iGEM 2022 — Team bfsu-icunited

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![](https://static.igem.wiki/teams/4400/wiki/main/pic5.jpg)

April

Week 1 (Mon Apr 18 22 - Sun April 24 22)

This is our first online lesson. As our team members are from different
schools, this is the first time we introduced ourselves. The teacher introuduced the basic parts of
igem, including the igem cycles and the competition itself

![](https://static.igem.wiki/teams/4400/wiki/notebook/pic1.jpg)

![](https://static.igem.wiki/teams/4400/wiki/notebook/pic2.jpg)

Week 2 (Mon Apr 35 22 - Sun May 1 22)

We looked at the website ourselves, including examplary projects, namely
engineering, human practice, and wiki.

May

Week 3 (Mon May 2 22- Sun May 8 22)

As not all member is familiar to synthetic biology, our teacher did a short
presentation informing of the general information such as what is DNA,cloning, genetic engineering, etc.

![](https://static.igem.wiki/teams/4400/wiki/notebook/pic3.jpg)

Week 4 (Mon May 9 22-Sun May 15 22)

We began brainstorming our project.

Week 5 (Mon May 23 22-Sun May 29 22)

We determined the content of the project and began to plan the experiment.

June

Week 6 (Mon May 30 22 - Sun Jun 5 22)

From May 30th, every member came to Beijing in order to began doing lab work. This is
our reference experiment.

Aim

Activate E. coli DH5a/pSB1A3-mRFP, E. coli DH5a/pMD18T-insert

Preparation

1. Prepare medium and required materials

2. Clean bench, materials sterilization

3. Prepare diluted primer

4. Prepare configuration for amp mother solution (100mg/mL)

Experiment procedures

1. Spraying alcohol after wearing gloves before entering clean bench. Ignite alcohol lamp after
drying gloves.

2. For sterilization, when we open/close the test tubes or bottles, we have to heat the opening of
the test tubes or bottles with the alcohol lamp.

3. Using pipette, adding 5 mL LB medium.

4. Using pipette, adding 2.5μL Amp antibiotics

5. Using pipette, adding E. coli DH5a/pSB1A3-mRFP（or E. coli DH5a/pMD18T-insert）100μL

![](https://static.igem.wiki/teams/4400/wiki/notebook/pic4.jpg)

Week 7 (Mon Jun 6 22 - Sun Jun 12 22)

Aim

Extracte plasmids

Amplify the DNA using PCR

Double enzyme digestion

Agarose gel electrophoresis

Gel extraction

Experiment procedures

1. Extract the plamid

a. we use the alkaline lysis method to break the E. coli cell the extract 4 tubes of pSB1A3-mRFP
and 1 tube of pMD18T-insert plasmids. This includes using the centrifuging machine to collect the
pure DNA instead of the other impurities and carbohydrate that is decanting and washing repeatedly
after centrifuging.

c.

![](https://static.igem.wiki/teams/4400/wiki/notebook/pic5.png)

d.Our result is reassured by examining dsDNA concentration and sample purity using Nanodrop.

e.

![](https://static.igem.wiki/teams/4400/wiki/notebook/pic6.png)

2. Proceeding PCR amplification program

3. Mix a. PrimeSTAR®️HS b. buffer c. primer 1 d. primer 2 e. template f. dNTP mixture g. sterile
water to form a mixture

4. Mix 5μL mixture with 1μL 6xPS buffer

5. Proceeding electrophoresis by putting the mixture and loading marker into agarose gel

6. After electrophoresis, observing the result displayed on gel imaging system

7. Cut the gel then put into centrifuge tube

8. Recycle the gel

Week 8 (Mon Jun 13 22 - Sun Jun 19 22)

Aim

1. Prepare competent cells

2. Ligation

3. Transformation of linking products

Preparation

Precooling solutions: a. 0.1M CaCl2 b. 15% 0.1M CaCl2 in 4℃ fridge
Experiment procedures

Experimrnt procedures

1. Ligation

2. Mix 1μL T4 DNA ligase, 6μL insert DNA, 1μL linear plasmid, 1μL 10 x DNA ligase buffer

3. Put the mixture in to PCR machine, at 16℃ for an hour

4. Prepare competent cells

5. Separate the E. coli solution, pour the LB meidum

6. Equally seprate the E. coli into two centrifuge tubes, then add 1 mL CaCl2 for each tube, mix
the tube

7. Ice-bath fot 30 min

8. Centrifuge, pour the waste

9. Resuspend the E. coli with CaCl2

10. Add 1μL E.coli + CaCl2 solution to each centrifuge tube which contain ligation system

11. Ice-bath for 20 min

12. Water-bath at 42℃ for 1 min

13. Ice-bath for 10 min

14. Use glass spreading rod to apply on the medium evenly, then

15. seal

Results

![](https://static.igem.wiki/teams/4400/wiki/notebook/pic7.jpg)

Week 9 (Mon Jun 20 22 - Sun Jun 26 22)

Aim

Obtain positive cloning result

Preparation

1. sterile water 240μL

2. DNTP mix 24μL

3. Buffer 3μL

4. Primer 1 6μL

5. Primer 2 6μL

Experiment procedures

1. Taq mix: mix all the solutions from prepartions

2. Sperate the mixture into several centrifuge tubes

3. Mix 49μL taq mix and 1μL polymerase, then pick template DNA in the centrifuge tube

4. Add 0.6g agarose to 1Xbuffer, then heat

5. Add gene green into agarose solution, dyeing the gene

6. Use PCR system for electrophoresis

7. Select valid target gene fragments

8. Add ammonia benzyl to LB meidum

9. Add 5-10 Ml meidum with antibiotoic into the test tubes

10. Add the same template DNA into the test tubess

11. Sterlise the test tubes

12. Put test tubes into shaker

Week 10 (Mon Jun 27 22 - Sun Jul 3 22)

Aim

Extract the plasmid from E.coli prepared.

Tranfer the target gene to E.coli BL 21

Look under fluorescence microscope and examine the results

Experiment procedures

1. Extract plasmid from bacterial solution validated for the correct DHSa/ PSB1A3-target fragment

2. Transform the pSB1A3-fragment into E. coli BL21

3. Validation experiments by fluorescence microscopy

Results

Through observation under fluorescence microscope, we found that the fluorescent
protein was successfully expressed, which reveals the experiment was successful.

![](https://static.igem.wiki/teams/4400/wiki/notebook/pic8.jpg)

![](https://static.igem.wiki/teams/4400/wiki/notebook/pic9.jpg)

![](https://static.igem.wiki/teams/4400/wiki/notebook/pic10.jpg)

July

Week 11 (Mon Jul 4 22 - Sun Jul 10 22)

In the meantime, we began carrying out our human practices, interviewing experts and
carrying surveys.

Week 12 (Mon Jul 11 22 - Sun Jul 17 22)

Our team participated in the Junior Synthetic Biology Exchange Meeting on July 17th

7.17 meeting

Any further information can be found on our wechat official account.

![](https://static.igem.wiki/teams/4400/wiki/notebook/pic11.png)

Week 13 (Mon Jul 18 22 - Sun Jul 24 22)

We created a team WeChat official account, which is part of our education.
We began to send igem science popularization and team and project introduction on it.

![](https://static.igem.wiki/teams/4400/wiki/notebook/pic12.jpg)

Week 14 (Mon Jul 25 22 - Sun Jul 31 22)

We completed CCIC's poster production and Pre video recording, and successfully uploaded
it.

![](https://static.igem.wiki/teams/4400/wiki/notebook/pic13.png)

![](https://static.igem.wiki/teams/4400/wiki/notebook/pic14.jpg)

August

Week 15 (Mon Aug 1 22 - Sun Aug 7 22)

The mug design in human practice has been completed and found a manufacturer to make it.

![](https://static.igem.wiki/teams/4400/wiki/notebook/pic15.jpg)

Week 16 (Mon Aug 8 22 - Sun Aug 14 22)

On this week we host our first online meeting with the LZU-HS-China team. We
first learned of each other's project during the 7.17青少年合成生物学交流大会 that is the July 17th Junior Synthetic
Biology Exchange Meeting.

Week 17 (Mon Aug 15 22- Sun Aug 21 22)

We attended the online opening ceremony of CCiC.

Week 18 (Mon Aug 22 22- Sun Aug 28 22)

We completed the two-minute video production and dubbing，and uploaded it.

September

Week 19 (Mon Aug 29 22 - Sun Sep 4 22)

We held weekly meetings to analyze the progress of the project, and improved it.

Week 20 (Mon Sep 5 22 - Sun Sep 11 22)

Our team members continue to complete the parts they are responsible for, constantly
modifying and improving quality.

Week 21 (Mon Sep 12 22 - Sun Sep 18 22)

In the opening ceremony of our school, our group members

Week 22 (Mon Sep 19 22 - Sun Sep 25 22)

Our team participated in a online lecture on probiotics and discussed many microbial
roblems with Mr. Zeng.

![](https://static.igem.wiki/teams/4400/wiki/notebook/pic16.jpg)

![](https://static.igem.wiki/teams/4400/wiki/notebook/pic17.jpg)

Week 23 (Mon Sep 26 22 - Sun Oct 2 22)

Make a 15-minute speech video. We also participated in a human practice workshop.

![](https://static.igem.wiki/teams/4400/wiki/notebook/pic18.png)

![](https://static.igem.wiki/teams/4400/wiki/notebook/pic19.png)

![](https://static.igem.wiki/teams/4400/wiki/notebook/pic20.png)

October

Week 24 (Mon Oct 3 22 - Sun Oct 9 22)

All the team members will carry out centralized training, complete their respective
tasks, and discuss the existing problems for further improvement.

![](https://static.igem.wiki/teams/4400/wiki/foot.jpeg)

© 2022 - Content on this site is licensed under a [Creative Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).
The repository used to create this website is available at [gitlab.igem.org/2022/bfsu-icunited](https://gitlab.igem.org/2022/bfsu-icunited).

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![](https://static.igem.wiki/teams/4400/wiki/main/pic5.jpg)

The first round

The project design

BPA was detected by tyrosinase. In the presence of dissolved oxygen, tyrosinase catalyzes the conversion of BPA into o-quinones, which can be reduced and release electrons, which can be detected in an external band circuit through a galvanic cell.

build

1.Synthesis of tyrosinase gene by direct DNA synthesis.

2.The tyrosinase gene was inserted into pSB1A3 plasmid by molecular cloning.

3.Prepare competent cells for transformation.

4.Molecular cloning and screening were carried out to obtain positive clones, and the labeled DH5α/pSB1A3 target fragment colonies were selected for colony PCR with Tap enzyme.

detection

1. The successful transformation of E. coli to Tyr was confirmed by Sanger sequencing. E. coli BL21 containing pET23b was used as control

2. Determination of tyrosinase activity According to a previous spectrophotometric method, this method measures the conversion of L-dopa to dopa red, a red oxidation product. The initial rate of reaction is proportional to the concentration of enzyme. The enzyme activity was determined by centrifugation at 8000rpm for 10 min

3. Electrochemical measurements were performed using a Chi660C Electrochemical workstation (ChenhuaCo, Shanghai., China) with a conventional single chamber and three electrode cells with an internal volume of 5 mL. CV reactions were obtained in a 3 mL reaction mixture of 0.1MPBS, pH=7.0 and BPA at a scanning rate of 100mV/s. The immobilized electrode was used as the working electrode, and the Ag/ silver chloride and planar platinum electrodes were used as the reference and counter electrodes, respectively.

Learning

The detection reaction is not obvious, because these chemical molecules need to be transported across the membrane, which may hinder the efficiency of the reaction. When the concentration of BPA is low, the corresponding concentration is not well detected. Consider using surface display technology to increase the chances of BPA molecules coming into contact with the bio detector, thereby increasing sensitivity.

Second Round

design

Based on tyrosinase, an electrochemical biosensor was developed to detect bisphenol A by cell surface display technology. The BPA-recognizing Tyrosinase was fused with the carrier protein INP, and the INP was used to anchor Tyrosinase to the surface of E.coli cells.

Construction

1. Obtain the target gene - Tyrosinase

2. The target gene was implanted into E.coli DH5α/ pSB1A3-MRFP

3. The plasmid was extracted, DNA was amplified by PCR, and double digestion and gel recovery experiments were performed

4. Competent cells were prepared, and the engineered vector pSB1A3-target fragment was transformed into the expression host E.coli BL21

5. Molecular cloning and screening were performed to obtain positive clones, and the labeled DH5α/pSB1A3 target fragment colonies were selected for colony PCR with Tap enzyme.

Test

1. Electrochemical detection Electrochemical measurements were performed using a Chi660C Electrochemical workstation (ChenhuaCo, Shanghai., China) with a conventional single chamber and three electrode cells with an internal volume of 5 mL. CV reactions were obtained in a 3 mL reaction mixture of 0.1MPBS, pH=7.0 and BPA at a scanning rate of 100mV/s. The stationary electrode was used as the working electrode, and the Ag/ silver chloride and planar platinum electrodes were used as the reference electrode and counter electrode, respectively. In the amperometric study, the optimized potential was applied to the enzyme electrode in 50mmPBS solution (pH 7.0) to obtain a stable current response

2. Electrochemical detection of BPA in real samples The performance of the biosensor was investigated by selecting three commercial canned tea and fruit juice as samples containing BPA to detect BPA concentration. HPLC assays were performed in parallel as described above to test the accuracy of the biosensor. The recovery rate of BPA was calculated by randomly adding different final concentrations of pure BPA solution to the sample. All tests were performed in triplicate and their mean values were considered. Use OriginPro8.0 software

Learning

A functional biosensor with LOD, accuracy, and sensitivity compared to other biosensors was constructed by simple adsorption of cells on GC electrodes. These results indicate that adsorption is a feasible method to immobilize living cells with highly active and surface-displayed tyrosinase. Further development of this biosensor should focus on improving its capability and durability

![](https://static.igem.wiki/teams/4400/wiki/foot.jpeg)

© 2022 - Content on this site is licensed under a [Creative Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).
The repository used to create this website is available at [gitlab.igem.org/2022/bfsu-icunited](https://gitlab.igem.org/2022/bfsu-icunited).