iGEM 2023 — Team sustech-oce

# Notebook

![illustrations](https://static.igem.wiki/teams/4809/wiki/leaf-bg-top.png)
![illustrations](https://static.igem.wiki/teams/4809/wiki/dots-group-sm.png)
![illustrations](https://static.igem.wiki/teams/4809/wiki/leaf-yellow.png)
![illustrations](https://static.igem.wiki/teams/4809/wiki/leaf-orange.png)
![illustrations](https://static.igem.wiki/teams/4809/wiki/dots-group-cyan.png)
![illustrations](https://static.igem.wiki/teams/4809/wiki/leaf-cyan-lg.png)

* [Human Practices Diary](#1)
* [Experimental Diary](#2)
* [Modeling Diary](#3)

## Human Practices Diary

### Event 1: Meeting with Professor Zhang Weiwen

**Event Purpose:** Exchange ideas with Professor Zhang Weiwen, obtain expert
advice, and enhance the research proposal.

**Event Plan:**Discuss biosafety, project direction, and receive expert guidance.

**Expected Event Outcomes:**Improve the project proposal, consider biosafety and
feasibility, and lay the foundation for project development.

### Event 2: SUSTech Deep Blue Charity Bazaar

**Event Purpose:** Organize a charity fair, unite two universities to raise funds
for the "Sunshine Little Library · Child Growth" project.

**Event Plan:**Set up unique stalls, plan engaging activities and merchandise, and
collect donations for charity.

**Expected Event Outcomes:**Raise public awareness of the iGEM competition and
synthetic biology, and support charitable causes.

### Event 3: Meeting with Associate Professor Wang Chen

**Event Purpose:** Exchange ideas with Associate Professor Wang Chen, obtain
professional advice on indoor pollutants.

**Event Plan:**Discuss sources of indoor air pollutants, models for pollutant gas
concentration, Henry's Law calculations, and detection methods.

**Expected Event Outcomes:**Purchase suitable measurement instruments, conduct
on-site measurements of indoor pollutants, and understand international testing
standards.

### Event 4: Joint Presentation at SUSTech and Medical School

**Event Purpose:** Present a joint lecture to introduce synthetic biology and the
iGEM competition to the university community, fostering cooperation among
participating teams.

**Event Plan:**Organize a joint presentation by two iGEM teams, sharing their
respective projects and engaging in discussions.

**Expected Event Outcomes:**Increase awareness of synthetic biology and the iGEM
competition, encouraging more collaboration and interaction.

### Event 5: 7th South China Exchange Meeting

**Event Purpose:** Gather iGEM teams from South China to provide a platform for
collaborative interaction and exploration of synthetic biology.

**Event Plan:**Showcase projects of 16 teams under the theme "Villagarnival,"
interact, and learn from each other.

**Expected Event Outcomes:**Promote team collaboration, knowledge sharing, and
create a conducive research environment.

### Event 6: Qianlinshan Primary School Outreach

**Event Purpose:** Conduct a science outreach program for primary school students
to introduce them to microbiology and spark their interest in the natural world.

**Event Plan:**Present topics such as microbiology and indoor air pollution
purification systems, organize interactive activities, and engage in hands-on craft
making.

**Expected Event Outcomes:**Ignite students' curiosity about biodiversity and
ecosystems, and encourage them to apply concepts of sustainable development.

### Event 7: 7.7 CCiC Conference

**Event Purpose:** To facilitate interaction and learning among Chinese iGEM
teams, promoting the development of synthetic biology.

**Event Plan:**Held at Hainan University, the conference includes academic
lectures, team presentations, and special workshops.

**Expected Event Outcomes:**Strengthen interaction within the Chinese iGEM
community, encourage innovation and collaboration, and provide more resources and
support for synthetic biology.

### Event 8: 9.4 2023 Guangdong Science Center SynBio & SDG Outreach

**Event Purpose:** To communicate the concepts of synthetic biology and
sustainable development to the public through science outreach exhibitions.

**Event Plan:**Conduct a science outreach exhibition at the Guangdong Science
Center, including a survey questionnaire, creative product displays, and engaging
experiments.

**Expected Event Outcomes:**Raise public awareness of synthetic biology and
sustainable development, stimulate interest in indoor air quality and environmental
protection.

### Event 9: Daily Updates on WeChat Public Account

**Event Purpose:** To convey the work of the SUSTech-OCE team and knowledge about
synthetic biology to the public.

**Event Plan:**Create a specialized column called "SUSTech-OCE Daily Blog" on
WeChat, posting daily images and content to document the team's experimental
progress, science outreach activities, and events.

**Expected Event Outcomes:**Increase public understanding of synthetic biology,
raise the team's profile, and share information related to environmental protection
and sustainability.

### Event 10: Literature Review 1-3

#### Literature Review 1: Indoor Formaldehyde Pollution

**Event Purpose:** To investigate the status of indoor formaldehyde pollution in
China, understand its impact on public health, and assess the importance of
sustainable solutions.

**Event Plan:**Conduct literature research on formaldehyde production and
consumption in China, its concentration in indoor air, and the factors influencing
formaldehyde release.

**Expected Event Outcomes:**Gain insights into the severity of indoor formaldehyde
pollution, assess its long-term effects, and emphasize the significance of
sustainable solutions for reducing indoor formaldehyde concentrations.

#### Literature Review 2: Formaldehyde and Health

**Event Purpose:** To explore the connection between formaldehyde exposure and
health issues, emphasizing the potential risks of indoor formaldehyde pollution.

**Event Plan:**Investigate international classifications of formaldehyde as a
carcinogen, explore its effects on lung function, and discuss the economic
implications of formaldehyde-related diseases.

**Expected Event Outcomes:**Highlight the health risks associated with
formaldehyde exposure and the financial burdens imposed by formaldehyde-related
diseases.

#### Literature Review 3: Indoor Air Purification Solutions

**Event Purpose:** To review existing methods for indoor air purification,
focusing on their limitations and need for innovation.

**Event Plan:**Research common air purification methods such as natural
ventilation, activated carbon, and indoor plants, and evaluate their effectiveness
in real-life scenarios.

**Expected Event Outcomes:**Illustrate the challenges of traditional indoor air
purification methods and emphasize the importance of innovative solutions for
tackling indoor air pollution effectively.

### Event 11: Research on Hospitals: Pollutants and Health

**Event Purpose:** To understand the connection between pollutants and health,
emphasizing the impact of indoor air quality.

**Event Plan:**Conduct interviews with Shandong Provincial Hospital to gather data
and information about the health hazards associated with air pollution, focusing on
indoor pollutants such as formaldehyde, aromatic substances, and rare gases.

**Expected Event Outcomes:**

Highlight the potential health hazards of pollutants like formaldehyde,
benzene-related substances, and rare gases, including risks of leukemia, oxidative
stress damage, and lung cancer.

Emphasize the importance of improving indoor air quality for public health.

This research aimed to investigate the links between pollutants and health by
collecting data from Shandong Provincial Hospital, with a particular focus on indoor
air quality and its impact on well-being. The expected results emphasize the
potential health risks associated with indoor pollutants and advocate for necessary
measures to enhance indoor air quality.

### Event 12: 7.31 Furniture City Survey

**Event Purpose:**

Increase Public Awareness of Indoor Formaldehyde Pollution: The primary goal of this
activity is to raise public awareness of the issue of indoor formaldehyde pollution,
emphasizing the potential risks associated with formaldehyde in furniture.

Understand the Sources and Proportion of Formaldehyde in Furniture: This activity
aims to investigate the sources and proportion of formaldehyde in furniture,
providing a scientific basis for taking measures to reduce indoor formaldehyde
concentrations.

**Event Plan:**

On-Site Research by Team Members: Team members Yang Jiayi and Yu Le will conduct
on-site research by visiting furniture stores.

Survey a Variety of Furniture: The team will conduct surveys on a wide range of
furniture, with a specific focus on panel furniture and custom-made furniture,
recording formaldehyde data.

Purchase Precision Formaldehyde Concentration Measuring Instruments: Precision
instruments for measuring formaldehyde concentrations will be acquired to ensure the
accuracy of the data.

Data Integration and Chart Creation: Data collected will be integrated, and charts
will be created to highlight the correlation between formaldehyde levels in
furniture and health risks.

**Expected Event Outcomes:**

Raise Public Awareness of Formaldehyde in Furniture: The activity is expected to
increase public awareness of the issue of formaldehyde in furniture, encouraging the
purchase of eco-friendly furniture.

Provide Real Data to Emphasize Formaldehyde Risks: By providing real and accurate
data, this activity aims to emphasize the risks associated with formaldehyde in
furniture. This, in turn, will assist individuals in taking measures to reduce
indoor formaldehyde concentrations and improve indoor air quality.

Encourage Manufacturers to Use Eco-Friendly Materials: The activity also seeks to
encourage manufacturers to adopt more environmentally friendly materials, reducing
formaldehyde content in furniture. This will benefit indoor air quality and overall
human health.

### Event 13: Picture Book Creation and Exchange

**Event Purpose:** The purpose of this activity is to introduce the public to the
new methods of synthetic biology for addressing environmental issues through the
creation of picture books. The goal is to generate interest and curiosity among the
public about this field.

**Event Plan:**In collaboration with another team, OUC-Haide, a series of adorable
picture books will be created, featuring mascots as the main characters. These
picture books will serve as a means of conveying scientific information in an
engaging and easily understandable manner.

**Expected Event Outcomes:**

The expected outcomes of this activity include:

Increased Public Understanding of Synthetic Biology Methods: The activity aims to
increase public understanding of the methods of synthetic biology for addressing
environmental challenges.

Encouraging the Adoption of Scientific Methods: By creating awareness and interest
in synthetic biology, the activity encourages people to consider and utilize
scientific methods to improve their quality of life.

### Event 14: Hardware Design and Production

**Event Purpose:** This activity aims to design the hardware components to support
the implementation of the project, including cultivating blue-green algae and
monitoring pollutant gases.

**Event Plan:**We will design a glass tank and related hardware, including
filters, air pumps, etc., for cultivating blue-green algae and monitoring the
fluorescence of Escherichia coli.

**Expected Event Outcomes:**The expected results include support for project
implementation, improved experimental efficiency, and monitoring of indoor pollutant
concentrations.

### Event 15: Meeting with Professor Zhang Weiwen

**Event Purpose:** The purpose of this activity is to understand the public's
level of awareness regarding indoor air pollution and synthetic biology, and to
provide data support for the project.

**Event Plan:**We will distribute surveys in two rounds to different groups of
people to collect data.

**Expected Event Outcomes:**The expected results include an understanding of the
level of awareness among different age groups regarding indoor air pollution and
synthetic biology, providing guidance for the project.

These activities are planned to increase public awareness of indoor air pollution,
synthetic biology, and sustainability, promoting the dissemination of scientific
knowledge, and providing support for environmental improvement and health.

## Experimental Diary

2023.6.16
Yidong

Wang
,
Jiulin

Chen

Preparation of
LB
solid medium

400ml
\*1
：
10g LB + 6g Agar
+ 400ml
ddH
2
O

Preparation of
LB liquid medium

400ml \*1
：
10g LB + 400ml
ddH
2
O

Formaldehyde solution preparation

Formaldehyde mother
solution

(37 wt.%) MW
：
30.03
；
concentration
：
1 \* 37% / 30.03 / 1 = 12.321mol/L

Formaldehyde stock solution

(
1mol/L
)
：
8.13ml
m
other solution
+ 91.87ml
ddH2O

Preparation of kanamycin mother
solution

Weigh
500mg

kanamycin solid in a 15ml centrifuge tube and add deionized water to

10ml

(
50mg/mL
)

U
se a filter membrane with a pore size of 0.22
μ
m to filter the mother
solution
, and divided it into 10 1.5ml EP tubes, which were stored in the refrigerator at -20
℃
.

Preparation of LB solid medium containing kanamycin

Heat the prepared 400ml LB solid medium until the
agar
is completely melted and cool at room temperature
.

When cool to not hot (about 50°C), add 400μl kanamycin mother liquor (50μg/mL), mix well, and pour the medium into a disposable plate for use.

Plasmid introduction of

pET28a-FrmR-fdh-fghA
&pET28a-FrmR-sfGFP

1.

C
entrifuge

t
he synthesized target plasmid dry powder sample (2μg)

at 12000rpm for 1 minute.

2.

A
dd
200μ
l
ddH2O
，
c
lose the tube cover and vortex to dissolve it fully
。

3.

Take 1 tube (100
μl
) of
competent cell
s and place them on ice for 20-30 min to melt them
,

a
dd
5μl dissolved target plasmid (50ng), mix gently, and leave on ice for 30 min.

4.

42
℃

water bath heat shock 45~60 s, quickly transferred to the ice bath, standing for 2 min (do not shake the sample during the ice standing process, otherwise it will reduce the conversion efficiency).

5.

Add
700
μL

of
LB medium without antibiotics
into the centrifuge tube, mix,
incubate
at 37
℃

at 200 rpm for 60 min.

6.

A
bsorbed 100μL
culture
solution, evenly coat it on LB medium containing kanamycin, and place the plate upside down in an incubator at 37 °C for overnight culture
.

Note: Plate 1, 1 (H) is the imported pET28a-FrmR-sfGFP engineering bacteria, and 2, 2 (H) is the imported pET28a-FrmR-fdh-fghA engineering bacteria

2023.6.17
Yidong

Wang
,
Jiulin
Chen

Prepare
100
μmol
/L formaldehyde solution in a watering can: 1μl
stock
solution + 10 mL deionized water

Plate fluorescence test

Plate 1 (H) was sprayed with 100
μ
M formaldehyde three times and incubated at 37
℃

for 1h. Results: No obvious fluorescence was observed
.

Plate 1 (H) added 100
μ
M formaldehyde 100
μ
L
and incubated at 37
℃

for 3h.

Results: No obvious fluorescence was observed
.

Plate 1 (H) added 100
μ
M formaldehyde 100
μ
L
and incubated at 37
℃

for
4.5
h.

Results: No obvious fluorescence was observed
.

Plate 1 (H) was simultaneously added with 1mM, 10mM, 100mM formaldehyde 10
μ
l and incubated at 37
℃

overnight.

PCR

verification

S
elect 2 single colonies from each plate and name them 1a, 1b, 1c, 1d, 2a, 2b, 2c, 2d. Dissolve in 20μ
L
sterile water as PCR template.

P
repare the PCR system on ice.

|  |  |
| --- | --- |
| Taq PCR Master Mix | 12.5  μ L |
| Forward Primer (10uM) | 1  μ L |
| Reverse Primer (10uM) | 1  μ L |
| Sterile ddH 2 O | 9.5  μ L |
| T emplate | 1  μ L |
| Total | 25  μ L |

Note: Primers for 1,

1 (H) were pET28-R &28aF;

Primers for
2
,

2
(H)

were

pET28-R &pET28-F

Set up the PCR program, put the PCR tube into the instrument, and start to run PCR.

95
℃
，
3min

![](avywx-5w09a.001.png)
95
℃
，
15s

30
×

55
℃
，
15s

72
℃
，
60s / 100s

72
℃
，
5min

4
℃
，
forever

Note: 1, 1 (H) extension time is 60s; 2, 2 (H) extension time is 100s
.

Verification by agarose gel electrophoresis

Weigh 0.72 g agarose, add 60ml 1×TAE solution (1.2%), heat to dissolve agarose. Add 6μL SYBR dye, pour into the mold, cool at room temperature.
Absorb
5µL PCR product, add
it
to the gel hole, and
turn on

the
electrophoresis apparatus. After the agarose gel electrophoresis,
view
the results on the photometer and photograph
.

R
esult
:

There are multiple strips and they do not match the length of the target fragment.

Prepar
e
formaldehyde in various concentrations
:

100mM
,

10mM
,

1mM
,

100μM
,

10μM

Plate streak

S
treak s
ingle colonies 1a, 2b, 1c
with
inoculating loop
on LB plates containing kanamycin

2023.6.18
Yidong

Wang

Streak single colonies 1a, 2b, 1c
again
，
a
nd perform liquid culture with 10mL

tube (4mL LB + 4
μL

kanamycin mother
solution
).

P
reparation of culture medium

LB
（
s
olid
）
400ml \*1

LB
（
l
iquid
）
200ml \*2

R
ename the single colony 1a, 2b, 1c plates marked by plates the previous day as 1aI, 2bI, 1cI, and perform liquid culture with 10ml tube (4ml LB + 4μL kanamycin mother
solution
).

2023.6.19
Yidong

Wang
,
Zhiang
Chen

PCR

verification

Samples
：
1a
，
2b
，
1c
，
2d
，
1aI
，
2bI
，
1cI

T
a
k
e
100μL of liquid culture bacteria solution, centrifuge at 12000rmp for 1min, remove the supernatant, add 30μL NaOH solution and resuspend, and then add

70μL Tris as template.

P
repare the PCR system on ice.

|  |  |
| --- | --- |
| Taq PCR Master Mix | 12.5  μ L |
| Forward Primer (10uM) | 1  μ L |
| Reverse Primer (10uM) | 1  μ L |
| Sterile ddH 2 O | 9.5  μ L |
| T emplate | 1  μ L |
| Total | 25  μ L |

Note: The primers used were pET28-R &28aF

Set up the PCR program, put the PCR tube into the instrument, and start to run PCR
。

95
℃
，
3min

![](avywx-5w09a.001.png)
95
℃
，
15s

30
×

55
℃
，
15s

72
℃
，
60s / 100s

72
℃
，
5min

4
℃
，
forever

Note: 1, 1 (H) extension time is 60s; 2, 2 (H) extension time is 100s
.

Verification by agarose gel electrophoresis

Result: The test was successful. Seven samples showed a single band, which was consistent with the target size.

P
reservation
：
A
bsorb 700μL of liquid culture solution +

300μL of 50% glycerol for
culture

preservation and store it in a -80
℃

refrigerator
.

Pre-experiment of green fluorescence induced by formaldehyde

Take
28 1.5ml EP tubes and add them to 1ml LB medium and 1μL kanamycin mother solution, then inoculate 7 tubes with 1μL 1a, 1c, 1aI, 1cI bacterial solution at 37 ° C and 200rpm.

W
hen
OD600
≈
0.
4,
Add formaldehyde to concentrations as shown in the following table
：

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| S ample | 1a | 1c | 1aI | 1cI |
| Formaldehyde concentration （ μ M ） | 0 | 0 | 0 | 0 |
| 5 | 5 | 5 | 5 |
| 10 | 10 | 10 | 10 |
| 25 | 25 | 25 | 25 |
| 50 | 50 | 50 | 50 |
| 100 | 100 | 100 | 100 |
| 250 | 250 | 250 | 250 |

Continue cultivation at 37 degrees, 200rpm. Every 10min,
observe
the green fluorescence by naked eye under ultraviolet lamp.

Results: The difference of 4 samples began to appear after 30min, and the difference was obvious after 1h, and the fluorescence intensity increased with the concentration gradient of formaldehyde. The difference between samples 1aI and 1c is the most obvious.

2023.6.20
Yidong

Wang
,
Zhiang
Chen

M
ade the
bacterial solution of sample 1aI induced by 250μM formaldehyde yesterday
into a temporary plate and observed under a
fluorescence microscope
,
we found that there was obvious fluorescence
.

2023.6.21
YidongWang

S
treak

strain
1aI

a
nd
1c.

2023.6.29
Yidong

Wang
,
Jiulin
Chen
,
Xin Yang

Culture s
train 1aI in a conical flask, 100ml LB medium +100μL kanamycin mother solution, and inoculated with 100μL 1aI activation solution
.

Culture at 37 ° C, 200rpm for 2-3h, take 100ul to black transparent bottom 96-well plate, add 1mmol/L formaldehyde 0.5μl, 1μl, 2.5μl;

10mmol/L formaldehyde 0.5μL, 1μL, 2.5μl; 100 mmol/L formaldehyde 0.5μL, 1μL
.
The final concentrations were 0,

5,

10,

25,

50,

100,

250,

500,

1000
μmol
/L.

Make three parallels for each concentration gradient.
Use
100μ
L
of the same length to the early index stage of Escherichia coli bacteria containing no fluorescent gene

as background fluorescence blank and
use
100μ
L
of sterilized LB liquid medium as OD600 blank.

Make three parallels
.
M
easure
t
he fluorescence intensity and OD600

every 10min with an ELIASA
.
Set the temperature 37
℃

and the maximum speed in the

ELIASA
.

Results:
The lid of the b
lack transparent bottom 96
-
well plate
was
fog
ged
, detection failure.

2023.6.30
Jiulin
Chen
,
Xin Yang

Culture s
train 1aI

in a conical flask, 100ml LB medium +100μL kanamycin mother solution, and inoculated with 100μL 1aI activation solution.

Carry out t
he fluorescence indication experiment of formaldehyde
.

Results:

The temperature was set incorrectly and the experiment failed
.

2023.7.03
Yidong

Wang

A
ctivate

strain
1aI
in
4m
L
LB liquid medium
.

2023.7.04
YidongWang

Activate
strain
1aI
in
4m
L
LB liquid medium
.

Activate
strain

BL21

(
No plasmid was introduced
)
i
n 4mL LB liquid medium

a
s a negative control
.

2023.7.05
Jiulin
Chen
,
Xin Yang

Culture strain 1aI
, BL21
in a conical flask, 100ml LB medium +100μL kanamycin mother solution, and inoculated with 100μL 1aI
, BL21
activation solution.

Carry out t
he fluorescence indication experiment of formaldehyde
.

Result:
The experiment
succeeded
.

2023.7.06
YidongWang

Zhiang
Chen Shuang Liang

T
est the formaldehyde metabolism of strains 2b, BL21 and LB

medium
.

Results: Strain 2b proved to be able to metabolize formaldehyde, but the metabolic capacity was smaller than that of BL21
.

2023.7.13
Yidong

Wang

I
noculate

2b
，
BL21
.

2023.7.15
Yidong

Wang
,

Zhiang
Chen
,
Shuang Liang

I
ncrease

the initial formaldehyde concentration to 500μM, and repeated the formaldehyde metabolism test for strains 2b, BL21 and LB
.

Results: The metabolic capacity of strain 2b was still lower than that of BL21.

2023.7.23
Yidong

Wang
,
Xin Yang

R
eceive
Polycoccus
PCC7942 strain from Shanghai University of Science and Technology
.

Spectral scanning of the bacterial solution revealed an absorbance peak at 685nm.

Prepare
BG11
medium
800ml \*1

I
noculate

100μL PCC7942 bacterial solution in 4ml BG11 medium in a light incubator at 30
℃
and 1500lux.

P
our

the plate
BG11 BG11+Kan

2023.7.24
Yidong

Wang

PCC7942

coating
,

LB
plate,
37
℃
，
cultured in the dark

BG11 30
℃
，
cultured in the
light

2023.7.25
Yidong

Wang
,

Zhiang
Chen
,
Shuang Liang

Repeat t
he formaldehyde metabolism test for strains 2b, BL21 and LB
.

Results: The metabolic capacity of strain 2b was still lower than that of BL21
.

2023.7.26
Yidong

Wang

P
repare medium:
LB

(liquid)
400ml\*1

Prepare sodium alginate solution
：
100ml
d
dH
2
O
+ 2.0g
s
odium alginate

2023.7.27
Yidong

Wang

P
repare medium:

CoB
BG11
liquid medium

S
treak
+

Liquid culture
:
1aI, 1c

2023.7.28

Yidong

Wang
,

Jiulin
Chen

D
etecte
t
he absorbance of PCC7942 OD685 and 1aI, 1c OD600
.

Sodium alginate embedding first attempt

10ml

sodium alginate solution
+ 1ml 1aI
b
acterial solution

S
lowly drop
t
he mixtur
e

into 0.2M sterilized CaCl2 solution using a syringe with the needle removed
,
and le
ave
for 5h to make the sodium alginate embedment fully
calcify
.
Then equally divided

the embedding into LB medium and
CoB
BG11 medium at 25
℃
and 200rpm for culture.

The bacteria solution of PCC7942 coated on July 24 did not grow on BG11 medium, but plaque grew on LB medium cultured in the dark, indicating that the cyanobacteria bacteria solution was contaminated by stray bacteria.

S
elect

single colony
for streaking.

Prepare
0.2M PBS 50ml\*
2, r
emove bacteria by filtration

D
issolve sodium alginate embeddings with PBS according to protocol method and measured OD600.

2023.7.29
Jiulin
Chen

M
easure the OD600 of 1aI in LB medium and
CoB
BG11 medium respectively.

2023.7.30
Jiulin
Chen

Measure the OD600 of 1aI in LB medium and
CoB
BG11 medium respectively.

2023.7.31
Jiulin
Chen

Measure the OD600 of 1aI in LB medium and
CoB
BG11 medium respectively.

2023.8.01
Jiulin
Chen

Measure the OD600 of 1aI in LB medium and
CoB
BG11 medium respectively.

2023.8.02
Jiulin
Chen

Measure the OD600 of 1aI in LB medium and
CoB
BG11 medium respectively.
A
lso measured
t
he pH value of the medium
.

2023.8.03
Jiulin
Chen

Measure the OD600 of 1aI in LB medium and
CoB
BG11 medium respectively.
A
lso measured
t
he pH value of the medium
.

C
onduct plasmid transformation experiments on PCC7942 according to protocol.

2023.8.04
Jiulin
Chen

Measure the OD600 of 1aI in LB medium and
CoB
BG11 medium respectively.
A
lso measured
t
he pH value of the medium
.

Prepare
BG11 plate 200ml\*1

Heat, add 200u
L
kanamycin, pour plate

2023.8.05
Jiulin
Chen

Take
100u
L
of the transformed bacterial solution and coated it on BG11 plate and cultured it in a light incubator.

2023.8.18
Yidong

Wang
,

Jiulin
Chen

Prepare

medium

LB plate
400ml\*1

LB
plate
200ml\*1

LB liquid medium
200ml\*1

BG11

plate
500ml\*1

BG11
liquid medium
200ml\*1

I
noculate the transformed strain ABC on
8.
3 into 4ml BG11+4μL Kan and culture it with light
.

I
noculate 1ml of the strain inoculated on
7.23
into 4ml BG11 and cultured it by light
.

P
our plate

2023.8.19
Yidong

Wang

C
onduct secondary purification of PCC7942 contaminated strains growing on LB medium
.

I
ntroduce
t
he
SacC
plasmid and
CobFDB
plasmid

into BL21 according to protocol.

2023.8.20
Yidong

Wang

Import failed the day before. No colony has grown on the plate.

2023.8.21
Yidong

Wang

Sodium alginate embedding
second
attempt

10ml
Sodium alginate solution
+1ml 1aI

bacterial solution

D
rip

the calcium chloride solution and incubate it in 4ml LB medium and 4ml
CoB
BG11 medium after standing for 5h.

2023.8.22
Yidong

Wang

A
dd formaldehyde into yesterday's sodium alginate embedding culture system to 250 and 500μM and continued to culture
at
37 ℃
and
200rpm

for 1h.

Place the vibrating tube under ultraviolet light for observation
, i
t was found that E. coli coated with sodium alginate could be successfully induced by formaldehyde to emit green fluorescence.

Import
SacC
and
CobFDB
back to BL21.

2023.8.23
Yidong

Wang
,

Jiulin
Chen

C
olon
ies grew on the plate.
S
elect
ed two single colonies from
the Escherichia coli plate with
SacC
plasmid
, streaked
and named as Sa and Sb
. Select t
wo single colon
ie
s on the E. coli plate with
CobFDB
plasmid
, streaked and
named Ca and
Cb
.

Try again to introduce the
CscB
plasmid into PCC7942.

Prepare 0.1M sodium bicarbonate solution and filter to remove bacteri
a.

Prepare 0.1M
c
alcium chloride solution and filter to remove bacteri
a.

Inoculate 1ml of PCC7942 into 4ml BG11 + 0.8ml 0.1M sodium bicarbonate solution
.

2023.8.24
Yidong

Wang

Sa
，
Sb
，
Ca
，
Cb

w
ere
successfully verified by PCR
.

Streak

Sa
，
Sb
，
Ca
，
Cb

C
oat
PCC7942
on
BG11 &BG11+Kan

Inoculate
PCC7942 with 50μL bacterial solution in 4ml BG11 + 1ml 0.1M sodium bicarbonate solution + 5μL kanamycin mother solution
;
a
nd
50μL bacterial solution in 4ml BG11 + 1ml sodium bicarbonate solution of 0.1M.

I
noculate
Sa
in 4ml LB+4μL kanamycin mother solution.

Inoculate
S
b
in 4ml LB+4μL kanamycin mother solution.

2023.8.25
Yidong

Wang

Inoculate
Sa
in 4ml LB+4μL kanamycin mother solution.

Inoculate
Sb
in 4ml LB+4μL kanamycin mother solution.

Inoculate
BL21(DE3)
i
n
4ml LB
.

2023.8.26
YidongWang
,
Xin Yang

Preserve

Sa
，
Sb
，
Ca
，
Cb

C
onduct

sucrose decomposition efficiency

experiment

(
S
ample
：
BL21
、
Sa
、
Sb
)
.

See protocol for details
.

Inoculate Sa in 4ml LB+4μL kanamycin mother solution.

Inoculate
Sb
in 4ml LB+4μL kanamycin mother solution.

Inoculate
BL21(DE3) i
n
4ml LB.

Inoculate
PCC7942-1 i
n
4ml LB.

Inoculate
PCC7942-
2
i
n
4ml LB.

2023.8.27
Yidong

Wang

Inoculate Sa
\*2
in 4ml LB+4μL kanamycin mother solution.

Inoculate
BL21(DE3) i
n
4ml LB.

2023.8.28
Yidong

Wang
,
Xin Yang

Conduct second sucrose decomposition efficiency experiment

(
S
ample
：
BL21
、
Sa
、
Sa + IPTG
)
.
See protocol for details
.

2023.9.04
Yidong

Wang

Sor
,

SQR-SDO

Plasmid enzyme digestion, ligand assembly
.

See protocol for details.

Select the target strip,
conduct
gel extraction
.

See protocol for details.

2023.9.05
Yidong

Wang

C
onduct

gel extraction
again
. See protocol for details.

T
est t
he formaldehyde metabolism of strains Ca,
Cb
, BL21 and LB
.

Results: The metabolic capacity of strain Ca and
Cb
was still lower than that of BL21
.

2023.9.06
Yidong

Wang

Inoculate
Strain
Cb
in LB medium with formaldehyde concentration of 500M to screen out strains with strong formaldehyde resistance (high expression of formaldehyde metabolism genes).

Inoculate

AB
(
sorAB
)

puncture bacteria

in 4ml LB+4μL kanamycin mother solution.

Inoculate

SS

(
SQR-SDO
)

puncture bacteria

in 4ml LB+4μL kanamycin mother solution
.

DNA is connected overnight for 16 hours

2023.9.07
Yidong

Wang

I
ntroduce
t
he connected plasmid into Escherichia coli DH10B,
electro revolution
\*2, calcium revolution \*1
.
See protocol for details.

I
ntroduce p
lasmid m (H2S indicator plasmid)

into Escherichia coli BL21, calcium
revolution.

Extract
the DNA of
PCC7942-1 &2 DNA
.

Perform
PCR

with
bacterial universal primers 27F
and 1942R, and send the PCR product to sequencing companies for sequencing.

Results: The contaminated strain was nitrate-reducing bacterium
.

Inoculate
Cb
4ml \*2
.

2023.9.08
Yidong

Wang

9.7

Conversion result
:

E
lectro revolution
:

Only a few colonies have grown

C
alcium revolution
:

Colony count is normal

Plasmid m:
Colony count is normal

S
elect
electro revolution
colony, name it SA1 (
electro
)
.

S
treak
+

4ml

liquid culture

S
elect
calcium
revolution
colony, name it SA
1
(
Ca
2+
)

&SA2
(
Ca
2+
)
. S
treak
+

4ml
liquid culture

Select
m
colony, name it
M1
&
M2
. Streak + 4ml liquid culture

Inoculate s
train
Cb
to
a
new LB medium with formaldehyde concentration of 500M to screen out the strains with strong formaldehyde resistance (high expression of formaldehyde metabolism genes).

2023.9.09
Xin Yang

P
reserve

SOCE316 \* 3
PCC7942 contaminated strain
——

nitrate
-
reducing bacteria

2023.9.12
Yidong

Wang

PCR verification
.

S
ample
:

SA1
(
Ca
2+
),

SA2
(
Ca
2+
)
,
M1
,

M2
. Succeeded.

Inoculate
PCC7942
i
n
BG11+NaHCO
3
medium
,
and
measure OD685.

BG11+NaHCO
3
m
edium
(
BG11
：
0.1M NaHCO
3
= 4:1
)

Inoculate
PCC7942

in
BG11+NaHCO
3
m
edium and add

with different concentrations of ampicillin to detect the growth.
(
Amp
:

25,

50,

75,

100 mg/L
)

2023.9.14
Yidong

Wang

Measure
PCC7942

bacterial solution

OD685

Inoculate
M1
,

M2
,

SA1

(
Ca
2+
),

SA2

(
Ca
2+
)

Culture s
train
Cb
in
a
new LB medium with formaldehyde concentration of 1000M to screen out the strains with strong formaldehyde resistance (high expression of formaldehyde metabolism genes).

2023.9.15
Yidong

Wang

Measure
PCC7942
bacterial solution

OD685

PCC7942 second conversion experiment
.

See protocol for details.

M
1 &M2
gradient

S
2-

concentration indication test

Divid
M1 and M2 bacterial solution into 1ml in 1.5ml EP tube, and
add
Na
2
S solution until the final concentration was 10
,
1, 0.1, 0
μ
g/ml. Mix well and culture overnight at 37
℃
.

2023.9.16
Yidong

Wang

Measure
PCC7942
bacterial solution

OD685

Prepare

LB
liquid medium
100ml \*10

Prepare
BG11
plate
200ml \*2

Simplify the
H
2
S

detection process

Simplified process
：

①

150
μ
l

liquid to be tested
+ 150
μ
l

reagent 1
+ 150
μ
l

reagent 1
,
thoroughly mix

②

Add
1
50
μ
l
reagent
4,
thoroughly mix

③

Add
30
μ
l
reagent
5
,

Leave for 20 minutes
, m
easure
OD665

Results: After simplification: 1.799Abs Original process: 1.800Abs

9.15 M1 &M2
gradient

S
2-

concentration indication test

Prepare t
he bacteria solution into temporary plates and observed under fluorescence microscope. No red fluorescence was found.

2023.9.17
Yidong

Wang

Measure
PCC7942
bacterial solution

OD685

Extract plasmid
.

S
ample
：
SA1
,
SA2
. See protocol for details.

Result
：

|  |  |  |  |
| --- | --- | --- | --- |
|  | ng/ μ l | A260/280 | A260/230 |
| SA1 | 73.9 | 2.04 | 2.32 |
| SA2 | 85.9 | 2.07 | 2.32 |

2023.9.19
Yidong

Wang
,

Zhiang
Chen

Formaldehyde metabolism test
.

S
ample
：
Cb
,

Cb+Kan
,

LB
,

BL21
.

Succeed.

Measure
PCC7942
bacterial solution

OD685

2023.9.20
Yidong

Wang

Re-inoculate PCC7942 BG11 + Na2CO3 to a concentration of 1.2g/L

Measure
PCC7942
bacterial solution

OD685

2023.9.22
Yidong

Wang

Measure
PCC7942
bacterial solution

OD685

The cyanobacteria added with Na
2
CO
3
in 9.20
did not grow
.

I
noculated with BG11+ NaHCO3 to a concentration of (0.02M).

Inoculate M1 &M2

2023.9.23
Yidong

Wang

M1 &M2
gradient

S
2-

concentration indication
second
test

Divid
M1 and M2 bacterial solution into 1ml in 1.5ml EP tube, and
add
Na
2
S solution until the final concentration was 10
,
1, 0.1, 0
μ
g/ml. Mix well and culture overnight at 37
℃
.

2023.9.25
Yidong

Wang

Measure
PCC7942
bacterial solution

OD685

Inoculate
Sa
&
Sb

2023.9.26
Yidong

Wang

Measure
PCC7942
bacterial solution

OD685

P
repare
CoB
+
m
other solution
：

Weigh
6.200g NaCl
，
0.214g NH
4
Cl
，
2g Sucrose
，
Dissolve in 50ml sterile water and filter to remove bacteri
a
.
Add 50ml/L when using.

2023.9.27
Yidong

Wang

PCC7942
third
conversion experiment
.

See protocol for details.

Measure
PCC7942
bacterial solution

OD685

2023.9.28
Yidong

Wang

Measure
PCC7942
bacterial solution

OD685

Name t
he transformed PCC7942, PA,

PB,

PC, coat
them
on BG11 plate with kanamycin added, and inoculate in liquid culture with kanamycin added, at 30
℃
, with supplementary light culture.

2023.9.30
Yidong

Wang

Measure
PCC7942
bacterial solution

OD685

Inoculate
SS
、
M1
、
M2

2023.10.02
Yidong

Wang

Measure
PCC7942
bacterial solution

OD685

SS &M1 &M2 coculture
S2
indication test

Add
450μl SS bacterial solution and 450μl M1 bacterial solution into 1.5ml EP tube,
a
dd IPTG until the concentration is 1mM
, add Na
2
S until the concentration is 10, 1, 0.1, 0
μ
g/ml
，
o
vernight culture at 37
℃
.

Add
450μl SS bacterial solution and 450μl M
2
bacterial solution into 1.5ml EP tube,
a
dd IPTG until the concentration is 1mM
, add Na
2
S until the concentration is 10, 1, 0.1, 0
μ
g/ml
，
o
vernight culture at 37
℃
.

2023.10.03
Yidong

Wang

Measure
PCC7942
bacterial solution

OD685

Prepare t
he SS-M1\SS-M2 bacterial solution into temporary plates and observed under fluorescence microscope.

Results: A small amount of fluorescence was observed in SS-M2 (10μg/ml), and the rest was not observed.

Place t
he co-culture solution in a shaker for further culture.

2023.10.04
Yidong

Wang

Measure
PCC7942
bacterial solution

OD685

Prepare t
he SS-M1\SS-M2 bacterial solution
cultured for 2 days
into temporary plates and observed under fluorescence microscope.

Results: A small amount of fluorescence was observed at each concentration gradient.

2023.10.05
Yidong

Wang

Measure
PCC7942
bacterial solution

OD685

2023.10.06
Yidong

Wang

Measure
PCC7942
bacterial solution

OD685

2023.10.07
Yidong

Wang

Measure
PCC7942
bacterial solution

OD685

T
he plasmid PCC7942 was transformed successfully, and the
colony
was successfully grown in the medium containing kanamycin.

Experiment of cyanobacteria-Escherichia coli co-culture system
.

Mix cyanobacteria and E. coli in 10ml sodium alginate solution
.
See protocol for details.

2023.10.08
Yidong

Wang

Measure
PCC7942
bacterial solution

OD685

M
easure

OD of cyanobacteria and Escherichia coli in the co-culture system and sucrose concentration and pH in the medium.

2023.10.09
Yidong

Wang

Measure
PCC7942
bacterial solution

OD685

Measure

OD of cyanobacteria and Escherichia coli in the co-culture system and sucrose concentration and pH in the medium.

## Modeling Diary

2023.5.8
:
Recruited team members, including three students specializing in Bioinformatics, Statistics, and Data Science, ensuring a diverse skill set for the project.
(Gengshang Dong)

2023.5.15
:
Gained a comprehensive understanding of the iGEM competition rules and available resources through the official iGEM website.

2023.6.2
:
Established the overall goals and project vision; initiated the learning process for synthetic biology principles and conducted an initial literature review to comprehend previous work in the relevant field.

2023.6.16
:
Began brainstorming and developed initial ideas for the modeling approach, focusing on visualizing E. coli metabolic pathways (specifically, the production of dehydrogenase).

2023.6.23
:
Commenced the construction of the mathematical model, identifying the reaction equation related to formaldehyde metabolism.
(Songshuo Jia)

2023.7.18
:
Received the first set of experimental data from the wet lab team and used it to simulate the relationship between biosensor fluorescence intensity and formaldehyde concentration.
(Songshuo Jia, Kaisheng Zheng)

August
, 2023
:
Initiated collaboration with the wet lab teams to obtain experimental data, ensuring a strong integration between the modeling and experimental components.
(Together)

2023.8.
29:
Adjusted and optimized the model based on the experimental results obtained from the co-culture of Escherichia coli and Cyanobacteria
.

2023.9.
2:
Refined the mathematical model by conducting data analysis and optimizing parameters, drawing from the literature. Conducted simulated experiments
on the computer
to validate the model.
(Kaisheng Zheng)

2023.10.
10:
Derived ordinary differential equations to represent all reaction processes within the model.
（
Songshuo Jia
）

2023.8.1 – 2023.8.30

Learning the basic mechanics of FBA and setting up the algorithm framework.(Kaisheng Zheng)

2023.9.1 – 2023.10.7

Learning FEM and choosing an appropriate object to model.(Kaisheng Zheng)

Finish building the model of “alginate beads with microorganism embedded”, and setting up the algorithm of finite element analysis on MATLAB, confirming the idea of the pump’s installation.(Kaisheng Zheng)

2023.9.21 – 2023.9.25

Analyzing the formaldehyde detection data from the wet lab, met the result from the previous FBA result.(Kaisheng Zheng)

2023.10.10
:
Successfully solved the ordinary differential equations using a program developed by Songshuo J
ia
, ensuring the accuracy and completeness of the mathematical model.

2023.10.
8:
Completed the first version of the model and prepared an initial draft of the competition report, encompassing the model's development and key findings.
(Kaisheng Zheng)

2023.10.
10:
Finalized all content and materials required for the iGEM wiki submission, ensuring alignment with the competition's documentation and presentation guidelines.
(Gengshang Dong)

# Engineering

![illustrations](https://static.igem.wiki/teams/4809/wiki/leaf-bg-top.png)
![illustrations](https://static.igem.wiki/teams/4809/wiki/dots-group-sm.png)
![illustrations](https://static.igem.wiki/teams/4809/wiki/leaf-yellow.png)
![illustrations](https://static.igem.wiki/teams/4809/wiki/leaf-orange.png)
![illustrations](https://static.igem.wiki/teams/4809/wiki/dots-group-cyan.png)
![illustrations](https://static.igem.wiki/teams/4809/wiki/leaf-cyan-lg.png)

* [Overview](#10)
* [1. Formaldehyde indicating system](#20)
* [2. Formaldehyde metabolic system](#30)

+ [First cycle](#31)
+ [Second try](#32)

* [3. Hydrogen sulfide indicating system](#40)
* [4. Hydrogen sulfide metabolic system](#50)
* [5. co-culture system](#60)

+ [5.1 Sucrose production](#61)
+ [5.2 Sucrose decomposition](#62)

* [Reference](#70)

## Overview:

Indoor air pollution is a common problem worldwide. Since then, our team has performed
gene editing on *Escherichia coli*, which can absorb and remove the main indoor
pollution
gases formaldehyde(HCHO) and hydrogen sulfide(H2S), and can emit different
fluorescence
indicators when different pollutants exceed the standard. In order to keep the metabolic
system running continuously, we added engineered *Synechococcus elongatus* to the
system
as producers. *S. elongatus* absorb carbon dioxide through photosynthesis to
produce
sucrose to provide carbon and energy sources for *E. coli*. Through this project,
we hope
to provide a new green and sustainable way to treat indoor air pollution.

---

### 1. Formaldehyde indicating system

**Design**

FrmR is a repressor protein from *E. coli*. In the absence of formaldehdye,
FrmR binds
to the promoter region, preventing transcription. In the presence of formaldehyde,
the nucleophilic Cys36 of the FrmR reacts with formaldehyde and causes a
conformational change that results in dissociation from the promoter[1].
Based on
this, we can construct the downstream gene expression system induced by formaldehyde
through FrmR and corresponding promoter sequences（figure 1）.

![](https://static.igem.wiki/teams/4809/wiki/pic1.png)

Figure 1: FrmR working diagram

In order to improve the sensitivity of *E. coli* to formaldehyde, we selected
the
promoter pTR47M4 optimized by Woolston, BM. et al[1]. The downstream gene
was then
placed with sfGFP, a high-brightness green fluorescent protein. The genome company
was commissioned to synthesize the entire sequence and construct it into the
skeleton plasmid pET28a. Here is our plasmid map (figure 2):

![](https://static.igem.wiki/teams/4809/wiki/pic2.png)

Figure 2: Construction of pET28a-FrmR-sfGFP

**Build**

After receiving the synthetic plasmid from a genetics company, the plasmid is
transformed into *E. coli* B21 (DE3). And we performed colony PCR
validation（figure 3）
for checking if transformation succeed. The PCR products were sent to a gene company
for further sequencing verification.

![](https://static.igem.wiki/teams/4809/wiki/pic3.png)

Figure 3: Colony PCR of recombinant *E. coli* strain 1aI for
Formaldehyde
indicating. P.S.:
Parallel sample； N.C.: Negative control; P.C.:
Positive control

**Test**

We added formaldehyde solution to *E. coli* strain 1aI cultured overnight,
continued
to culture for one hour, and observed under a fluorescence microscope, and found
that *E. coli* emitted obvious green fluorescence.

![](https://static.igem.wiki/teams/4809/wiki/pic4.jpg)

Figure 4: *E. coli* strain 1aI under fluorescence microscope.
Formaldehyde
induced concentration: 250μM

We added formaldehyde solution with different concentration gradients into the
bacterial solution of *E. coli* strain 1aI (OD≈0.4) at the beginning of the log
stage,
and read the fluorescence signal every 5 minutes in the microplate reader（figure 5）.

![](https://static.igem.wiki/teams/4809/wiki/pic5.png)

Figure 5: The change of fluorescence intensity induced by different
concentrations of formaldehyde

**Learn**

We found that under the induction of formaldehyde, the fluorescence intensity of *E.
coli* strain 1aI increased with time and gradually reached saturation. At the
same
time, the fluorescence intensity increased with the increase of concentration in the
range of 0-250μM. After the concentration is greater than or equal to 500μM, the
cell no longer grows, the fluorescence no longer increases, and E. coli strain 1aI
may not be able to tolerate formaldehyde above this concentration.

---

### 2. Formaldehyde metabolic system

**Design**

Formaldehyde is a very common secondary metabolite in living organisms, so many
organisms have the ability to metabolize formaldehyde, which also includes our
chassis microbes - *E. coli*. However, the formaldehyde metabolism capacity of
wild-type *E. coli* is not enough to meet our needs. Therefore, we decided to
transform formaldehyde dehydrogenase and formate dehydrogenase into *E. coli*
BL21
(DE3) to improve the ability of *E. coli* to metabolize formaldehyde.

#### First cycle

**Design**

At first, for formaldehyde dehydrogenase, we chose fdh gene from *Methylorubrum
extorquens[2]*.

*fdh*(formaldehyde dehydrogenase) protein can oxidize formaldehyde to formic
acid in
two steps. In the first step, reduced glutathione(GSH) reacts with formaldehyde in
an enzyme-independent manner to form S-hydroxymethyl glutathione, which is then
transported to the cell solute, S-Formylglutathione is formed as a substrate of *fdh*.
Then S-Formylglutathione is hydrolyzed by hydrolase to form formic acid and
glutathione.[2]

For formate dehydrogenase, we referred to the 2019 iGEM team ZJUT-China and chose
*lbfdh*(BBa\_K2936004) gene from *Lactobacillus buchneri* they used.

We placed *fdh* gene and *lbfdh* gene after *PFrmR*
promoter and FrmR gene(figure 6), so
that in the absence of formaldehyde, corresponding proteins would not be expressed
to affect the growth of *E. coli* itself. In the presence of formaldehyde,
formaldehyde dehydrogenase and formate dehydrogenase are expressed to completely
oxidize formaldehyde to carbon dioxide and water to complete the removal of
formaldehyde.

![](https://static.igem.wiki/teams/4809/wiki/pic6.png)

Figure 6: Gene pathway of Formaldehyde metabolic system at first try

Here is our plasmid map(figure 7):

![](https://static.igem.wiki/teams/4809/wiki/pic7.png)

Figure 7: Construction of pET28a-FrmR-*fdh-lbfdh*

**Build**

After receiving the synthetic plasmid from a genetics company, the plasmid is
transformed into *E. coli* B21 (DE3). And we performed colony PCR validation
for
checking if transformation succeed. The PCR products were sent to a gene company for
further sequencing verification.

**Test**

We tested the formaldehyde metabolizing ability of *E. coli* strain 2b(with
pET28a-FrmR-*fdh-lbfdh* ) and wild-type *E. coli* BL21(DE3). (figure 8)

![](https://static.igem.wiki/teams/4809/wiki/pic8.png)

Figure 8: Concentration of HCHO in liquid environment with different
bacteria.
(2b: *E. coli* BL21(DE3) with pET28a-FrmR-*fdh-lbfdh*;

BL21:
wild-type
*E. coli* BL21(DE3); Control: Blank)

**Learn**

As is shown in the figure, the results of this attempt are not satisfactory. The
metabolic capacity of E. coli with the gene related to formaldehyde metabolism was
lower than that of wild-type *E. coli*. By detecting the pH value of the
medium, we
found that after overnight culture, the pH of the medium of strain 2b decreased
significantly (ph≈6). Therefore, we speculated that the reason for the slow
metabolism was that the oxidation rate of formic acid was slower than that of
formaldehyde, resulting in the accumulation of formic acid, which reduced the pH of
the system and the activity of E. coli. Based on this, we made a second try.

#### Second try

**Design**

We chose *CboFDH* to replace *lbfdh*(figure 9).

*CboFDH* is a kind of NAD+-dependent formate dehydrogenase from *Candida
boidinii*,
which could catalyzes the reversible conversion of formate to carbon dioxide using
as coenzyme the NAD+/NADH system[3]. We selected *CboFDH* optimized
after
Site-saturation mutagenesis by Weihua Wu et al.[4], which has a higher
catalytic
efficiency.

![](https://static.igem.wiki/teams/4809/wiki/pic9.png)

Figure 9: Gene pathway of Formaldehyde metabolic system at second try

Here is our plasmid map（figure 10）:

![](https://static.igem.wiki/teams/4809/wiki/pic10.png)

Figure 10: Construction of pET28a-FrmR-*fdh-CboFDH*

**Build**

After receiving the synthetic plasmid from a genetics company, the plasmid is
transformed into *E. coli* B21 (DE3). And we performed colony PCR validation
for
checking if transformation succeed. The PCR products were sent to a gene company for
further sequencing verification.

**Test**

We tested the formaldehyde metabolizing ability of *E. coli* strain Cb(with
pET28a-FrmR-*fdh-CboFDH*) and wild-type *E. coli* BL21(DE3).(figure 11)

![](https://static.igem.wiki/teams/4809/wiki/pic11.png)

Figure 11: Concentration of HCHO in liquid environment with different
bacteria.(Cb: E. coli BL21(DE3) with pET28a-FrmR-*fdh-CboFDH*;
BL21: wild-type
*E. coli* BL21(DE3); Control: Blank)

**Learn**

As is shown in figure 11 , we achieved a satisfactory result this time. Cb has
stronger formaldehyde metabolism capacity than wild-type *E. coli* BL21(DE3).
Cb
removed 93.0% of formaldehyde within 30min, while wild-type *E. coli* BL21(DE3)
without the corresponding gene only removed 56.7% of formaldehyde. We succeeded in
increasing the ability of *E. coli* to metabolize formaldehyde.

---

### 3. Hydrogen sulfide indicating system

**Design**

We selected 3 genes: sqrR, SQR, mCherry for this system.

SqrR is a repressor protein. In the absence of hydrogen sulfide, C41 and C107 of
SqrR are in the reduced form. This form of SqrR binds the promoter region and
represses the expression of downstream gene. In the presence of hydrogen sulfide,
the resultant cellular RSS(highly oxidized sulfur species,termed reactive sulfur
species) promotes the formation of a di-, tri-, or tetrasulfide bond between C41 and
C107, inhibiting the ability of SqrR to bind to the promoter region. RNA polymerase
binds DNA and subsequently induces gene expression[5].

SQR(Sulfide quinone oxidoreductase) is a disulfide oxidoreductase, which oxidizes
S2- to zero-valent S(Sn). And Sn inhibits the
ability of SqrR to bind to the
promoter region as a kind of RSS, then downstream genes were expressed.

mCherry is a red fluorescent protein that is widely used in biotechnology as a
tracer.
In our scenario, in the absence of H2S, the sqrR protein binds to the
promoter
region and prevents downstream mCherry expression; When hydrogen sulfide is present,
SQR oxides S2-, generates Sn, prevents sqrR from binding to
the promoter, and the
downstream mCherry is normally expressed and emits red fluorescence.

Here is our plasmid map（figure 12）:

![](https://static.igem.wiki/teams/4809/wiki/pic12.png)

Figure 12: Construction of pET28a-SQR-mCherry-sqrR

**Build**

After receiving the synthetic plasmid from a genetics company, the plasmid is
transformed into *E. coli* B21 (DE3). And we performed colony PCR
validation（figure
12） for checking if transformation succeed. The PCR products were sent to a gene
company for further sequencing verification.

![](https://static.igem.wiki/teams/4809/wiki/pic13.png)

Figure 13: Colony PCR of recombinant *E. coli* for H2S
indicating.
P.S.: Parallel sample； N.C.: Negative control； P.C.: Positive control

**Test**

Unfortunately, the hydrogen sulfide indicating system we designed did not work as we
expected. After we added sodium sulfide, the *E. coli* didn't turn red as expected.

**Learn**

After talking with the team members and our Instructor, we suspect that the reason
may be that the expression of the SQR gene is too low to activate the downstream
pathway.

To test this hypothesis, we cultured H2S indicated *E. coli* strain M with
another *E. coli* strain SS(with SQR gene) overnight after adding sodium
sulphide. After that,
the bacterial solution was made into a temporary slide and observed under a
fluorescence microscope, and it was successfully found that some *E. coli*
cells
emitted red fluorescence(figure 13). And it was often broken *E. coli* cell
that
glowed intensely red under the fluorescence microscope, which confirmed our
conjecture.

![](https://static.igem.wiki/teams/4809/wiki/pic14.jpg)

![](https://static.igem.wiki/teams/4809/wiki/pic15.jpg)

Figure 14: E. coli strains M and SS under fluorescence microscope with same
field. Left: under exciting light; Right: under white light.

---

### 4. Hydrogen sulfide metabolic system

**Design**

In this system, we refer to the work of Team Tongji\_China in 2021 and make
modifications on this basis. We selected two genes from Tongji\_China(2021),
SQR(BBa\_K3823001) and SDO(BBa\_K3823002).

As mentioned above, SQR can oxidize S2- to Sn. Then SDO(Sulfur
dioxygenase) is a
GSH-dependent homodimer and the molecular mass of each subunit is approximately 23
kDa. It could catalyze the oxidation of S0 to sulfite in the presence of reduced
glutathione (GSH) by using Fe3+ or molecular oxygen as electron acceptor.

Different from Tongji\_China(2021), we selected two other enzymes to oxidize sulfite
to sulfate, which are sorA and sorB.

SOR(sulfite:cytochrome c oxidoreductase) is the main enzyme system for oxidizing
sulfites in bacteria. It is encoded by two genes, sorA and sorB, and generally forms
heterodimeric. SorA is a large subunit containing Mo (about 40.2kDa), while SorB is
a small subunit containing Heme c. It's only 8.8 kDa. SorAB is a kind of SDH(sulfite
dehydrogenases) distributed in periplasmic space. SorAB in bacteria can oxidize
sulfites directly to sulfate[6]. We selected sorA (GenBank accession
number
AAZ62443.1) and sorB (GenBank accession number AAZ62442.1) genes from *Cupriavidus
pinatubonensis* JMP134[6].

Here is our plasmid map（figure 15）:

![](https://static.igem.wiki/teams/4809/wiki/pic16.png)

Figure 15： Construction of pET28a-SQR-SDO-*sorA-sorB*

**Build**

After receiving the synthetic plasmid from a genetics company, the plasmid is
transformed into E. coli B21 (DE3). And we performed colony PCR validation for
checking if transformation succeed. The PCR products were sent to a gene company for
further sequencing verification.

**Test**

Unfortunately, the hydrogen metabolic indicating system we designed did not work as
we expected.

---

### 5. co-culture system

#### 5.1 Sucrose production

**Design**

In order to keep the metabolic system running continuously, we added engineered
*Synechococcus elongatus* to the system as producers.

When *S. elongatus* are cultured in an environment with high salt
concentration, they
can synthesize and secrete sucrose as an osmolyte. However, *S. elongatus* do
not
actively release sucrose outside the cell. On this basis, *S. elongatus* were
designed
to express sucrose permease(CscB), a sucrose/proton symporter, which could enable
*S.
elongatus* to secrete sucrose into the growth medium and support the growth
of E.
coli in the absence of other carbon sources[7].

We collaborated with the iGEM team shanghaitech-china(2022), who generously donated
the plasmids from their project last year. Here's a map of the plasmids they shared
with us (Figure 16).

![](https://static.igem.wiki/teams/4809/wiki/pic17.png)

Figure 16: Construction of pUC57-AmpR-NS3-2-CscB-LacI-KanR-NS3-1

**Build**

After receiving the plasmids shanghaitech-china(2022) shared with us, the plasmid is
transformed into *S. elongatus PCC7942*.

**Test**

Unfortunately, we only transformed the *cscB* gene into *S. elongatus* two
days before
the freeze day because *S. elongatus PCC7942* were contaminated with other
bacteria.
So we didn't have time to test our strain.

**Learn**

The slow growth rate of S. elongatus PCC7942 is the main reason that restricts the
progress of our experiment. We learned that another S. elongatus strain, UTEX2973,
has a very similar genome and traits to PCC7942, but has a faster growth rate and is
a better territorial microbe. However, unfortunately, there are no commercial
companies selling this strain in China mainland. So we didn't get this strain.

#### 5.2 Sucrose decomposition

**Design**

Because wild-type E. coli did not have good use of sucrose, we have expressed a
β-fructofuranosidase (SacC, BBa\_K4115017) in
*E. coli*, after referring to the scheme
of Team shanghaitech-china(2022). SacC is secreted into the intercellular space to
hydrolyze sucrose into glucose and fructose. Then these two simple sugars can then
be efficiently absorbed and utilized by *E. coli*.

Here is our plasmid map(figure 17):

![](https://static.igem.wiki/teams/4809/wiki/pic18.png)

Figure 17: Construction of pET28a-SacC

**Build**

After receiving the synthetic plasmid from a genetics company, the plasmid is
transformed into *E. coli* B21 (DE3). And we performed colony PCR validation for
checking if transformation succeed. The PCR products were sent to a gene company for
further sequencing verification.

**Test**

*E. coli* with SacC gene and wild-type Escherichia coli were cultured in LB
medium
containing the same concentration of sucrose, and the sucrose concentration in the
medium was measured every 1.5h(figure 18).

![](https://static.igem.wiki/teams/4809/wiki/pic19.png)

Figure 18: Concentration of sucrose in liquid environment with different
bacteria.

**Learn**

The concentration of sucrose in culture medium of *E. coli* with SacC gene decreased
obviously, indicating that *E. coli* was absorbing and utilizing sucrose. However, the
sucrose concentration in the culture medium of wild-type *E. coli* increased, which
was unreasonable, because *E. coli* itself did not produce sucrose. We speculated that
*E. coli* produced some metabolites with similar structure to sucrose in the metabolic
process, which affected the measurement of sucrose concentration by the kit.

---

### Reference

[1] Benjamin M. Woolston, et. al. (2018) Development of a formaldehyde biosensor with
application to synthetic methylotrophy. *Biotechnology and Bioengineering*.
115:206–215.

[2] 马凯 (2022) 甲醛高效降解菌的筛选驯化及其关键酶的研究(安徽农业大学)

[3] Bulut, H., et. al. (2021) Conserved Amino Acid Residues that Affect Structural
Stability of Candida boidinii Formate Dehydrogenase. *Applied Biochemistry and
Biotechnology*. 193:363–376.

[4] Weihua Wu et al (2019) Site-saturation mutagenesis of formate dehydrogenase from
Candida bodinii creating effective NADP+-dependent FDH enzymes. *Journal of
Molecular
Catalysis B: Enzymatic*. Volume: 61 157–161.

[5] Shimizu, T., et al (2017) Sulfide-responsive transcriptional repressor SqrR
functions as a master regulator of sulfide-dependent photosynthesis. *PNAS*.
Volume:114 Issue:9 Page:2355-2360 DOI10.1073/pnas.1614133114.

[6] Yufeng Xin., et al(2020) The Heterotrophic Bacterium *Cupriavidus
pinatubonensis*
JMP134 Oxidizes Sulfide to Sulfate with Thiosulfate as a Key Intermediate. *Applied
and Environmental Microbiology*. Volume:86 Issue:22 e01835-20.

[7] Cui, YX., et al (2022) Construction of an artificial consortium of Escherichia
coli and cyanobacteria for clean indirect production of volatile platform
hydrocarbons from CO2. *FRONTIERS IN MICROBIOLOGY* volume: 13. DOI:
10.3389/fmicb.2022.965968.

# Experiments

![illustrations](https://static.igem.wiki/teams/4809/wiki/leaf-bg-top.png)
![illustrations](https://static.igem.wiki/teams/4809/wiki/dots-group-sm.png)
![illustrations](https://static.igem.wiki/teams/4809/wiki/leaf-yellow.png)
![illustrations](https://static.igem.wiki/teams/4809/wiki/leaf-orange.png)
![illustrations](https://static.igem.wiki/teams/4809/wiki/dots-group-cyan.png)
![illustrations](https://static.igem.wiki/teams/4809/wiki/leaf-cyan-lg.png)

* [Culture Medium Preparation](#1)
* [Transformation and Coating](#2)
* [Plasmid assembly](#3)
* [Formaldehyde indication system experiment](#4)
* [Formaldehyde metabolism system Experiments](#5)
* [hydrogen sulfide metabolism system Experiments](#6)
* [Sucrose decomposition system Experiments](#7)

Culture Medium Preparation

LB liquid medium

Reagent: LB broth powder(
Sangon Biotech A507002-0250
), deionized water

Device: Measuring cylinder, Electronic balance, Medicine spoon

Procedure(for 1 L volume):

1. Accurately weigh 25 g of LB broth powder and then add 1 L of deionized water.
2. High-pressure steam sterilization at 121℃ for
   20
   min.

Note: If antibiotics need to be added, they must be added after autoclaving, otherwise the antibiotics will be ineffective. Antibiotics need to be filtered separately for sterilization
.

|  |  |
| --- | --- |
| Component | |
| Tryptone | 10g/L |
| Yeast extract | 5g/L |
| NaCl | 10g/L |

LB plate

Reagent:
LB broth powder(Sangon Biotech A507002-0250), deionized water
,
A
gar powder
(HuanKai Microbial 028990A)
.

Device: Measuring cylinder, Electronic balance, Medicine spoon

Procedure(for 1 L
volume
with 1.5% agar):

1.
Accurately weigh 25 g of LB broth powder
, 15g
agar powder
and then add 1 L of deionized water.

2.High-pressure steam sterilization at 121℃ for 20 min.

Note: If a plate with antibiotics is required, the antibiotics should be added after autoclaving and before the liquid begins to set, and thoroughly mixed
.
Antibiotics need to be filtered separately for sterilization
.

|  |  |
| --- | --- |
| Component | |
| Tryptone | 10  g/L |
| Yeast extract | 5  g/L |
| NaCl | 10  g/L |
| A gar powder | 15 g/L |

BG11 liquid medium

Reagent: BG11
(Coolaber AML201-100L)
,
deionized water

Device: Measuring cylinder, Electronic balance, Medicine spoon

Procedure(for 1L
volume
):

1. A
   dd
   988ml
   of deionized water
   and then add 10ml ingredient A , 1ml ingredient B, 1ml ingredient C of
   BG11
   (Coolaber AML201-100L).
2. High-pressure steam sterilization at 121℃ for
   20
   min.

Note: If antibiotics need to be added, they must be added after autoclaving, otherwise the antibiotics will be ineffective. Antibiotics need to be filtered separately for sterilization
.

Do not mix
ingredient
A,

ingredient
B,

ingredient
C directly, or it will precipitate

|  |  |
| --- | --- |
| Component | |
| NaNO 3 | 1500 mg/L |
| K 2 HPO 4 | 40 mg/L |
| MgSO 4 | 36.6 mg/L |
| CaCl 2 | 27.2 mg/L |
| Citric acid | 6 mg/L |
| Ammonium ferric citrate | 6 mg/L |
| EDTANa 2 | 1 mg/L |
| Na 2 CO 3 | 20 mg/L |
| H 3 BO 3 | 2.86 mg/L |
| MnCl 2 ·4H 2 O | 1.81 mg/L |
| ZnSO 4 | 0.22 mg/L |
| CuSO 4 ·5H 2 O | 0.08 mg/L |
| Na 2 MoO 4 ·2H 2 O | 0.39 mg/L |
| CoCl 2 ·6H 2 O | 0.0409 mg/L |

BG11 plate

Reagent: BG11
(Coolaber AML201-100L)
,
deionized water
,
A
gar powder(HuanKai Microbial 028990A).

Device: Measuring cylinder, Electronic balance, Medicine spoon

Procedure(for 1L
volume

with 1.5% agar
):

1. A
   dd
   988ml
   of deionized water
   and then add 10ml ingredient A , 1ml ingredient B, 1ml ingredient C of
   BG11
   (Coolaber AML201-100L) and then add 15g agar powder.
2. High-pressure steam sterilization at 121℃ for
   20
   min.

Note: If a plate with antibiotics is required, the antibiotics should be added after autoclaving and before the liquid begins to set, and thoroughly mixed
.
Antibiotics need to be filtered separately for sterilization
.

Do not mix
ingredient
A,

ingredient
B,

ingredient
C directly, or it will precipitate
.

|  |  |
| --- | --- |
| Component | |
| NaNO 3 | 1500 mg/L |
| K 2 HPO 4 | 40 mg/L |
| MgSO 4 | 36.6 mg/L |
| CaCl 2 | 27.2 mg/L |
| Citric acid | 6 mg/L |
| Ammonium ferric citrate | 6 mg/L |
| EDTANa 2 | 1 mg/L |
| Na 2 CO 3 | 20 mg/L |
| H 3 BO 3 | 2.86 mg/L |
| MnCl 2 ·4H 2 O | 1.81 mg/L |
| ZnSO 4 | 0.22 mg/L |
| CuSO 4 ·5H 2 O | 0.08 mg/L |
| Na 2 MoO 4 ·2H 2 O | 0.39 mg/L |
| CoCl 2 ·6H 2 O | 0.0409 mg/L |
| A gar powder | 15 g/L |

CoB
BG11 liquid medium

Reagent: BG11
(Coolaber AML201-100L)
,
deionized water
,
NaCl
, NH
4
Cl, sucrose.

Device: Measuring cylinder, Electronic balance, Medicine spoon

Procedure(for 1L
volume
):

1. A
   dd
   988ml
   of deionized water
   and then add 10ml ingredient A , 1ml ingredient B, 1ml ingredient C of
   BG11
   (Coolaber AML201-100L).
2. Add
   6.2g

   NaCl
   ,
   0.214g

   NH
   4
   Cl
   , and
   20g

   sucrose
   .
3. High-pressure steam sterilization at 121℃ for
   20
   min.

Note: If antibiotics need to be added, they must be added after autoclaving, otherwise the antibiotics will be ineffective. Antibiotics need to be filtered separately for sterilization
.

Do not mix
ingredient
A,

ingredient
B,

ingredient
C directly, or it will precipitate
.

|  |  |
| --- | --- |
| Component | |
| NaCl | 6.200g/L |
| NH 4 Cl | 0.214g/L |
| S ucrose | 20g/L |
| NaNO 3 | 1500 mg/L |
| K 2 HPO 4 | 40 mg/L |
| MgSO 4 | 36.6 mg/L |
| CaCl 2 | 27.2 mg/L |
| Citric acid | 6 mg/L |
| Ammonium ferric citrate | 6 mg/L |
| EDTANa 2 | 1 mg/L |
| Na 2 CO 3 | 20 mg/L |
| H 3 BO 3 | 2.86 mg/L |
| MnCl 2 ·4H 2 O | 1.81 mg/L |
| ZnSO 4 | 0.22 mg/L |
| CuSO 4 ·5H 2 O | 0.08 mg/L |
| Na 2 MoO 4 ·2H 2 O | 0.39 mg/L |
| CoCl 2 ·6H 2 O | 0.0409 mg/L |

Transformation and Coating

1. C
   entrifuge the synthesized target plasmid dry powder sample (2μg) in a tube at 12000rpm for one minute.
2. Add 200μL ddH
   2
   O, close the tube cover, and vortex shock to dissolve it fully.
3. Take 100 μL (1 tube) receptive cells melted on ice, add 5μL dissolved target plasmid (50ng), gently mix, and leave on ice for 30 min.
4. 42 ℃ water bath heat shock 45~60 s, quickly transferred to the ice bath, standing for 2 min (do not shake the sample during the ice standing process, otherwise it will reduce the conversion efficiency).
5. A
   dd 700 μL LB medium without antibiotics into the centrifuge tube, mix it well,
   place it
   at 37 ℃ and 200 rpm for 60 min.
6. Absorb 100μL resuscitation solution and evenly apply it to LB medium containing kanamycin, and place the plate upside down in 37 °C incubator for overnight culture.
7. S
   elect several single colonies for confirmation by PCR amplification.

7.1 Culture the selected
single colonies
overnight using LB medium containing kanamycin.

7.2 Take 1ml of the bacterial solution cultured overnight, centrifuge it at 8000rpm for 3 minutes, remove the supernatant, suspend it with 30
μ
l NaOH (20 mM), then add 70
μ
l Tris (1M), mix it well, and take it as the template DNA.

7.3 Set up the PCR system on the ice.

|  |  |
| --- | --- |
| Green Taq Mix(Vazyme P131) | 12.5  μ L |
| Forward Primer (10uM) | 1  μ L |
| Reverse Primer (10uM) | 1  μ L |
| Sterile ddH 2 O | 9.5  μ L |
| Template DNA | 1  μ L |
| Total | 25  μ L |

7.4 Set up the PCR program, put the PCR tube into the instrument, and start to run.

95
℃
，
3min

![](https://static.igem.wiki/teams/4809/wiki/adepu-lnjt7-001.png)
95
℃
，
15s

30
×
55
℃
，
15s

72
℃
，
60s

72
℃
，
5min

4
℃
，
forever

Note: The annealing temperature should be adjusted according to the Tm of the primers, and the extension time should be adjusted according to the length judged by the amplification.

8. Verification by agarose gel electrophoresis.

8.1 Weigh 0.36g agarose, add 30ml 1
×
TAE solution (1.2%), heat to dissolve agarose.

8.2 Add 3uL SYBR dye(biosharp BS354B), mix it well, pour into the gel tank and cool at room temperature.

8.3 Add 2μl DNA marker
(biosharp BL102A)
into the loading pore. Add 5 μl PCR product into the loading pore.

Record the position of the sample added.

8.4 Turn on the electrophoresis apparatus and start running.

8.5 After running, check the result on the gel imager, take a photo and record.

8.6 Sent the PCR products to a genetic company for sequencing and verification.

Plasmid assembly

1. C
   entrifuge the synthesized target plasmid dry powder sample (2μg) in a tube at 12000rpm for one minute.
2. Add 200μL ddH
   2
   O, close the tube cover, and vortex shock to dissolve it fully.
3. Working on ice, add the following reagents.

|  |  |
| --- | --- |
| 10X cutsmart | 5  μ L |
| Plasmid （ 2 μ g ） | 40  μ L |
| EcoRI-HF | 2.5  μ L |
| XbaI | 2.5  μ L |
| Total | 50  μ L |

Note: The enzyme activity of EcoRI-HF and XbaI is 20000 unit/ml. 1U is the restriction enzyme amount required for complete enzyme digestion of 1μg DNA within 1h at 37℃ in a 50ul system. Using 2.5 μL of the restriction enzyme is equivalent to 50 units, or 50μg of DNA per hour.

4. Mix
   it
   well and place
   it
   in an incubator at 37℃ for more than 3h.
5. C
   onfigured the agarose gel,
   then
   electrophoresis, and f
   ind
   the target fragment after digestion based on the band position.

Note: The target fragment length of sorAB is 1942bp and the target fragment length of SQR-SDO is 7139bp. The difference between the target fragment of SQR-SDO and the plasmid fragment is only 22bp, and the target fragment can be isolated by reducing the agarose ratio, extending the electrophoresis time, and using the plasmid fragment comparison.

6. Precisely cut the gel containing the target bands with a scalpel under the UV transilluminator.
7. Accurately weigh the pieces of gel. Put them into 1.5 ml EP tubes respectively.
8. According to the instructions in the
   Gel Extraction Kit(OMEGA D2500-01)
   recover the DNA fragment until the last step.
9. Take the PCR tube and add the reagent according to the following ratio.

|  |  |
| --- | --- |
| Pet 28-SQR-SDO （ 7139bp ） | (0. 03 - 0. 08 pmol) |
| sorAB （ 1942bp ） | (0.1 - 0.3 pmol) |
| Sterile purified water | Up to 5  μ L |

Note: The molar ratio of the carrier to the insertion sequence needs to be between 1:2 and 10, and the specific volume needs to be adjusted according to the recovered DNA concentration.

10. Add 1 volume (10 μL) of Solution I (DNA Ligation Kit <Mighty Mix >(Cat. #6023)) and mix completely.
11. Incubate at 16℃
    overnight
    .
12. After incubation, transform the assembled plasmid into
    E
    .
    coli
    DH10B, marked the plate with antibiotics, and screened out the successfully introduced plaque.
13. S
    elect the
    single
    clone for PCR amplification and confirmation, and sent the PCR product to a gene company for sequencing verification
    .
14. After verification, the amplified Plasmid was extracted using the NucleoSpin® Plasmid and Mini kit
    .
15. The extracted plasmid DNA was stored in a -20 ° C refrigerator
    .

Formaldehyde indication system experiment

1. Selecte verified
   E. coli
   strains and inoculated them into LB medium containing kanamycin, culture them at 37
   ℃
   and 200 rpm.

2.
When the growth of
E. coli
reaches the early exponential stage (OD about 0.4), add 100ul into the 96-well black transparent bottom plate, and then add methanol to the final concentration of 0,

5,

10,

25,

50,

100,

250,

500,

1000 μmol/L, respectively. Make three parallels for each concentration gradient.

3. Add 100ul wild-type
E. coli
of the same
early exponential stage (OD about 0.4)
as the background fluorescence blank and 100ul sterilized LB liquid medium as the OD600 blank.
Make three parallels for each concentration gradient.

4. Teste the fluorescence intensity and OD600 every 10min with multifunctional microplate reader. Set the temperature 37
℃
and the maximum speed in multifunctional microplate reader.

F
ormaldehyde metabolism system

Experiments

1. Take 9 1.5ml EP tubes and configured formaldehyde gradient solution with final concentrations of 0, 5, 10, 20, 50, 100, 200, 500, 1000µM, 350ml each. Note: LB medium was used to configure formaldehyde gradient solution.

2. Set up the experimental group:
E. coli
BL21 with Metabolically associated plasmids; Control group: wild type
E. coli
BL21; Blank group: LB.
E. coli
was cultured in conical bottles to the stable stage, and add formaldehyde to the final concentration of 500 µmol/L and cultured at 37℃ and 200rpm.

3. Take 600µl bacterial solution every 5 or 10min. Immediately return the cone to the shaker after sampling.

4. Centrifuge them at 13000 rpm for 2 min, and transfer 350µl supernatant to 1.5ml EP tube as test liquid.

5
.
Add 350µl acetyl acetone color developing solution(OKA GCTC6108) to each sample, mix well, heat in a constant temperature water bath at 40℃ for 30min, remove and cool at room temperature for 10min.

6. Measure OD412 on
microplate reader
, making three parallels for each sample.

7. Use
the data
of
hole sample
s

which have
determine the
formaldehyde
concentration to prepare a standard curve, and calculated the
formaldehyde
concentration of other hole samples according to the standard curve.

8. According to the formaldehyde content of different time, make the formaldehyde content change diagram of different samples.

hydrogen sulfide
metabolism system

Experiments

1. Take 9 1.5ml EP tubes and configured
   Na
   2
   S
   gradient solution with final concentrations of 0, 0.5, 1, 2.5, 5, 10, 25, 50, 100 µg/ml, 550ml each. Note: LB medium was used to configure formaldehyde gradient solution.
2. Set up the experimental group:
   E. coli
   BL21 with Metabolically associated plasmids; Control group: wild type
   E. coli
   BL21; Blank group: LB.
   E. coli
   was cultured in conical bottles to the stable stage, and add
   Na
   2
   S
   to the final concentration of
   50
   µ
   g/ml
   and cultured at 37℃ and 200rpm.
3. Take 1000µl bacterial solution every 30 min. Immediately return the cone to the shaker after sampling.
4. Centrifuge them at 13000 rpm for 2 min, and transfer 150µl supernatant to 1.5ml EP tube as test liquid.
5. Hydrogen sulfide detection kit
   (bioleaper BR5000292)
   reagents are added to each sample in the following order
   .
   1. Add 150
      µ
      L of reagent 1 and 150
      µ
      L of reagent 3 to each sample and mix thoroughly.
   2. Add 150

      µ
      L
      of reagent 4 and mix thoroughly
      , let stand at 4
      ℃
      for 10 minutes.
   3. Add 30

      µ
      L
      of reagent 5, thoroughly shake and mix, and let stand at room temperature for 20 minutes
      .
6. Measure OD
   665
   on microplate reader, making three parallels for each sample.
7. Use
   the data
   of
   hole sample
   s

   which have
   determine the Na
   2
   S concentration to prepare a standard curve, and calculated the Na
   2
   S concentration of other hole samples according to the standard curve.
8. According to the
   Na
   2
   S
   content of different time, make the formaldehyde content change diagram of different samples.

Sucrose decomposition

system

Experiments

1. Take 8 1.5ml EP tubes and configured sucrose gradient solution with final concentrations of
   0,
   1.0,

   2.5
   ,
   5
   ,
   10
   ,
   15, 20,

   25 mg/ml
   , 60ml each. Note: LB medium was used to configure formaldehyde gradient solution.
2. Set up the experimental group:
   E. coli
   BL21 with plasmid
   pET28a-SacC
   ; Control group: wild type
   E. coli
   BL21; Blank group: LB.
   E. coli
   was cultured in conical bottles to the stable stage, and add
   sucrose
   to the final concentration of
   20mg/mL, add IPTG
   to the final concentration of

   1 mM
   and
   then
   cultured at 37℃ and 200rpm.
3. Take 100µl bacterial solution every 1.5h. Immediately return the cone to the shaker after sampling.
4. Centrifuge them at 13000 rpm for 2 min, and transfer 60µl supernatant to 1.5ml EP tube as test liquid.
5. Plant Sucrose Content Assy
   kit
   (boxbio AKPL006M)
   reagents are added to each sample in the following order
   .
   1. Add 30µl of reagent 1,
      mix thoroughly
      , incubate at 95℃ for 5 minutes, and cool to room temperature.
   2. Add 420ul reagent
      2
      and 120µl reagent
      3
      ,
      mix thoroughly,
      incubate at 95℃ for 30 minutes, and cool to room temperature.
6. Measure OD
   480
   on microplate reader, making three parallels for each sample.
7. Use
   the data
   of
   hole sample
   s

   which have
   determine the
   sucrose
   concentration to prepare a standard curve, and calculated the
   sucrose
   concentration of other hole samples according to the standard curve.
8. According to the sucrose content of different time, make the formaldehyde content change diagram of different samples.

# Results

![illustrations](https://static.igem.wiki/teams/4809/wiki/leaf-bg-top.png)
![illustrations](https://static.igem.wiki/teams/4809/wiki/dots-group-sm.png)
![illustrations](https://static.igem.wiki/teams/4809/wiki/leaf-yellow.png)
![illustrations](https://static.igem.wiki/teams/4809/wiki/leaf-orange.png)
![illustrations](https://static.igem.wiki/teams/4809/wiki/dots-group-cyan.png)
![illustrations](https://static.igem.wiki/teams/4809/wiki/leaf-cyan-lg.png)

* [Pollution gas indicator system](#1)
* [Pollution gas metabolism system](#2)
* [Co-culture system](#3)

Our ABS system consists of three subsystems,
pollution gas indicator system
, pollution gas metabolism system, co-culture system. Next we will show the success and failure of the experiment on the three subsystems
.

P
ollution gas indicator system

In this subsystem, we designed two sets of schemes to indicate two different common indoor pollution gases,
HCHO
and
H
2
S.

1. HCHO

We transformed the designed and synthesized plasmid pET28a-FrmR-sfGFP into
E. coli
B21 (DE3) and verified the transformation by colony PCR.

![](https://static.igem.wiki/teams/4809/wiki/a629a-uqlen-001.png)

Figure
1
：
Colony PCR of recombinant
E. coli
strain 1aI

for Formaldehyde indicating
.
P.S.：Parallel sample； N.C.：Negative control
；
P.C.: Positive control

We added formaldehyde solution to
E. coli
strain 1aI cultured overnight, continued to culture for 1 hour, and observed under a fluorescence microscope, successfully found that E. coli emitted obvious green fluorescence.

![1aI 250uM 12](https://static.igem.wiki/teams/4809/wiki/a629a-uqlen-003.jpeg)

![1aI 250uM 13](https://static.igem.wiki/teams/4809/wiki/a629a-uqlen-002.jpeg)

Figure
2
：
E. coli
strains
1aI
under fluorescence microscope with same field. Left: under exciting light; Right: under white light.

Under excitation light irradiation,
E. coli
with different concentrations of formaldehyde also showed different degrees of green fluorescence

![QQ图片20230706164813](https://static.igem.wiki/teams/4809/wiki/a629a-uqlen-004.png)

Figure
3
：
Under the induction of different concentrations of formaldehyde,
E. coli
emitted different degrees of green fluorescence

We then take more accurate measurements. Different concentrations of formaldehyde were added to strain 1aI, and the changes of fluorescence intensity and OD600 over time were detected under microplate reader.

![Change of fluorescence intensity of strain 1aI](https://static.igem.wiki/teams/4809/wiki/a629a-uqlen-005.png)

Figure
4
:

The change of fluorescence intensity induced by different concentrations of formaldehyde

Strain 1aI successfully displayed different fluorescence signals for different concentrations of formaldehyde, indicating that the pathway we designed worked successfully
.

We normalized the fluorescence intensity by dividing it by OD600
(figure 5).

![Changes of relative fluorescence intensity of strain 1aI](https://static.igem.wiki/teams/4809/wiki/a629a-uqlen-006.png)

Figure
5
:
N
ormalized
fluorescence intensity induced by different concentrations of formaldehyde.

The results indicate that

under the induction of formaldehyde, the fluorescence intensity of E. coli strain 1aI increased with time and gradually reached saturation. At the same time, the fluorescence intensity increased with the increase of concentration in the range of 0-250μM. After the concentration is greater than or equal to 500μM, the cell no longer grows, the fluorescence no longer increases, and E. coli strain 1aI may not be able to tolerate formaldehyde above this concentration.

2. H
   2
   S

We transformed the designed and synthesized plasmid pET28a-FrmR-sfGFP into
E. coli
B21 (DE3) and verified the transformation by colony PCR.

![](https://static.igem.wiki/teams/4809/wiki/a629a-uqlen-007.png)

Figure
6
:
Colony PCR of recombinant
E. coli
for
H
2
S
indicating
.
P.S.：Parallel sample； N.C.：Negative control
；
P.C.: Positive control

Unfortunately, the hydrogen sulfide indicating system we designed did not work as we expected. After we added sodium sulfide, the
E. coli
didn't turn red as expected.

After talking with the team members and our Instructor, we suspect that the reason may be that the expression of the SQR gene is too low to activate the downstream pathway.

To test this hypothesis, we cultured H2S indicated E. coli strain M with another E. coli strain SS(with SQR gene) overnight after adding sodium sulphide. After that, the bacterial solution was made into a temporary slide and observed under a fluorescence microscope, and it was successfully found that some E. coli cells emitted red fluorescence(figure 7). And it was often broken E. coli cell that glowed intensely red under the fluorescence microscope, which confirmed our conjecture. However, due to time constraints, we have not been able to further improve this part of the system.

![ss-M2 10 W 2](https://static.igem.wiki/teams/4809/wiki/a629a-uqlen-008.jpeg)

![ss-M2 10 R 2](https://static.igem.wiki/teams/4809/wiki/a629a-uqlen-009.jpeg)

Figure
7
:
E. coli strains M and SS under fluorescence microscope with same field. Left: under exciting light; Right: under white light.

pollution gas metabolism system

In this subsystem, we also designed two sets of schemes to metabolize two different common indoor pollution gases, HCHO and H
2
S.

1. HCHO

We configured a series of
HCHO
solutions with concentration gradient and tested them with detection reagents according to certain methods. The standard curve obtained is ideal. It can be considered that our detection method can accurately reflect the relative content of sulfide in the solution within this concentration range
(figure 8)
.
Note: Limit the fitting curve to pass through the zero point, otherwise the error near the zero point will be too large.

![Standard Curve of Methanal](https://static.igem.wiki/teams/4809/wiki/a629a-uqlen-010.png)

Figure
8
:
Standard curve of HCHO

At first,

we chose fdh gene from Methylorubrum extorquens
and
lbfdh
(BBa\_K2936004) gene from
Lactobacillus buchneri
to metabolize
HCHO. But the results were not good(figure 9).

![Changes of methanal v2.0](https://static.igem.wiki/teams/4809/wiki/a629a-uqlen-011.png)

Figure
9
:
Concentration of HCHO in liquid environment with different bacteria.(2b:
E. coli
BL21(DE3)
with
pET28a-FrmR-
fdh
-
lbfdh;
BL21
：
wild-type

E. coli
BL21(DE3)
； Control： Blank)

The metabolic capacity of E. coli with the gene related to formaldehyde metabolism was lower than that of wild-type E. coli. By detecting the pH value of the medium, we found that after overnight culture, the pH of the medium of strain 2b decreased significantly (ph≈6). Therefore, we speculated that the reason for the slow metabolism was that the oxidation rate of formic acid was slower than that of formaldehyde, resulting in the accumulation of formic acid, which reduced the pH of the system and the activity of E. coli. Based on this, we made a second try.

A
t second time,
We chose
CboFDH
to replace
lbfdh.

![Changes of methanal v4.0](https://static.igem.wiki/teams/4809/wiki/a629a-uqlen-012.png)

Figure
10
:
Concentration of HCHO in liquid environment with different bacteria.(Cb:
E. coli
BL21(DE3)
with
pET28a-FrmR-
fdh
-
CboFDH;
BL21
：
wild-type

E. coli
BL21(DE3)
； Control： Blank)

As is shown in figure 9 , we achieved a satisfactory result this time. Cb has stronger formaldehyde metabolism capacity than wild-type
E. coli
BL21(DE3). Cb removed 93.0% of formaldehyde within 30min, while
wild-type
E. coli
BL21(DE3)
without the corresponding gene only removed 56.7% of formaldehyde. We succeeded in increasing the ability of
E. coli
to metabolize formaldehyde.

Note: Before testing the formaldehyde metabolism capacity of engineered Escherichia coli, it is best to use LB medium containing some formaldehyde for a few days. This would allow the modified strain to adapt to the formaldehyde environment and increase the number of plasmids containing formaldehyde metabolism genes
.

2. H
   2
   S

We did not succeed in this part of the experiment. After successfully transforming the plasmid,
E. coli
did not show significant metabolic capacity for S
2-
. The reason is unknown.

co-culture system

In this system, we introduced
Synechococcus elongatus

PCC7942 to provide energy and carbon source for
E
.
coli
, and introduced
SacC
gene into
E
.
coli
to enhance its ability to metabolize sucrose
.

5. c
   oli

   with

   SacC

We configured a series of
sucrose
solutions with concentration gradient and tested them with detection reagents according to certain methods. The standard curve obtained is ideal. It can be considered that our detection method can accurately reflect the relative content of sulfide in the solution within this concentration range.

![Standard Curve of Sucrose](https://static.igem.wiki/teams/4809/wiki/a629a-uqlen-013.png)

Figure
11
:
Standard curve of sucrose

![Changes of sucrose v2.0](https://static.igem.wiki/teams/4809/wiki/a629a-uqlen-014.png)

Figure
12
:
Concentration of
sucrose
in liquid environment with different bacteria

The concentration of sucrose in culture medium of
E. coli
with SacC gene decreased obviously, indicating that SacC gene successfully enhanced the ability of
E. coli
to absorb and utilize sucrose.

However, the sucrose concentration in the culture medium of wild-type E. coli increased, which was unreasonable, because E. coli itself did not produce sucrose. We speculated that E. coli produced some metabolites with similar structure to sucrose in the metabolic process, which affected the measurement of sucrose concentration by the kit.

After the sucrose metabolized by E. coli was homogenized, the data of the two experiments were in good agreement(figure 13).

![Sucrose consumption](https://static.igem.wiki/teams/4809/wiki/a629a-uqlen-015.png)

Figure
13
:
Sucrose consumption of
E. coli
BL21 with SacC

S
.
elongatus
PCC7942
with
CscB

S. elongatus
PCC7942 Slow growth rate
(figuer 14)
and
Nitratireductor sp.

contamination events in the process

have prevented us from completing the original plan. We completed the conversion of the cscB only two days before freeze day, so there was no time to test the ability of S. elongatus PCC7942 to produce sucrose
.

![Growth curve of PCC7942 v2](https://static.igem.wiki/teams/4809/wiki/a629a-uqlen-016.png)

Figure
14
:
Growth curve of
S. elongatus
PCC7942