3rd, 4th, 7th,
and 8th colonies had a remarkable
inhibitory effect on the growth of PAO1. Consistent with this result, supernatants from all the
*G.
hansenii* isolates have formed inhibition zone on the plate spread with PAO1, yet with
different
sizes.
The 3rd and 4th isolate displayed the most significant antipseudomonal effect,
while the control,
the
9th colony had no effect. Taken together,  **these results show that expression of
SE protein was
successfully induced in *G. hansenii* ATCC 53582. Besides, by adjusting the RBS upstream
the
SE-encoded
sequence, we screened and identified that the 4th strain,
pR-RBS300-SE-B1006-J23118-RBSII-IMM-rrnB
T1-pSEVA331-*G. hansenii* ATCC 53582-4# exhibited the optimal antipseudomonal
effect.**

Figure 4. (a) Optical density of bacterial culture of PAO1 treated by the supernatant of
engineered strains for 790 minutes. (b) Inhibition zone test. (c) Strains are used for Figure(a) and
Figure(b).

3.4 Video shooting

[ ](https://static.igem.org/mediawiki/2021/2/20/T--SZPT-CHINA--Engineering-video-1.mp4)

Video 1. Video shooting of the zone of inhibition of 4th bacteria:
pR-RBS300-SE-J23118-RBSII- IMM-rrnB T1-pSEVA331-*G. hansenii* ATCC 53582-4#.

II. C-di-GMP signaling and BC film production module ([BBa\_K3740030](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3740030))

1. Design

This c-di-GMP signaling and BC film production module has two submodules. One is the
diguanylate cyclase sub-module, which regulates the synthesis of c-di-GMP. The bacterial phytochrome
BphS is utilized to synthesize c-di-GMP in a light-dependent manner, thereby controlling BC film
production in *G. hansenii* ATCC 53582. The other is c-di-GMP phosphodiesterase sub-module,
which
regulates the hydrolysis of c-di-GMP. By screening different parts, the level of c-di-GMP in
bacteria
can remain in a low level or even close to zero under the dark condition.

2. Strategy

A series of plasmids derived from J23100-B0034 were constructed and transferred into
*G.
hansenii* ATCC 53582, in which the BC film production was quantified. Finally, fcsR ([BBa\_K3740022](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3740022))
was
selected as the c-di-GMP phosphodiesterase encoding gene. Different promoters were then used to
direct
the expression of FcsR. Based on the screening results, [BBa\_K3740030](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3740030),
[BBa\_K3740031](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3740031) and
[BBa\_K3740043](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3740043)
were
constructed and introduced into *G. hansenii* ATCC 53582 through electroporation. Finally, BC
film
production was assessed in these strains.

Figure 5. The gene circuit of J23100-B0034-bphS-pET RBS-bphO-rrnB T1-J23109-B0034-fcsR-
rrnB
T1 ([BBa\_K3740030](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3740030)).

3. Experimental results

3.1 Agarose gel electrophoresis

As shown in Figure 6 (a), c-di-GMP phosphodiesterase-encoded genes ([BBa\_K3740065](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3740065)),
([BBa\_K3740062](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3740062))
and ([BBa\_K3740066](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3740066)) were
identified successfully by PCR amplification and c-di-GMP diguanylate
cyclase-encoded genes ([BBa\_K3740047](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3740047)) was
identified by PCR amplication; As shown in Figure 6(b),
composite
parts including ([BBa\_K3740030](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3740030)), ([BBa\_K3740031](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3740031))
and ([BBa\_K3740043](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3740043)) were
identified successfully by PCR.

Figure 6. (a), (b)The agarose gel electrophoresis image of the constructed plasmid after
PCR
amplification. (c)Plasmids are used for Figure(a) and Figure(b).

3.2 Film production verification experiment

3.2.1 Film production verification experiment of bphS and fcsR

Method: The constructed plasmids were introduced into *G. hansenii* ATCC 53582 and
cultured at
30°C. Then a single colony was picked to launch its liquid culture. Then the culture was distributed
in
two copies of 12-well plates, with one under NIR light illumination and the other under dark
condition
for 4 days. (For more detailed steps, please click [Experiments](https://2021.igem.org/Team%3ASZPT-CHINA/Experiments))

As shown in Figure 7 (a), BC production in J23100-fcsR-rrnB T1-pSEVA331- *G. hansenii* ATCC
53582 and
the
control group pSEVA331- *G. hansenii* ATCC 53582 were different, indicating that FcsR was
capable of
hydrolyzing c-di-GMP in *G. hansenii*. While, J23100-bphS-pET RBS-bphO-rrnB T1-pSEVA331-*G.
hansenii*
ATCC
53582, produced a higher level of BC film under near-infrared light than that under dark condition,
indicating that BphS in *G. hansenii* ATCC 53582 can synthesize c-di-GMP.  **To
summarize, BphS
and FcsR
were
opted as the c-di-GMP synthetase and hydrolase in our system, respectively.**

Figure 7. (a)BC yield by J23100-B0034-fcsR-rrn B T1-pSEVA331-*G. hansenii* ATCC
53582,
J23100-B0034-yhjH-rrnB T1-pSEVA331-*G. hansenii* ATCC 53582, J23100-B0034-rocR-rrnB
T1-pSEVA331-*G.
hansenii*
ATCC 53582 and the vehicle control pSEVA331-*G. hansenii* ATCC 53582; (b) BC yield by
J23100-bphS-pET
RBS-bphO-rrnB T1-pSEVA331-*G. hansenii* ATCC 53582 under NIR light illumination and dark
condition.

3.2.2 Film production verification experiment of bphS-fcsR

A significant difference in BC film production under NIR light and dark conditions were
observed for those strains, including J23100-B0034-bphS-pET RBS-bphO-rrnB T1-J23109-B0034-fcsR-rrnB
T1-pSEVA331-*G. hansenii* ATCC 53582, J23100-B0034-bphS-pET RBS-bphO-rrnB
T1-J23110-B0034-fcsR-rrnB
T1-pSEVA331-*G. hansenii* ATCC 53582, however J23100-B0034-bphS-pET RBS-bphO-rrnB
T1-J23119-B0034-fcsR-rrnB
T1-pSEVA331-*G. hansenii* ATCC 53582 had no difference in BC film production under NIR light
and dark
conditions,  **these results showed that J23100-B0034-bphS-pET RBS-bphO-rrnB
T1-J23109-B0034-fcsR-rrnB
T1-pSEVA331-*G. hansenii* ATCC 53582 and J23100-B0034-bphS-pET RBS-bphO-rrnB
T1-J23110-B0034-fcsR-rrnB
T1-pSEVA331-*G. hansenii* ATCC 53582 can be used as our gene circuit.**

Figure 8. (a) BC film production by different strains, (b) Strains are used for
Figure(a).

3.3 Video shooting

[ ](https://static.igem.org/mediawiki/2021/5/59/T--SZPT-CHINA--Engineering-video-2.mp4)

Video 2. Video shooting of J23100-B0034-bphS-pET RBS-bphO-rrnB T1-J23109-B0034-fcsR-rrnB
T1-pSEVA331-*G. hansenii* ATCC 53582 bacterial BC production.

4. Learning:

In our film production assay, the film production by engineered bacteria under dark
condition was expected to remain at a very low level. Surprisingly, a substantial amount of BC film
was
still found without NIR light induction, even though the level was lower than that in the
NIR-induced
group. In the future, we will solve this problem and refine the genetic circuit.

III. Safety and drug release module ([BBa\_K3740044](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3740044))

1. Design

This module is a dual design of both safety control and drug release. We use pDawn, a
blue-light-responsive promoter to direct the expression of the lytic proteins, which would cause the
lysis
and death of the engineered bacteria. Then, SE protein expressed within the engineered bacteria will
be
released.
We have constructed a set of plasmids which include pDawn to induce the expression of an
anti-holin-free
Lambda lysis cassette (S105), φX174 lysis gene (X174 E), and LKD16 lysis cassette (LKD16). They were
chosen
to lyse our chassis bacteria. After screening, X174 E ([BBa\_K2656015](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2656015)) was
selected as the final part
in
our
safety and drug release module.

2. Strategy

RFP was firstly used to verify the responsiveness of the pDawn promoter to blue light.
Then,
random primer guided mutagenesis method to modulate the strength of the RBS ([BBa\_B0034](http://parts.igem.org/wiki/index.php?title=Part:BBa_B0034)) located
upstream
of
X174 E. Finally, the pDawn-RBSNNN-X174 E-rrnB T1 ([BBa\_K3740044](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3740044)) plasmid
was constructed and
introduced
into
*E. coli*. *E. coli* isolates that grow normally in the dark and cannot grow under blue
light
were
screened
out.
Finally, the plasmid was extracted, and further introduced into *G. hansenii* ATCC 53582, in
which the
responsiveness of pDawn to blue light was also verified.

Figure 9. The gene circuit of pDawn-RBSNNN-X174 E-rrnB T1 ([BBa\_K3740044](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3740044)).

3.Experimental results

3.1 The pDawn promoter responds to blue light verification in *G. hansenii* ATCC
53582

Red fluorescence could be observed in the pDawn-B0034-RFP-rrnB T1-pSEVA331-*G.
hansenii*
ATCC
53582 under 470-nm blue light, but there was no red fluorescence under dark condition,**suggesting
that
the pDawn promoter could respond to blue light and induce gene expression in *G. hansenii*
ATCC
53582.**

Figure 10. Red fluorescence photo of the pDawn-B0034-RFP-rrnB T1-pSEVA331-*G.
hansenii*
ATCC
53582 cultured under 470-nm blue light (a) or dark condition (b) for 24 hours.

3.2 Batch screening of pDawn-RBSNNN- X174 E-rrnB T1-pSEVA331 in response to blue light
lysis
in *E. coli*

Method: We use the random primer method to modulate the strength of the RBS ([BBa\_B0034](http://parts.igem.org/wiki/index.php?title=Part:BBa_B0034)) located
upstream
of X174 E. After introducing into *E. coli* DH5α, a drop plate assay was performed to screen
the
bacterial
isolate that can grow normally in the dark but cannot under the blue light irradiation.

As shown in Figure 11, the 4th isolate that grew normally in the dark but did not under blue light,
 **indicating that we successfully expressed the cleavage protein X174 E in *E. coli.***

Figure 11：The growth status of pDawn-RBSNNN-X174 E-rrnB T1-pSEVA331-DH5α under blue
light
(a)
and dark condition (b).

3.3 pDawn-RBSNNN-X174 E-rrnB T1-pSEVA331-7# in response to blue photolysis in *G.
hansenii*
ATCC 53582

Method: We launched the liquid culture of pDawn-RBSNNN-X174 E-rrnB T1-pSEVA331-DH5α,
extracted the plasmid and introduced into *G. hansenii* ATCC 53582. After 2 days of incubation,
we
selected 20 monoclonal to spot on two parallel new plates, which were incubated for another 2 days,
with
one under blue light and the other in the dark. The strains with obvious growth difference under
different illumination conditions were preserved. The drop plate assay using this strain was
repeated to
verify the lysis effect of X174 E on *G. hansenii* ATCC 53582.

As shown in Figure 12 (a), *G. hansenii* ATCC 53582 strains under the dark condition exhibited
better
growth than those under blue light irradiation; (b) pDawn-RBSNNN-X174 E-rrnB T1-pSEVA331-*G.
hansenii*
ATCC
53582-7# showed a stable lysis effect under blue light illumination but not in the dark, indicating
that
we successfully expressed the cleavage protein X174 E in *G. hansenii* ATCC 53582.

Figure 12. (a) The growth status of pDawn-RBSNNN-X174 E-rrnB T1-pSEVA331-*G.
hansenii* ATCC
53582-4# under blue light (1) and dark conditions (2), (b)The growth status of pDawn-RBSNNN-X174
E-rrnB
T1-pSEVA331-*G. hansenii* ATCC 53582-7# under blue light (1) and dark conditions (2).

In our safety and drug release module, we verified the lysis effect of ([BBa\_K3740024](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3740024)) and
([BBa\_K3740032](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3740032)), however
their effect was inferior to that of ([BBa\_ K2656015](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2656015)). If you
are interested in
our
other two parts, please Click: ([BBa\_K3740033](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3740033)) and ([BBa\_K3740051](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3740051)).

Reference

[1] Gupta S, Bram E E, Weiss R. Genetically programmable pathogen sense and destroy. [J]. Acs
Synthetic
Biology, 2013, 2(12): 715-723.
[2] Jin F, Gao YM, Huang YJ, et al. Current therapies for *Pseudomonas aeruginosa* infection
[J].
Journal
of Integration Technology, 2021, 10(4): 50-66.
[3] Michel-Briand Y, Baysse C. The pyocins of *Pseudomonas aeruginosa.* [J]. Biochimie, 2002,
84(5-6): 499-510.
[4] Denayer S, Matthijs S, Cornelis P. Pyocin S2 (Sa) Kills *Pseudomonas aeruginosa* Strains
via the
FpvA
Type I Ferripyoverdine Receptor [J]. Journal of Bacteriology, 2007, 189(21): 7663.
[5] Ryu M H, Gomelsky M Near-infrared Light Responsive Synthetic c-di-GMP Module for Optogenetic
Applications [J]. Acs Synthetic Biology, 2014, 3(11): 802
[6] Elaheh Sajadi,Seyed Safa-Ali Fatemi,Valiollah Babaeipour,Ali Asghar Deldar,Bagher
Yakhchali,Mohammad
Saberi Anvar. Increased cellulose production by heterologous expression of bcsA and B genes from
Gluconacetobacter xylinus in *E. coli* Nissle 1917 [J]. Bioprocess and Biosystems
Engineering, 2019, 42(12):
2023-2034.
[7] Jin X, Riedel-Kruse I H. Biofilm Lithography enables high-resolution cell patterning via
optogenetic
adhesin expression [J]. Proceedings of the National Academy of Sciences of the United States of
America,
2018: 3698-3703.
[8] Ceyssens P J, Lavigne R, Mattheus W, et al. Genomic Analysis of *Pseudomonas aeruginosa*
Phages
LKD16
and LKA1: Establishment of the KMV Subgroup within the T7 Supergroup [J]. Journal of Bacteriology,
2006.
[9] Yibo S, Jianhe S, Shi Y, et al. Current advance in the topological structure and function of
holin
encoded by bacteriophage lambda [J]. Acta Microbiologica Sinica, 2012, 52(2): 141-145.
[10] Witte A, Wanner G, Sulzner M, et al. Dynamics of PhiX174 protein E-mediated lysis of
*Escherichia
coli* [J]. Archives of Microbiology, 1992, 157(4): 381-388.
[11] Ohlendorf R, Vidavski R R, Eldar A, et al. From dusk till dawn: one-plasmid systems for
light-regulated gene expression [J]. Journal of Molecular Biology, 2012, 416(4): 534-542.

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# Team:SZPT-CHINA/Experiments

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### Experiments

Engineering Experiments

Prototype Experiments

● Medium and Antibiotic

Materials

· Antibiotic
· Double distilled water (ddH2O)
· Tryptone
· NaCl
· Yeast Extract
· Glucose
· Peptone
· H25Na2O16P
· C6H10O8
· Agar
· Absolute ethanol

Procedure

1.1. Medium:

|  |  |  |
| --- | --- | --- |
| Medium | Materials | Mass/g |
| LB Medium (1000mL) | Tryptone | 10 |
| NaCl | 5 |
| Yeast Extract | 5 |
| LB Agar (1000mL) | Tryptone | 10 |
| NaCl | 5 |
| Yeast Extract | 5 |
| Agar | 15 |
| HS Medium (500mL) | Glucose | 20 |
| Peptone | 5 |
| Yeast Extract | 5 |
| H25Na2O16P | 6.8 |
| C6H10O8 | 1.5 |
| HS Agar (500mL) | Glucose | 20 |
| Peptone | 5 |
| Yeast Extract | 5 |
| H25Na2O16P | 6.8 |
| C6H10O8 | 1.5 |
| Agar | 15 |

1.2. Antibiotic:

1.2.1. Keep the antibiotics until room temperature, label the
centrifuge
tubes, microfiltration membranes and syringes, calculate and record the mass of the antibiotics;

\* Note: Calculation method: Preparation Volume (mL) \* Concentration (mg/mL) = Mass (mg)

1.2.2. When antibiotics return to room temperature, start weighing clean and dry centrifuge
tubes
and samples with an analytical balance;

1.2.3. After weighing, use 1mL pipetting gun to suck ultrapure water. Do it faster in front, and
slow it down at the last 0.5mL. Stop adding water when the liquid level reaches 10mL;

1.2.4. After dissolution, centrifuge at 5000rpm for 30sec and then suck ultrapure water with a
100μL
pipetting gun. After replenishing the liquid level to 10mL again, pass through the membrane to
another
dry, clean and labeled centrifuge tube and store it at 4℃.

\* Note: Chloramphenicol should be dissolved in absolute ethanol.

● Preparation of Competent Cells

Materials

· Target strain DH5α
· BT medium
· BT Buffer A
· BT Buffer B
· Sterilized LB medium and LB Agar plate

Procedure

2.1. Take out the bacteria strain from the refrigerator, streak LB plate and culture
it
in a 37℃ incubator.

 2.2. Select a monoclonal colony from the plate and inoculate it into the sterilized LB medium,
and
culture it in a shaker at 37℃, 250rpm for 12h-16h.

2.3. Dilute the bacteria in step 2 into BT medium at the ratio of 1:100, shake the bacteria, and
detect OD600 to 0.4-0.7, and the shaking time is about 2-2.5h.

\* Note: BT medium shall use conical flasks larger than 250mL, and the shaker shall exceed
250rpm.

2.4. Transfer the bacterial solution to 50mL centrifuge tube and place them on ice for 5-10min.

2.5. Centrifuge at 5000rpm for 5min at 4℃, sip up the waste liquid with pipetting gun and
collect
precipitation.

2.6. Each 50mL bacterium sediment is re-suspended gently with 16mL ice-precooled BT Buffer A.

2.7. Repeat the step 5.

2.8. Each 50mL bacterium sediment is resuspended gently with 4mL ice-precooled BT Buffer B.

2.9. Each 100μL is individually packed into a 1.5mL centrifuge tube. After packing, fasten down
the
cover and put it on the ice. When it is almost enough for a box, quickly put it in the box and store
it
at -80℃.

\*Note: All operation must be done on the ice.

● Preparation of Cellulase:40 mg/mL

Materials

· Cellulase powder

· ddH2O

Procedure

3.1. Weigh 1g cellulase powder into a 50mL centrifuge tube and add 20mL ddH2O
to
dissolve
it. Ultrasonic can accelerate dissolution. After dissolution, there will be a lot of foam in the
centrifuge tube, which will affect our final volume calibration.

 3.2. Therefore, centrifuge at 8000rpm for 10min. The centrifugal time depends on the foam
volume so
that it does not affect volume calibration. You can take out the centrifuge tube to observe every
10min.

3.3. After there is not much foam, calibrate the volume to 25mL and then pass through the
membrane.

● Minimum Inhibition Concentration

Materials

· HS medium
· 0.2% cellulase
· Antibiotic

Procedure

4.1. *Gluconacetobacter hansenii* ATCC 53582 stored in glycerol is inoculated in a 50mL
centrifuge
tube of
10mL HS medium and 0.2% cellulase, and cultured at 230 rpm, 30℃ for 24-72h until OD600 >
0.7.

4.2. *Gluconacetobacter hansenii* ATCC 53582 in step 1 is inoculated to 50mL centrifuge tube
containing 10mL
HS medium and 0.2%(v/v) cellulose, and cultured overnight at 230rpm, 30℃ until OD600=
0.4-0.7. Then
centrifuge at 3200rpm for 14min, discard supernatant solution and resuspend with 1mL HS medium.

4.3. The activated *Gluconacetobacter hansenii* ATCC 53582 is inoculated with a transferring
loop
and divided
into the corresponding antibiotic plate. After culturing at 30℃ for 24-72h, you can observe and
record
the
growth of bacterial plaque.

● Plasmid Construction

Materials

· Template and primer
· ddH2O
· Buffer, dNTP, enzyme (it is different between PCR and ligation)
· GelRed
· Binding Buffer and SPW Wash Buffer
· Competent cells
· LB medium and antibiotic
· solution I, solution II, solution III
· HBC Buffer and DNA Wash Buffer
· glycerol

Procedure

5.1. Add 25μL 2 × Phanta Max Buffer, 20μL ddH2O, 1μL dNTP Mix (10 mM each), 1μL Phanta Max
Super-Fidelity DNA Polymerase, 1μL templates (need to dilute to 30ng/μL) and corresponding primers,
oscillate and send them to the PCR thermal cycler (ProFlex 3x32-Well).

5.2. The amplified products were analyzed by 1% agarose gel electrophoresis, compare BP and cut
their correct range. Then carry out gel recovery:

5.2.1. Add 300μL Binding Buffer, centrifuge at 13000rpm for 1min, discard waste liquid——repeat
twice;

5.2.2. Add 700μL SPW Wash Buffer, centrifuge at 13000rpm for 1min, discard waste liquid——repeat
twice;

5.2.3. Centrifuge at 13000rpm for 2min——in order to remove alcohol;

5.2.4. Add 30μL ddH2O and stand for 2 min, centrifuge at 13000rpm for 1min;

5.2.5. Measure the PCR concentration.

5.3. Ligation:

Add 2μL 5×CE Multis Buffer, 1μL enzyme (Exnase Multis), appropriate volume of fragment, carrier
and
ddH2O. And send them to the PCR thermal cycler.

\*Calculation:

(1) The volume of fragment and carrier=(20×BP)/Concentration of PCR;

(2) The volume of ddH2O=10μL-the volume of all solutions expect ddH2O.

5.4. Transformation:

5.4.1. Thaw the competent cells (take ice), add the bacterium liquid ligated and apply ice for
30min. Heat it for 90sec at 42℃ in a water bath, then apply ice again for 2min;

5.4.2. Add 500μL LB to bacterium liquid, shake it at 37℃ and 220 rpm for 1h in a shaker, then
centrifuge at 13000rpm for 1 min;

5.4.3. Discard the supernatant solution, resuspend the plasmid with a pipetting gun, take 100μL
to
the plate (LB and antibiotic), evenly spread with a cell spreader and culture it overnight at 37℃.

5.5. The PCR identification:(If PCR identification is not required, pick the bacteria and spot
separately on the plate, compare their growth under blue light and dark conditions)

5.5.1. Pick bacterial plaque, crack at 95℃ for 10min in a water bath and centrifuge at 13000 rpm
for
2min;

5.5.2. Take 2μL supernatant solution, add 5μL buffer, 2μL ddH2O,0.25μL dNTP, 0.25μL
enzyme and
0.25μL primers, oscillate and send to the PCR thermal cycler.

5.5.3. The amplified products were analyzed by 1% agarose gel electrophoresis, compare BP and
record
the correct bacterial. Then pick and inoculate the bacterial to medium (including antibiotic), shake
them at 37℃ and 250rpm for 12-16h in a shaker.

5.6. Plasmid extraction:

5.6.1. Take 1.5mL bacterial liquid and centrifuge at 13000rpm for 1min, discard waste
liquid——repeat
three times;

5.6.2. Add 250μL solution I and resuspend; Add 250μL solution II and shake gently; Add 250μL
solution III and shake quickly, then centrifuge at 13000rpm for 15min;

5.6.3. Take the supernatant solution to the absorption tube and centrifuge at 13000rpm for 1min,
discard the waste liquid;

5.6.4. Add 500μL HBC Buffer and centrifuge at 13000rpm for 1min, discard the waste liquid;

5.6.5. Add 700μL DNA Wash Buffer and centrifuge at 13000rpm for 1min, discard the waste
liquid——repeat twice;

5.6.6. Centrifuge at 13000rpm for 2min;

5.6.7. Add 50μL ddH2O, stand for 2min and centrifuge at 13000rpm for 1min;

5.6.8. Measure the concentration.

5.7. Preserve the bacterial liquid to glycerol and send the plasmid to sequence.

● Electrotransformation

Materials

· HS medium + 0.2% cellulase
· 1mM HEPES buffer
· 15% glycerol
· HS medium
· HS Agar plate (including antibiotic)

Procedure

6.1. Inoculate *Gluconacetobacter hansenii* ATCC 53582 to 50mL centrifuge tube of 5mL HS medium
and
0.2%(v/v) cellulase, shake it at 30℃ and 180rpm for 24-72h in a shaker until OD600>0.7.

6.2. Inoculate to 10mL HS medium and 0.2% cellulase in a 250mL conical flask, shake it at 180rpm
and
30℃ for overnight until OD600=0.4-0.7.

6.3. Precool centrifuge to 4℃, prepare ice bucket and ice and put 1mM HEPES buffer and 15%
glycerol
into the ice.

6.4. Take out the reaching OD600 bacterial to ice for 10min.

6.5. Centrifuge at 4100rpm and 4℃ for 12min, discard the supernatant solution.

\*Note: Pour out the supernatant solution carefully but not the particles. *Gluconacetobacter
hansenii*
ATCC 53582 cannot form pellet as easily as *Escherichia coli (E.coli)*, most likely due to the
buffering
effect of cellulase. If the pellet is not attached to the tube wall after centrifuging, the pellet
is
smeared on the wall and centrifuged for a long time. After pouring the supernatant solution, gently
sip
it up or buckle on the tissue paper.

6.6. Resuspend with 10mL HEPES buffer.

\*Note: Firstly resuspend with 1mL HEPES buffer, then add 9mL HEPES buffer with an electric
pipetting
gun.

6.7. Centrifuge at 4100rpm and 4℃ for 12min, discard the supernatant solution. Repeat steps 6.5.
to
6.7.

6.8. After resuspending with 5mL 15% glycerol, place in a 4℃ centrifuge, 4100rpm, 14min and
discard
the supernatant solution.

6.9. After resuspending particles with 1mL 15% glycerol (glacial), each 100μL is packed into
1.5mL
centrifuge tubes individually.

\*Note: After freezing, the efficiency of the active battery may decrease, so immediate use may
produce the highest efficiency. 100μL, that is, the volume of plasmid plus *Gluconacetobacter
hansenii*
ATCC 53582 is 100μL, and the final resuspension volume depends on the number of samples.

6.10. Add 600ng plasmid to competent cells and gently flick, then suck it into (0.1cm)
electroporation cup and set 3000V and 5-8ms. After finishing, take immediately the bacterial to
500mL HS
medium and cellulase, and culture it at 180rpm and 30℃ for 16h.
Calculation: the volume of plasmid=600ng/Concentration (no more than 10μL.)

6.11. After centrifuging for 1min, resuspend with 100μL medium and spread the HS Agar (including
antibiotic), the colony will grow plaque in 24-72h. It needs to do the PCR identification to ensure
the
correct plasmid.

● The Inhibition Zone Experiment of SE Protein

Materials

· LB Agar plate
· FAB medium
· *Pseudomonas aeruginosa* (PAO1)
· 1mM FeCl3
· 1mol glutamate
· FAB medium+1% Agar

Procedure

7.1. Streak the PAO1 to LB Agar plate.

 7.2. Select the monoclonal point and inoculate to FAB medium, FeCl3, glutamate (each 1mL FAB
medium+30μL 1mol glutamate+1μL 1mM FeCl3, the same as below) in a 250mL conical flask and shake it
until
OD600=0.5-0.8.

 \*Calculation:

(1) OD1×V1=OD2×V2(OD1—data measured, V1—data required, OD2—5, V2—volume required)

 (2) V2=V requirement +V loss

 7.3. Collect the bacterial(V1) from step 2 with 50mL centrifuge tube, centrifuge at 4000rpm for
5min and discard the waste liquid.

 7.4. Resuspend with FAB medium(V2), glutamate and FeCl3.

 7.5. Suck the volume required to other 50mL centrifuge tube, add FAB medium+1% Agar, FeCl3,
glutamate (the total volume of each plate is 5mL), mix, pour it in a plate and blow for 45min.

 \*Note: When OD600=0.5, the bacterial number is 109, the volume required is 2mL;
When OD600=5, the bacterial number is 5×109, the volume required is 1mL.

 7.6. Drip 3μL supernatant solution to the plate and observe the growth of the inhibition zone.

● Expression and Identification of SE Antibacterial
Protein of
*Gluconacetobacter hansenii* ATCC 53582

Materials

· 1×PBS buffer
· PMSF
· 1 mg/mL lysozyme
· HS medium+cellulase+Chl148
· 4×Protein Loading Buffer
· Coomassie brilliant blue

Procedure

8.1. Shake the Gluconacetobacter hansenii ATCC 53582 with different expression levels of correct SE.

8.2. Inoculate the bacterial (step 1) to HS medium+cellulase+Chl148(the ratio of 1:50), shake
the
bacterial until logarithmic phase (0.3-0.7).

8.3. Pack individually the bacterial liquid to 50mL centrifuge tube, centrifuge at 4000rpm for
5min.

8.4. Wash the bacterial with 1×PBS buffer and centrifuge at 4000rpm for 5min, discard the
supernatnt
solution.

8.5. Add 5mL 1×PBS buffer (corresponding 200mL bacterial liquid) and put it in -80℃ refrigerator
freeze-thaw once.

8.6. Take out the bacterial liquid from refrigerator and add 10μL PMSF, 1 mg/mL lysozyme, then
heat
it at 37℃ in a water bath.

8.7. Ultrasonic wave: Power is 50% (about 400W), working time is 1sec, interval time is 3sec,
and
total working time is 3min.

8.8. Centrifuge(high-speed) at 8000rpm and 4℃ for 20min.

8.9. Take the supernatant solution to pass through the membrane, then put the samples to 4℃
refrigerator.

8.10. Measure the absorbance of protein solution of collection tube at 280nm, estimate roughly
the
concentration of samples.

8.11. Take the high concentration of several tubes of protein samples and control group
bacterial
suspension, add 4×Protein Loading Buffer, put it at 98℃ for 5-8min and make it fully denatured. Make
SDS-PAGE electrophoresis, set 100V for about 2h to make bromophenol blue run to the bottom.

8.12. Coomassie brilliant blue dyeing for 30min.

8.13. Decolorize for overnight, or heat in the microwave.

8.14. Take some photos and judge the concentration and purity of the target protein.

● The Verification Experiment of BC Film Yield on
12-Well Cell Culture plates

Materials

· HS medium
· Cellulase
· Chl148
· ddH2O
· 0.9% NaCl

Procedure

9.1. After electrotransformation, spread the bacterial to the plate with a cell
spreader.
Select a monoclonal point to shake the bacterial with HS medium+Chl148+cellulase.

9.2. Shake until the stable phase and detect the OD600>0.8.

9.3. Dilute the stable phase bacterial liquid at the ratio of 1:75 to shake with a 10mL conical
flask, detect the OD600=0.2-0.8.

9.4. Before experiment, measure the final OD600 of each bacterium.

9.5. Calculate the OD600 of the final obtained volume of each bacterium is 0.5.

9.5.1. V obtainment=V HS medium + V corresponding antibiotic:
the
number of conditions × the number
of samples × 1.6mL (the volume required for each group), you need to calculate the volume of loss.

9.5.2. OD resuspension=0.5: HS medium + corresponding volume/25(dilution)

9.5.3. OD aim × V aim=OD measurement × V obtainment

9.6. After collecting the bacterial, suck up the supernatant solution with pipetting gun.

9.7. Resuspend with 1mL 0.9% NaCl solution.

9.8. Centrifuge at 10000rpm for 3min.

9.9. Repeat steps 7 and 8.

9.10. Resuspend with 1mL 0.9% NaCl solution and unify all the bacterial until
OD600=0.5.

9.11. Dilute the OD600=0.5 resuspension at the ratio of 1:25 to original solution (HS
medium+
corresponding antibiotic) and mix.

9.12. Suck accurately 3mL the mixture solution to 12-well cell culture plate and make three
parallel
samples of each bacterium under the same condition.

9.13. Culture stationarily at 30℃ for 16h under the red light and dark conditions.

\*Note: The bacterial must to sip up to ensure the variables of the volume, culture time,
temperature
(and so on) unchanged and the red light is a single variable.

● The Treatment of BC Film(Drying Method)

Materials

· 100mM NaOH solution
· ddH2O

Procedure

10.1. Put three parallels under the same condition to the same beaker with a tweezer, and label the
culture condition to avoid the confusion.

10.2. Shake and wash for 5min with ddH2O, repeat three times.

10.3. Soak in 100mM NaOH solution and boil until the BC film become transparent.

10.4. Shake and wash for 5min with ddH2O, repeat three times.

10.5. Place each membrane to a single beaker and label the culture condition.

10.6. Put them into 85℃ oven until constant weight.

\*Note: Weighting after beaker are cooled to room temperature.

10.7. m constant weight - m beaker =m membrane.

● Quantitative Fluorescence Intensity

Materials

· LB medium

Procedure

11.1. Shake the bacterial with LB medium for 12-16h.

11.2. Dilute the bacterial at the ratio of 1:100 to shake until OD600=0.2(about), and
dilute
them at the
bacterial at the ratio of 1:10 to shake until OD600=0.2(about).

 11.3. Unify all bacterial OD600 to 2, suck 180μL of each sample to 96-well cell
culture
plates and
do three parallel samples.

11.4. Measure three green fluorescence intensity and OD600.

# Prototype Experiments

● The Experiment of Freeze-drying Bacterial

Materials

· *Gluconacetobacter hansenii* ATCC 53582 and the bacterial are modified by synthetic biology

· Dry skim milk (WONDERSUN BRAND)
· HS medium and HS Agar
· PBS buffer
.Physio logical saline

Procedure

1.1. Oscillate the conical flask containing the bacteria that can produce the BC film on the vortex
mixer for 3-4min to shake the bacteria out of the film.

 1.2. Suck up 1mL bacterial liquid from the standing conical flask to inoculate to 100mL HS
medium,
shake it at 180rpm and 30℃ until OD600=0.8.

1.3. Suck up 3mL from bacterial liquid to 50mL centrifuge tube, centrifuge at 4100rpm for 12min,
discard quickly the supernatant solution and collect the bacterial.

 1.4. Wash twice with PBS buffer (pH is 7.2-7.4), and centrifuge to collect.

 1.5. Add 3mL protective agent (10% dry skim milk) and phosphoric acid buffer, and mix fully
with
the vortex mixer.

1.6. After pre-freezing at -80℃ for 3h, put the bacterial tubes to freezing box and cover with
tin-
foil, then put them into lyophilizer.

\*Note: You need to detect engine oil before using the lyophilizer, then use it.

1.7. After freezing at -80℃ for 24h, remove the centrifuge tubes from lyophilizer.

\*Note: You need to wait until the lyophilizer gets down to -40℃ then you can leave.

1.8. Pour the plates on the alternate days. Blow for 30min and UV irradiate for 30min.

1.9. Before putting the centrifuge tube in the super clean bath, wipe 50mL centrifuge tube wall
with
70% alcohol and resuspend with 3mL PBS buffer.

1.10. The determination of bacterial survival rate:

1.10.1. Before processing:

1.10.1.1. After shaking fully the liquid samples, suck to 25mL samples with sterile suction pipe
and
put into the sterile conical flask containing 225mL physio logical saline (place the appropriate
amount
of sterile glass beads), shake fully to make 1:10 sample solution.

1.10.1.2. Suck 1:10 sample solution to sterile test tube containing 9mL physiological saline
(add
slowly along the tube wall and the tip does not touch the dilution solution), shake the test tube to
mix
fully to make 1:100 sample solution.

1.10.1.3. Take another 1mL micropipette sucker. According to the above-mentioned operation, make
10
times increment sample solution. Replace 1mL sterile sucker with 1 dilution per increment. Make a
total
of 8 gradients.

1.10.1.4. Select 2-3 consecutive appropriate dilutions, suck 100μL sample solution of each
dilution
to culture plate to spread (5 plates per dilution) and affix the seal, culture at 30℃ for 48h in the
constant temperature incubator.

1.10.2. After resuspending:

1.10.2.1. Suck up 1mL sample solution with 1mL micropipette and add to sterile test tube
containing
9mL physiological saline (add slowly along the tube wall and the tip does not touch the dilution
solution), shake the test tube to mix fully to make 1:10 sample solution.

1.10.2.2. Take another 1mL micropipette sucker. According to the above-mentioned operation, make
10
times increment sample solution. Replace 1mL sterile sucker with 1 dilution per increment. Make a
total
of 4 gradients.

1.10.2.3. Select 2-3 consecutive appropriate dilutions, suck 100μL sample solution of each
dilution
to culture plate to spread (5 plates per dilution) and affix the seal, culture at 30℃ for 48h in the
constant temperature incubator.

1.11. Calculation:

1.11.1. Select the colony with a CFU between 30 and 300 without spreading colony growth, and
measure
the total number of colonies by plate counting method. If it is lower than 30CFU, record the
specific
number of colonies by plate counting method. If it is larger than 300CFU, it can be recorded as too
many
to count. The number of colonies per dilution should be averaged on two plate numbers.

1.11.2. Report on the number of colonies: When the number of colonies is less than 100CFU,
according
to a “rounding” principle revision, take the first two digits and replace them with 0. It is also
expressed by the exponential form of 10 and then according to the a “rounding” principle revision,
two
digits valid numbers shall be adopted.

The bacterial survival rate/%=

● The Experiment of Freeze-drying Bacterial

Materials

· *Gluconacetobacter hansenii* ATCC 53582 and the bacterial are modified by synthetic
biology
· Tween-80,Tween-20
· EHP
· Hydrogenated polyisobutene
· Sepigel 305
· HS medium
· Sterile water

Procedure

2.1.Oscillate the conical flask containing the bacteria that can produce the BC film on the vortex
mixer
for 3-4min to shake the bacteria out of the film.

2.2.Suck up 1mL bacterial liquid from the standing conical flask to inoculate to 100mL HS medium,
shake
at 180rpm and 30℃ until OD600=0.5-0.8.

2.3.The obtained volume OD600 is 3, based on the formula: c measure × V measure=c aim × V
aim.

2.4.Suck 3mL culture liquid to 50mL centrifuge tube, centrifuge at 4100rpm for 12min, discard
quickly
the supernatant solution and collect the bacterial.

2.5.Wash twice with sterile water and centrifuge to collect.

2.6.Add the volume of aim medium and corresponding antibiotic, resuspend and mix.

2.7.Under the sterile condition, add aqueous phase, surfactant and oil phase. After oscillating and
emulsify until becomes homogeneous phase, add thickener and shake fully to make it dissolve.

\*Note: If there is so much foam,you can centrifuge at 300rpm for 5min.

2.8.Under the red light and dark conditions,take 1g emulsion to 35mm culture plate with electronic
balance.

References

[1] Yang Jiaping, Li Zhixi, Jiang Xiaozhi, etc. Study on freeze-drying conditions of
acetic
acid bacteria strains [ J ]. Chinese brewing, 2008, 20: 49-52.

[2] Wang Na, Guo Shilei, Zhang Yongxiang, etc. Selection of cryoprotectants for acetic acid bacteria
[ J
].Food and fermentation industry, 2015, 41 ( 003 ): 135-139.

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### Results

Exploratory experiments

Antipseudomonal drug production module

C-di-GMP signaling and BC film production module

Safety and drug release module

● Exploratory experiments

1. MIC values

Firstly, we tested the minimal inhibitory concentrations (MIC) of 4 antibiotics against
growth of *Gluconacetobacter hansenii* ATCC 53582, and the result was detailed in Table 1. The
MIC
data here would be employed in the following experiments.

|  |  |  |
| --- | --- | --- |
| Number | Antibiotics | MIC(ng/mL) |
| 1 | Gentamicin | 300 |
| 2 | Kanamycin | 180 |
| 3 | Chloramphenicol | 148 |
| 4 | Ampicillin | 100 |

Table 1. MIC values of different antibiotics against *G. hansenii ATCC* 53582

2. Culture exploratory experiment

To identify the optimal culture medium for *G. hansenii* ATCC 53582, an array of
growth
media
was selected for testing the bacterial growth. HS culture medium is commonly used for *G.
hansenii*
ATCC
53582, while HS-6, HS-7, and HS-8 are also utilized in some previous studies. Additionally, due to
the
similarity of medium composition to HS (Table 2), LB and SOC medium were included in our assay here.
Altogether, 6 different bacterial growth
media were employed to culture the
wild type
*G. hansenii* ATCC
53582 and J23100-mcherry-pSEVA331-*G. hansenii* ATCC 53582.

|  |  |
| --- | --- |
| Medium | Formula |
| HS | 2% glucose,0.5% peptone,0.5% Yeast Extract ,0.68% NaHPO4·12H2O,0.15% Citric Acid Monohydrate |
| LB | LB Broth |
| SOC | SOC Broth |
| HS-6 | 8% glucose,1% peptone,0.2% Citric Acid Monohydrate,0.27% NaHPO4·12H2O,0.03% MgSO4·7H2O |
| HS-7 | 8% glucose,2% YeastExtract,0.2% NaHPO4,0.1% 12H2OKH2PO4,0.02% MgSO47H2O 1% C2H5OH |
| HS-8 | 2% sucrose, 0.5% peptone,0.5% Yeast Extract, 0.68% NaHP04·12H2O,0.15% Citric Acid Monohydrate |

Table 2. Formula of 6 different bacterial growth media used in our assay

Consistent with visual observation, the optical density of bacteria grown in HS, SOC and
HS-8 indicated bacterial growth, whereas bacteria in other media failed to grow, as illustrated by
low
or even negative values of OD600. In comparison, following 48 hours of culture, a higher
level of
biomass for bacteria grown in SOC, HS, LB and HS-8 was observed, with the highest OD600
observed in SOC
medium. Thus, SOC might be the optimal growth medium. Notably, OD600 of bacterial culture
in
HS-8 at 48
hours was lower than that in 24 hours, an indicative of transition into the death phase in HS-8 at
this
timing. **Taken together, based on the growth assay using a range of culture media, SOC and HS
were
opted
for the further experiments concerning *G. hansenii* ATCC 53582.**

Figure 1. (a) Liquid culture of J23100-mcherry-pSEVA331-*G. hansenii* ATCC 53582
after
24
hours of agitation, (b) liquid culture of the wild type *G. hansenii* ATCC 53582 after 24 hours
of
agitation, (c) optical density of J23100-mcherry-pSEVA331-*G. hansenii* ATCC 53582 and wild
type *G.
hansenii* ATCC 53582 in different culture media after 24 hours of agitation, (d) liquid
culture of
J23100-mcherry-pSEVA331-*G. hansenii* ATCC 53582 after 48 hours of agitation, (e) liquid
culture of
*G.
hansenii* ATCC 53582 after 48 hours of agitation, (f) optical density of
J23100-mcherry-pSEVA331-*G. hansenii* ATCC 53582 and wild type *G. hansenii* ATCC 53582 in
different culture
media after 48 hours of agitation.

To quantify the bacterial cellulose (BC) production of *G. hansenii* ATCC 53582 in SOC and HS
medium, we
launched liquid culture of J23100-mcherry-pSEVA331-*G. hansenii* ATCC 53582 in HS and SOC
medium
with
different volumes (2mL, 2.5mL and 3mL) for 4 days. Then we measured the dry weight of the BC film,
which
was consistent with the BC thickness as visually observed (Figure 2. a, b). Intriguingly, bacteria
cultured in 3mL of HS medium yielded the highest amount of BC, even though the OD600 of
bacterial
culture in HS was lower than that in SOC.  **In the end, we chose HS medium to culture and
product
BC
film.**

Figure 2. (a) Visualization of bacterial cellulose production in 2mL, 2.5mL and 3mL of
HS and
SOC medium after 4 days of fermentation in 12-well plates, (b) Quantification of the bacterial
cellulose
dry weight.

3. Eletrotransformation condition exploratory experiment

In order to optimize the transformation efficiency of plasmid into *G. hansenii*
ATCC
53582,
we conducted electroporation assay with the different voltage values, either 2.5kV or 3kV. A larger
amount of bacterial transformants can be found on selective plates in the 3kV-treated group.
Therefore,
we chose 3kV to perform the electroporation of *G. hansenii* ATCC 53582 with plasmids in the
following
experiment.

4. Illumination intensity exploratory experiment

During the consult to the experts( For more detail, please click [Human\_Practices](https://2021.igem.org/Team%3ASZPT-CHINA/Human_Practices)),
suggestions
were
made to explore the effect of details intensity on *G. hansenii* ATCC 53582 product BC in the NIR
light
intensity in the c-di-GMP signaling and BC film production module. Then we cultured respectively
J23100-bphS-J23109-fcsR-pSEVA331-*G.hansenii* ATCC 53582 under 5， 10， 20， 50μW/cm2.
The
yield under 5, 10, 20, 50 μW/cm2 illumination intensity had no significant difference. Therefore, we still
chose 50μW/cm2
for film production experiment.

Figure 3. The BC yield at different illumination intensity

● Antipseudomonal drug production module

1. Goal

The function of this module is to produce a chimeric bacteriocin that can target
*Pseudomonas
aeruginosa* while having a broader antipseudomonal spectrum than pyocin S2. To enhance the
antipseudomonal effect, we further engineered the RBS strength to modulate the expression of SE
protein.

2. What work we have done

① Verify that the SE fusion protein has an antibacterial effect on *P.
aeruginosa*.
② Successfully construct a series of plasmids to express different levels of SE protein.
③ Through the inhibition zone test and growth curve test, screen *G. hansenii* ATCC
53582-derived
strains
with a pronounced antipseudomonal effect.

3. Proof

3.1 Antipseudomonal properties of SE protein

IPTG was added to a final concentration of 0.1 mmol/mL to induce SE protein expression
in
*E.
coli* BL21 with S2-PET28A or SE-PET28A at 25℃ for 12 hours. Supernatant containing SE or S2
proteins were
obtained by sonication and centrifugation. The crude protein extract was loaded into SDS-PAGE gel
and
stained by Coomassie Blue to verify the presence of target proteins ( For more detailed steps,
please
click [Experiments](https://2021.igem.org/Team%3ASZPT-CHINA/Experiments)). After
IPTG
induction, S2-PET28A-BL21 produced a 83.9 kDa
protein (lane 2) while
SE-PET28A-BL21 produced a 81 kDa protein (lane 3). In contrast, non-induced *E. coli* BL21
cells
did not
express S2 or SE protein (lane 1, lane 4). **The results showed that the constructed plasmids
S2-PET28A
and SE-PET28A could be used to express the fusion protein** (Figure 4. a).

Then, supernatant containing SE or S2 protein was respectively added into the culture of
*Pseudomonas
aeruginosa* PAO1, *P. aeruginosa* PAO1 Δ1150-1151（PAO1 Δ1150-1151）and *E. coli*
MG1655.
PBS
was used as a
negative control. Inhibitory effect of supernatant on the *P. aeruginosa* growth was determined
by
measuring OD600 using SYNERGY H1 micro-plate reader. **The result showed that the
SE-fusion protein could
prevent *P. aeruginosa* growth, irrespective of the presence of the PA1150-1151 region,
while
pyocin
S2
can only inhibit the PAO1 Δ1150-1151 mutant, but not the wild type strain. In addition, both SE
and
S2
proteins had no effect on *E. coli* growth** (Figure 4. b).

Figure 4. (a) SDS-PAGE analysis of protein expression in engineered *E. coli*. Lane
1
S2-PET28A BL21 without IPTG induction, lane 2 supernatant of S2-PET28A BL21 with induction by
0.1mmoL/mL
IPTG, lane 3 supernatant of SE-PET28A BL21 with induction by 0.1mmoL/mL IPTG, lane 4, SE-PET28A BL21
without IPTG induction, (b) Optical density of bacterial culture of PAO1, PAO1 Δ1150-1151 and *E.
coli*
MG1655 treated by the supernatant of S2-PET28A-BL21 or SE-PET28A-BL21 lysate for 790 minutes. PBS
was
used as the negative control.

3.2 Screen and identify an RBS to express SE protein with an optimal antipseudomonal
effect.

Using high-throughput screening, we engineered the strength of RBS that control the SE
protein expression level in *E. coli*, and the resultant plasmids were extracted and in turn
introduced
into the *G. hansenii* ATCC 53582. *G. hansenii* ATCC 53582 culture supernatant containing
SE
proteins that
were expressed under control of different RBS were obtained by sonication and centrifugation and
further purified by infiltration with micro-pored membrane. Then, supernatant in different groups
was
individually mixed with PAO1 culture, and incubated at 30℃ for 12 hours. SYNERGY H1 micro-plate
reader
was used to measure the OD600 to determine the effect on PAO1 growth. **As shown in
Figure 5 a,
supernatant of culture derived from the 3rd, 4th, 7th,
8th colony had significant
antipseudomal effect.**

3 μL of the supernatant containing SE protein was dropped on FAB plate with PAO1 evenly spread
(for
more
detailed steps, please click [Experiments](https://2021.igem.org/Team%3ASZPT-CHINA/Experiments)). Then, the
plates
were incubated at 30℃
for 12 hours.
Supernatants from all the *G. hansenii* isolates have formed inhibition zone, yet with
different
sizes.
Isolate 3rd and 4th displayed the most significant antipseudomonal
effect.**The results of the optical
density measurements of PAO1 growth and the inhibition zone prove that SE protein is
successfully
expressed in the engineered *G. hansenii*. A series of *G. hansenii* containing the SE
expression module
with difference RBS were constructed to screen for the strain with the best antipseudomonal
property.
As shown in Figure 5, the 4th strain pR-RBS300-SE-J23118-IMM-pSEVA331-*G.
hansenii*
ATCC
53582-4# is best
as it has the largest inhibition zone area.**

Figure 5. (a) Optical density of PAO1 treated with the supernatants of different
bacterial
strain lysates cultured after 12 hours of growth in PBS, (b) Drop plate assay to check the
inhibitory
effect of SE proteins on *Pseudomonas aeruginosa* PAO1, (c) Strains used in Figure (b).

● C-di-GMP signaling and BC film production module

1. Goal

As bacterial cellulose (BC) production in *G. hansenii* is regulated by the second messenger
c-di-GMP. Therefore, we aimed control BC film production by regulating c-di-GMP concentration,
*i.e*. so
that *G. hansenii* ATCC 53582 can produce BC under illumination of Near Infrared light (NIR
light)
at
680nm, but cannot under dark conditions.

2. What work we have done

① Construct the synthesis and hydrolysis modules of c-di-GMP in *G. hansenii* ATCC 53582.

② Screen the best modules for BC film production, and integrate them into one single plasmid.

3. Proof

We used bphS which is the coding part of the photo-activated diguanylate cyclase BphS as well as the
coding parts of c-di-GMP phosphodiesterase from different bacteria to construct the plasmids for
c-di-GMP synthesis module and c-di-GMP hydrolysis module in *E. coli* competent cells. Then the
plasmids
were extracted and transferred into the *G. hansenii* ATCC 53582.

Figure 6. (1) The genetic circuit for c-di-GMP synthesis module, (2) The genetic circuit
for
c-di-GMP hydrolysis module B, amplification of different regions by PCR, (c) Plasmids used in Figure
B.

According to the 12-well plate test (Figure 7. A, for more detailed steps, please
click
 [Experiments](https://2021.igem.org/Team%3ASZPT-CHINA/Experiments)), BC was dried
and
weighted. A marked difference in BC production between
J23100-fcsR-pSEVA331-*G. hansenii* ATCC 53582 and our control group pSEVA331-*G. hansenii*ATCC 53582 was
observed (Figure 7. B1). Therefore, J23100-fcsR-pSEVA331 was shown to have a marked hydrolytic
function.
On the other hand, J23100-bphS-bphO-pSEVA331-*G. hansenii* ATCC 53582 produced a higher level
of BC
under
the illumination of NIR light than the dark condition (Figure 7. B2). **Based on the above
results, we
chose BphS and FcsR as the c-di-GMP synthetase and hydrolase respectively in our
system.**

Figure 7. A. Verification experiment of BC film yield in 12-well plates B. (1) BC yield
by
J23100-fcsR-pSEVA331-*G. hansenii* ATCC 53582, J23100-yhjH-pSEVA331-*G. hansenii* ATCC
53582,
J23100-rocR-pSEVA331-*G. hansenii* ATCC 53582 and the vehicle control pSEVA331-*G.
hansenii*
ATCC 53582, (2)
BC yield by J23100-bphS-bphO-pSEVA331-*G. hansenii* ATCC 53582 under NIR light illumination and
dark
conditions.

We aim to construct a photo-activated system for production of BC film in *G.
hansenii*
ATCC
53582, where the amount of intracellular c-di-GMP (Figure 8. A) sustained at a low level under dark
condition, but can reach a high level under illumination of NIR light. To this end, we constructed
an
array of plasmids by assembling different synthesis and hydrolysis components of c-di-GMP. Finally,
these plasmids were introduced into the *G. hansenii* ATCC 53582 via electroporation to screen
the
optimal
isolate in BC yield (Figure 8. B).

We constructed three plasmids with different promoters that can control the c-di-GMP levels. Then
*G.
hansenii* ATCC 53582 were transformed with these plasmids. Then the BC film production in
12-well
plates
was assessed. Among the strains we tested, a significant difference in the BC yield under different
illumination conditions was found for J23100-bphS-bphO-J23109-fcsR-pSEVA331-*G. hansenii* ATCC
53582 and
J23100-bphS-bphO-J23110-fcsR-pSEVA331-*G. hansenii* ATCC 53582. Notably, a background level of
BC
production was always detected in all the strains under dark conditions.  **In the end, we
chose
J23100-bphS-bphO-J23109-fcsR-pSEVA331-*G. hansenii* ATCC 53582 which has a significant
difference in the
BC yield under different illumination conditions as the strain relatively good matches our
goal.**

Figure 8. (A) The genetic circuits for c-di-GMP signal transduction and BC growth module, (B) PCR
identification results, lane 1 J23100-bphS-bphO-J23109-fcsR-pSEVA331-*G. hansenii* ATCC 53582,
lane
2
J23100-bphS-bphO -J23110-fcsR-pSEVA331-*G. hansenii* ATCC 53582, lane 3
J23100-bphS-bphO-J23119-fcsR-pSEVA331-*G. hansenii* ATCC 53582, (C) BC film production by
different
strains, (D) Strains used in Figure C.

● Safety and drug release module

1. Goal

With the illumination of blue light, the expression of the lysis protein controlled by the pDawn
promoter would be activated. While the amount of the lysis proteins in the engineered bacteria
reaches a
certain threshold, the bacteria will be auto-lysed and the antipseudomonal proteins are released,
thus
antagonizing *P. aeruginosa* growth.

2. What have we done

① We have identified that the blue light-responsive promoter pDawn can activate the
expression of downstream genes in *G. hansenii* ATCC 53582 under blue light condition.
② We have constructed a set of plasmids which include pDawn to induce the expression of an
antiholin-free Lambda lysis cassette (S105), φX174 phage lysis gene (X174 E), and LKD16 lysis
cassette
(LKD16). After introducing these plasmids into *E. coli*, bacterial strains grow normally under
dark
conditions but do not under 470nm blue light.
③ The plasmids will be extracted from *E. coli* and subsequently introduced into the *G.
hansenii*
ATCC
53582, where the expression of X174 E can be activated under illumination of blue light

3. Proof

3.1 Functional verification of pDawn promoter in response to blue light in *G.
hansenii* ATCC 53582.

The plasmid pDawn-RFP-pSEVA331 was firstly constructed and introduced in *G.
hansenii*
ATCC
53582, where its function was tested. To be specific, bacteria were cultured under either the blue
light
at 470nm or dark conditions for 24 hours, and the intensity of red fluorescent signal was detected
using
fluorescence microscope. After 24 hours of growth, *G. hansenii* ATCC 53582 in the blue light
treated
group showed red fluorescence, but no fluoresces were observed for those under dark conditions
(Figure
9). Based on these results, pDawn in *G. hansenii* ATCC 53582 can be successfully activated by
blue
light
irradiation.

Figure 9. Red fluorescence photo of the pDawn-RFP-pSEVA331-*G. hansenii* ATCC 53582
cultured
under 470-nm blue light or dark conditions for 24 hours.

3.2 Functional verification of lysis protein in *G. hansenii* ATCC 53582

We employed the blue light-responsive promoter pDawn to control the expression of different lysis
genes,
including S105, X174 E and LKD16 (Figure 10). Random primer-guided mutagenesis on the target RBS
position
was performed ([BBa\_B0034](http://parts.igem.org/Part%3ABBa_B0034)) ( For more detailed
steps, please click [Improvement](https://2021.igem.org/Team%3ASZPT-CHINA/Improvement)). To verify
the lysis
effect of these components, we conducted the plate dot assay ( For more detailed steps, please
click
[Experiments](https://2021.igem.org/Team%3ASZPT-CHINA/Experiments)). Expression of
lysis genes
will be considered effective when bacterial
colonies can grow
normally in the dark, but not under blue light irradiation.

Figure 10. Genetic circuit for safety and drug release module.

To be specific, 20 single colonies on the culture plate were picked to spot on two parallel plates
(if
the number of single colonies on the culture plate is less than 20, picked all of them). The plates
were
incubated either in the dark or under blue light illumination for 24 hours to observe the colony
morphology, based on which we determine whether there is a lysis effect.

Although the colony of S105 could not grow under blue light its growth state was abnormal in the
dark.
We attributed this phenomenon to a basal expression level of lysis protein controlled by pDawn
promoter
(Figure 11. A (a1), (a2)). Of note, the 4th isolate of with pDawn-X174 E-pSEVA331-
*E. coli* grew
normally in
the dark but not under the blue light irradiation（Figure 11. A (b1) and (b2)）. Additionally, as
shown in
Figure 11. A (c1) and (c2), all the colonies of *E. coli* with pDawn-LKD16-pSEVA331- *E.
coli*
were
able to
grow under both conditions, whereas the growth of 4th, 9th,10th and
12th colonies was remarkably
inferior than those in the dark, indicating that pDawn-LKD16 allowed bacteria to be lysed in
response to
blue light, yet with a modest effect.

In order to construct *G. hansenii* ATCC 53582 strains with the capability of light-responsive
cell
lysis,
we firstly introduced the plasmids that have lysis effect in *E. coli* into the *G.
hansenii*
ATCC
53582,
and then 8 single colonies were picked to perform the drop plate assay. Two copies of plates were
separately incubated under the blue light and dark conditions at 30℃. The bacterial colony
morphology,
including the size and thickness, was observed every 24 hours to determine the lysis effect. After
introducing the plasmid containing S105, X174 E or LKD16 into the *G. hansenii* ATCC 53582,
they can
cause
similar colony-lysis phenotype. While the plasmids containing pDawn-S105 completely inhibited growth
of
*G. hansenii* ATCC 53582 under both conditions. (Figure 11. B (a), (b) and (c)). We thus
preserved
the
pDawn-X174 E-pSEVA331-*G. hansenii* ATCC 53582 and pDawn-LKD16- pSEVA331-*G. hansenii*
ATCC
53582 . And these
strains were spotted on the culture plates for screening. The strain pDawn-X174 E-pSEVA331-*G.
hansenii*
ATCC 53582-7# showed a stable lysis effect, while the others did not. (Figure 11. C). **In the
end, we
chose pDawn-X174 E-pSEVA331-*G. hansenii* ATCC 53582-7# which can be activated under
illumination
of blue
light as the goal strain.**

Figure 11. Drop plate assay to assess the inducible production the lysis protein by
470-nm
blue light illumination, (A) *E. coli* DH5α transformed with different lysis proteins, (B)
*G.
hansenii* ATCC
53582 transformed with different lysis proteins, (C) pDawn-X174 E-pSEVA331-*G. hansenii* ATCC
53582,
which
stably showed the lysis effect, (a1) and (a2), pDawn-S105-pSEVA331, (b1) and (b2), pDawn-X174
E-pSEVA331,
(C1) and (C2), pDawn-LKD16-pSEVA331,(a1), (b1) and (C1) are incubated under 470-nm blue light, (a2),
(b2) and (C2) are incubated under dark conditions.