iGEM 2021 — Team ECNUAS

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# Team:ECNUAS/Notebook

ECNUAS

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Notebook

2021-7-15

- Ice-breaking

- Initial Discussion on the project plan

![](https://static.igem.org/mediawiki/2021/e/e7/T--ECNUAS--notebook01.jpg)

2021-7-16

- Subject background survey

- Basic experimental operation

- Laboratory safety training

![](https://static.igem.org/mediawiki/2021/0/0a/T--ECNUAS--notebook02.jpg)

- Prepare growing medium (liquid culture medium)

![](https://static.igem.org/mediawiki/2021/e/e6/T--ECNUAS--notebook03.jpg)

2021-7-17

- Basic operate training

- Learning the concept of recombinant plasmids

- Prepare growing medium (solid culture medium)

• One type of Kanamycin
• One type of Ampicillin
• One type of Kanamycin and Ampicillin

- Dilute TAE buffer

2021-7-18

- Restriction endonuclease reaction

- DNA agarose gel electrophoresis identification

![](https://static.igem.org/mediawiki/2021/c/c2/T--ECNUAS--notebook04.jpg)

- DNA agarose gel reavers and purification

- DNA clip connection reaction

- Plasmid transformation

• Plasmid A (Atz sensor plasmid):pET28a(+)\_P7\_AtzR
• Plasmid B (Atz reporter plasmid): pUC57-pro\_Pprovoin5\_amilGFP

2021-7-19

- Found the plasmid B’s plate didn’t grow any colony, but Plasmid A grew

![](https://static.igem.org/mediawiki/2021/2/2f/T--ECNUAS--notebook05.jpg)

- Amplification of Atz sensor plasmid and leave it in shaker through night

- Then we start to think why plasmid B didn’t grow any flora

• We think that the plasmid transformation in day 7-18 might got some problem, so we do the DNA clip
connection and Plasmid transformation again by using day 7-18 DNA clip connection reaction.

- So we repeat experience again (but we didn’t get good stripes)

• restriction endonuclease reaction (only Pprovoin5)

- Increase the amount of sample to 15 μL
- Increase the amount of Pprovoin5 enzyme to 1.25 μL

• DNA agarose gel electrophoresis identification (all result get very light stripes)

1. First gel is from day 7-18 DNA clip connection reaction

![](https://static.igem.org/mediawiki/2021/f/ff/T--ECNUAS--notebook06.jpg)

2. Second gel is from day 7-19 restriction endonuclease digestion product

![](https://static.igem.org/mediawiki/2021/6/63/T--ECNUAS--notebook07.jpg)

- The potential cause why we don’t get stripes or very light stripes

A. turnip the gel 180 (may cause the stripe light)
B. When we’re adding more TAE, the TAE added might disperse light
C. Adding too much sample might disperse the stripes (because we change from 10 μL to 15 μL

- Preparing LB medium

2021-7-20

- Continue plasmid extraction

• By using the colonies from
day 7-19 (plasmid A)

- Restriction endonuclease reaction and DNA agarose gel electrophoresis
identification (only Pprovoin5) (twice because first one has no stripe)

1. No change

![](https://static.igem.org/mediawiki/2021/4/4d/T--ECNUAS--notebook08.jpg)

2. Changing the system from
50μL to 40μL by decrease water (10μL)

- DNA agarose gel electrophoresis identification result

1. No/very light stripe
2. Stripe appeared

![](https://static.igem.org/mediawiki/2021/5/5e/T--ECNUAS--notebook09.jpg)

- DNA clip connection reaction

- Plasmid transformation

A. Restriction endonuclease reaction product following the protocol (no resistance)
B. Restriction endonuclease reaction product following the protocol (with resistance)
C. 40 μL system restriction endonuclease reaction product
D. 40 μL system restriction endonuclease reaction product
E. Plasmid transformation from day 7-18

2021-7-21

- No conoly from day 7-20 (ABCDE)

![](https://static.igem.org/mediawiki/2021/a/ac/T--ECNUAS--notebook10.jpg)

- DNA agarose gel electrophoresis identification of day 7-20 DNA connection
reaction, found that there’s no stripes (very light)

![](https://static.igem.org/mediawiki/2021/d/d6/T--ECNUAS--notebook11.jpg)

- Clip connection

- Plasmid transformation (change from 10min RTP to 20 Celsius 1h)

![](https://static.igem.org/mediawiki/2021/6/66/T--ECNUAS--notebook12.jpg)

F. E. coli without transfer in plasmid in the non-resistant LB medium to make sure E. Coli sill
working
G. Transfer in Pprovoin5 plasmid
H. Plasmid transformation from 7-21
I. Plasmid transformation from 7-21
J. Plasmid transformation from 7-21

2021-7-22

- No conoly until afternoon second one from 7-21 has some colony, so plasmid
extraction 16h amplification

![](https://static.igem.org/mediawiki/2021/f/f4/T--ECNUAS--notebook13.jpg)

- Restriction endonuclease reaction

• Leave it in 37 celsius
through night

- DNA clip connection reaction (increase the amount of plasmid and leave 16 Celsius
through night)

• AmiGFP: Pprovoin5 = 4.5 μL :
1.5 μL

2021-7-23

- Plasmid extraction from day 7-22 amplification

• So we could have more
Pprovoin5 plasmid

- DNA agarose gel electrophoresis identification

• Marker changed

- 5 μL marker
- 0 μL loading buffer
- 2 μL Nucleic acid dyestuff

• Negative control (only amiGFP
plasmid because we don’t have enough Pprovoin5 plasmid)

2021-9

- Repeat the plasmid B constrcution
- Coloy PCR to verify the transformants of plasmid B and extract the plasmid for sequencing
- Prepare LB medium with two antibiotics (kana&amp)
- Extract the plasmid A and plasmid B to be transformed into DH5a to obtain bacteria C

![](https://static.igem.org/mediawiki/2021/3/3e/T--ECNUAS--notebook14.jpg)

2021-10-1~7

- Conduct function tests

![](https://static.igem.org/mediawiki/2021/1/1f/T--ECNUAS--notebook15.jpg)

- Cell-free extraction experiments

![](https://static.igem.org/mediawiki/2021/3/33/T--ECNUAS--chgt2.jpg)

Contact Info

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# Team:ECNUAS/Engineering

ECNUAS

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Engineering

Background

Atrazine is a herbicide compound commonly found in agriculture. It can diffuse into
soil and water. Atrazine can be degraded into cyanuric acid in this process, which leads to the accumulation
of cyanuric acid in the environment, and has harmful effects on all organisms in the environment. Although
Atrazine is currently banned in most European countries, the herbicide is still widely used in other parts
of the world. Atrazine in the environment can be monitored through various chemical analyses, including gas
chromatography and some physical and chemical detection systems. Although these methods can sensitively
measure the atrazine content in the environment, the steps are cumbersome, expensive, and time-consuming,
and require extensive infrastructure and technical expertise.

Design

Biosensor consisting of a cell-free systems that are sensitive and specific to
specific analytes. It can directly detect the atrazine in sample. Cell-free atrazine biosensor consists of
three to four components, which can promote gene expression and metabolism in vitro (Figure 1). (1) cell
lysate/extract, which contains the cellular mechanisms required for protein synthesis; (2) buffer mixture of
phosphorylated energy substrates, nucleoside triphosphates (NTP), amino acids, salts and other necessary
cellular cofactors; (3) DNA templates that define the genetic program to be performed in the reaction; and
the reaction Any other optional exogenous cofactors, substrates or inducers required. The biosensor detect
cyanuric acid (CYA), a degradation product of atrazine. After atzR binds to CYA, it activates the promoter
Provoin5, thereby activating downstream genes and inducing the expression of the reporter gene GFP.

![](https://static.igem.org/mediawiki/2021/8/88/T--ECNUAS--Engineering01.jpg)
Figure 1. Composition of the cell-free atrazine biosensor.

Build

![](https://static.igem.org/mediawiki/2021/8/82/T--ECNUAS--Engineering02.jpg)
Figure 2. Schematic maps of Ptat\_AtzR (sensor plasmid) and Pprovoin5\_amilGFP (reporter
plasmid).

Test

After the ice-breaking game, we played a short playlet.
We invited the children to take part in the drama. In the relaxed and humorous storyline, we gave the
children a preliminary introduction to the dangers of atrazine, and led them to our theme -- what do we know
about atrazine?

![](https://static.igem.org/mediawiki/2021/6/63/T--ECNUAS--Engineering03.jpg)
Figure 3. Histogram of the fluorescence intensity 4 hours after 30 μM CYA was added in the
culture.

In order to analyze the relationship between the concentration of cyanuric acid and
the fluorescence intensity, we designed the control groups and collected the data as showing above.
According to the histograms (Figure 4), the fluorescence intensity shows a decreasing trend with the
increase of concentration of cyanuric acid when we used the bacteria C for tests.

![](https://static.igem.org/mediawiki/2021/4/45/T--ECNUAS--Engineering04.jpg)
Figure 4. Histogram of the fluorescence intensity of bacteria C after 4 and 6 hours under different
concentration of the cyanuric acid.

Learn

In this project, we successfully atrazine biosensor in bacteria. However, cyanuric
acid might affect the growth of strains so that the higher the concentration of the cyanuric acid, the worse
the growth of the bacteria, the less of the amount of the effective “biosensor”. In order to fully eliminate
this impact, we introduced the concept of cell-free extraction and cell-free expression in the next stage of
our project.

Contact Info

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# Team:ECNUAS/Experiments

ECNUAS

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Experiments

Enzyme digestion

![](https://static.igem.org/mediawiki/2021/2/26/T--ECNUAS--Experiments01.jpg)

Restriction Endonuclease Reaction
1. Solutions are mixed in 50μl two pipettes and centrifugalized.
2. Pipettes are warmed in the PCR for 1 hour at 37 degrees for restriction to take place and the enzyme are
denatured 65 degrees for 20 minutes.

Electrophoresis

![](https://static.igem.org/mediawiki/2021/b/b0/T--ECNUAS--Experiments02.jpg)

DNA Agarose Gel Electrophoresis Identification

1. Configurate 1X TAE buffer.
2. Configurate and make electrophoresis gel.
3. Configurate DNA sample and marker.
4. Put the gel in appropriate place in the tank with 1X TAE buffer and sample the DNA sample and marker onto
the electrophoresis gel.
5. Run the electrophoresis.
Observe the DNA stripe using Ultra-thin ultraviolet platform.

DNA Agarose Gel Recovery and Purification

1. Cut down the parts of gel which contains the correct DNA and calculate the weight difference in the EP
tube from adding the gel by weighing the tube before and after adding the gel.
2. Add buffer DE-A, 3X gel volume, heat in water bath at 75 degrees until the gel melts.
3. Add buffer DE-B, 1.5X gel volume, and mix the solution until it is a transparent yellow color.
4. Transfer the liquid into a DNA preparation tube, centrifugalize at 12000 xg for 1 minute, and remove the
liquid below.
5. Add 500μl Buffer W1, centrifugalize at 12000 xg for 30 seconds, and remove the liquid below.
6. Add 700μl Buffer W1, centrifugalize at 12000 xg for 30 seconds, and remove the liquid below. Repeat this
process.
7. Centrifugalize at 12000 xg for 1 minute.
8. Add 25μl deionized water, which is pre-heated to 65 degrees, keep the tube in room temperature for 1
minute, and centrifugalize at 12000 xg for 1 minute.

T4 ligation

![](https://static.igem.org/mediawiki/2021/1/15/T--ECNUAS--Experiments03.jpg)

DNA Clip Connection Reaction
1. Configurate DNA clip connection sample.
2. Centrifugalize and react in room temperature for 10 minutes.
3. Denature the enzyme at 65 degrees for 10 minutes.

Prepare reagents

![](https://static.igem.org/mediawiki/2021/9/9f/T--ECNUAS--Experiments04.jpg)

Plasmid Transformation

1. Configurate LB solid medium, sterilize, cool, add ampicillin and kanamycin to each portion, spread it in
the plates, and wait for solidification.
2. Mix 2μL plasmid DNA and 20μl competent E. coli cell and put the tube on ice for 30 minutes.
3. Water bath the tube at 42 degrees for 90 seconds and ice bath for 2.5 minutes.
4. Add 1 mL resistance-free LB liquid medium to the tube and put it in the incubator at 37 degrees with 200
rpm for 45 minutes.
5. Centrifugalize for 2 minutes at 5000 rpm Remove 800μL supernatant. Resuspend the rest. Inoculate on the
plate, and incubate at 37 degrees for one night.

Plasmid Extraction

1. Pick up monoclonal colonies on the plate, inoculate in 10 mL LB liquid medium, and cultivate in incubator
for 12 hours to 16 hours at 250 rpm.
2. Add 500μL P1 to the CP3 adsorption column, centrifugalize at 12000 rpm for 1 minute to activate the
silicon membrane.
3. Take 1mL bacterial liquid. Centrifugate for 1 minute at 12000 rpm, and remove the supernatant.
4. Add 250μL P1 and resuspend the precipitate.
5. Add 250μL P2 and gently overturn the tube for 6 to 8 times.
6. Add 350μL P3. Gently overturn the tube for 6 to 8 times, and centrifugate for 10 minutes at 12000 rpm
after the white precipitates form.
7. Collect the supernatant to the column. Centrifugate for 30 to 60 seconds at 12000 rpm, and remove the
liquid.
8. Add 600μL PW. Centrifugate for 30 to 60 seconds at 12000 rpm. Remove the liquid, and repeat the process.

9. Centrifugate at 12000 rpm for 2 minutes.
10. Put the column into a new centrifuge tube, add 100μL EB. Keep the tube in room temperature for 2
minutes, and centrifugate at 12000 rpm for 2 minutes.

Colony PCR

1. Initial denaturation takes place in PCR for 5 minutes. Denaturation, annealing, and extension repeat 34
times. The final extension and the preservation ends the PCR process in 5 minutes.
2. Run the electrophoresis to test for the positive result.
3. Select the positive colonies and enlarge cultivation and add 5 milliliters medium to each tube.
4. Extract the plasmid and send them to the sequencing company.

Double-Antibiotics Medium Screening

1. Prepare fresh LB medium with two antibotics (kana&amp) and the concentrations are kan 50 ug/ml; amp
100ug/ml.
2. Extract plasmid A and plasmid B and transform into DH5a (Ref. Plasmid Extraction & Transformation ) and
culture in the double-antibiotics LB medium we prepared.

Function Test

1. Prepare the cyanuric acid solution
Take 103mg cyanuric acid powder, add 8ml ddH2O, 80°C water-bath to complete dissolution, and then obtain
100mm/L solution.
2. Functional test of bacteria C
Bacteria C was cultured and induced in a certain concentration of CYA and the fluorescence intensity would
be measured and collected.
The blank control in this experiment is the pure DH5a without any plasmids transformed.
3. Steps:
1）Select monoclonal C into 3ml LB medium (two antibiotics have been added) and is cultured in a constant
temperature shaker at 30 °C for 8-12 hours.
2）Dilute the seed by 100 times to 50 ml of new LB medium at 30 °C until the initial OD600 reaches 0.2
3）By adding CYA to different sample tubes to a certain concentration, observing at a constant temperature
30°C for a certain period of time, the fluorescence intensity was measured by Thermo instrument (excitation
wavelength is 503nm and the emission wavelength is 512nm), so as to explore the best induction condition and
the response time range of the biosensor
4）Notes: during the pre-experiments, we notice that the LB medium had a serious background effect in
measuring the fluorescence intensity, so all samples were measured after washing and resuspension.

Protein Extraction for Cell-Free Expression Reaction Use

\*2X YT+P media preparation: 6 g tryptone, 10 g yeast extract, 5 g sodium chloride, 7 g potassium phosphate
dibasic, 3 g potassium phosphate monobasic, and water to one liter.
1. Inoculate 30ml initial bacteria solution into 250ml flask, add antibiotics and culture overnight.
2. OD600 was measured after 16h culture to confirm whether the growth was saturated. 2X YT + P medium was
used as the blank control for measuring OD600 value. Then inoculate 20ml saturated fermentation medium into
1L growth medium to continue culture (2x YT + P needs to be preheated at 37°C 30mins in advance).
3. Culture for 3.5-5h at 37°C and 200 rpm until OD600 reaches 3. (after that, the solution, equipment and
utensils used in the ice step need to be chilled to 4 degrees in advance)
4. Add 400 uL 1M DTT to the wash buffer to make its final concentration 2mM. Take two clean 50ml Falcon
tubes, record the weight and keep them on ice.
5. Distribute the cell culture into two centrifuge tubes and balance them on ice—afterward, Centrifuge it at
4°C at 5000XG for 15 min. Carefully remove the supernatant and transfer the precipitate separately into a
clean, 50mL Falcon tube. Select 25mL each of wash buffer previously mixed with DTT for resuspension,
continue the process until no solid particles are present in the suspension
6. Centrifuge the solution at 4°C at 5000XG for 10 min, remove the supernatant and wash it with 25mL of wash
buffer
7. Centrifuge, the solution at 4°C 5000XG for 10 min, remove the supernatant and wash it with 25mL of wash
buffer
8. Centrifuge the solution at 4°C at 7000XG for 10 min, remove the supernatant, wipe all the exposed surface
and the remaining fluid inside the tube. Weight the mass of precipitation.
9. Flash-freeze the cells in liquid nitrogen and store them under -80°C. Let the cells unfreeze on ice for
one hour before attempting the next step.
10. Using Vortex oscillation, each gram of cell should be resuspended in 1mL of wash buffer.
11. Resuspend each pellet for 5 to 10 min before distributing 1.4mL of that suspension into four 1.7 mL
Eppendorf tube.
12. Sonicate the suspension on ice. The optimal treatment capacity should be at 350J. After sonication, the
suspension should have turned into a less sticky, brown liquid.
13. After lysis, add 4μL 1M DTT tp everu 1.7mL tube. Centrifuge the tube at 12,000XG, 4°C for 10 mins.
14. Pipette off the supernatants on top and combine them into two new Eppendorf tubes.
15. Cover the test tubes with aluminium foil and incubate them at 37°C, 200 rpm fro 80 min.
16. After incubation, the test tubes should turn opague. Centrifuge them at 12,000XG below 4°C and remove
all unrelevant protein.
17. Add 600uL of 1M DTT into the dialysis buffer to stabilize its concentration at 1mM and transfer it into
a beaker with a stirring rod. Soak the dialysis cassette (we used the Slide-A-Lyzer Dialysis Cassettes,
20.000 MWCO from Thermo Scientific) in the dialysis buffer for at least five minutes. Dry the cassette in a
dark bix without contaminating the membrane.
18. After centrifugation, pipette off the supernatants and relocate the clear extract into the dialysis
cassette.
19. 3 hours of dialysis should be conducted at 4 °C, timely change of buffer is not required.
20. After dialysis, remove the extract from the box and relocate them into two new, 1.7mL test tubes.
Centrifuge the tubes at 2000XG 4 °C for 10 min. When we obtain the extracted product -protein, relocate the
supernatant into a new testube and distribute it to multiple 35uL test tubes. Flash-freeze it on liquid
nitrogen and store at -80 °C.
21. Determine the concentration of the extracted product.

Contact Info

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# Team:ECNUAS/Results

ECNUAS

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Results

IConstruction of recombinant plasmid

![](https://static.igem.org/mediawiki/2021/d/d4/T--ECNUAS--result01.jpg)
Figure 1: The electrophoresis results of enzyme digestion and PCR.

Left:
Lane pUC57-amilGFP: Plasmid pUC57-mini-vector-amilGFP digested by Bsa1 and the band at around 750bp
(amilGFP: 703bp) was got.
Lane pUC57-Pprovoin5: Plasmid pUC57-kana-mini-Pprovoin5 (2097bp) digested by Bsa1.
Right:
Lane pUC57-amilGFP: amilGFP (703bp) was got by PCR method.

![](https://static.igem.org/mediawiki/2021/4/4c/T--ECNUAS--chgt3.jpg)
Figure 2: The electrophoresis (1) and sequencing (2) results of bacteria PCR.

Lane pUC57 -Pprovoin5-GFP-1 to 3: Bacteria PCR of monoclonals of amilGFP with size of 703bp. 1 to 3 were
positive monoclonals.
Extract 1 to 3 plasmids for sequencing.

![](https://static.igem.org/mediawiki/2021/9/98/T--ECNUAS--result03.jpg)
Figure 3: The bacteria C colony after transformation and selected by two antibiotics (kana&amp)
medium

Function tests

Table 1. Fluorescence intensity when the concentration of CYA equals to 30uM and the duration is 4
hours
![](https://static.igem.org/mediawiki/2021/f/f4/T--ECNUAS--result04.jpg)

![](https://static.igem.org/mediawiki/2021/9/96/T--ECNUAS--result05.jpg)
Figure 4. Histogram of the fluorescence intensity when the concentration of CYA equals to 30uM and the
duration is 4 hours

As seen from figure 4, comparing to the blank control,
bacteria C presents an obvious higher fluorescence reaction to the cyanuric acid, the derivative from
Atrazine. In such a case, it could indicate that our engineered bacteria could work for detecting cyanuric
acid.

Table 2. Fluorescence intensity of bacteria C when when the duration is 4 hours under different
concentration of the cyanuric acid
![](https://static.igem.org/mediawiki/2021/b/bc/T--ECNUAS--result06.jpg)

![](https://static.igem.org/mediawiki/2021/4/45/T--ECNUAS--result07.jpg)
Figure 5. Histogram of the fluorescence intensity of bacteria C when the duration is 4 hours under
different concentration of the cyanuric acid

![](https://static.igem.org/mediawiki/2021/b/b7/T--ECNUAS--result08.jpg)
Figure 6. Histogram of the fluorescence intensity of bacteria C when the duration is 6 hours under
different concentration of the cyanuric acid

In order to analyze the relationship between the concentration of cyanuric acid and
the fluorescence
intensity, we designed the control groups and collected the data as showing above. According to the
histograms (Fig. 5 and Fig. 6), the fluorescence intensity shows a decreasing trend with the increase of
concentration of cyanuric acid when we used the bacteria C for tests. Therefore, we speculate that the
cyanuric acid might affect the growth of strains so that the higher the concentration of the cyanuric acid,
the worse the growth of the bacteria, the less of the amount of the effective “biosensor”. In order to fully
eliminate this impact, we introduced the concept of cell-free extraction and cell-free expression in the
next stage of our project.

![](https://static.igem.org/mediawiki/2021/f/ff/T--ECNUAS--result09.jpg)
Figure 7. Curve of the fluorescence intensity of bacteria C against hours

In order to analyze the relationship between the fluorescence intensity and the
induction hours, we collected the data and drew the curves under various concentrations of cyanuric acid
(CYA) as showing above.

In figure 7, we can see that basically there is an increasing trend of the
fluorescence intensity of bacteria C as the induction hour increases. In addition, the curves also indicate
that the appropriate detection hour for our biosensor to detect cyanuric acid would be 4 hours later where
several curves tend to balance.

![](https://static.igem.org/mediawiki/2021/e/ee/T--ECNUAS--chgt4.jpg)

We followed the protocol and conducted the extract preparation for cell-free expression. In the end, we
obtained the extracted product, AtzR protein whose concentrations were measured by Nano 400 as shown in
table 3 with 1.5 ml each around.

Future Plan:

1. In order to further figure out the influence of cyanuric acid on the growth of bacteria C, there shall be
several control experiments to be conducted, such as to determine the growth curves of bacterial strains
under different concentrations of cyanuric acid.
2. As we just attempted the cell-free extraction once, this technique requires more experimental conditions
groping to solid this part of work and collect more products for future cell-free expression use.
3. Once we obtain enough products of cell-free extraction, we could step into the cell-free expression
experiments to ensure the performance of our biosensor without bacteria.
4. In order to achieve our final goal, the 3D print would be necessary for us to build the device which
could load our cell-free biosensor and conduct more function tests with it. In the meantime, as the buffer
in cell-free expression needs to be freshly configured, it is necessary to explore ways to preserve the
product if it is made into a portable detection device.
5. In the future, we hope to carry our device to detect the content of cyanuric acid in lake/river water and
other environmental water.

Contact Info

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