iGEM 2022 — Team szpt-china

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block bgimg %}notebook.jpg{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

{% include 'pages/wikimenu.html' %}

## Week 1-10

### Week 4

### Project

1. Built up a team on January 24th.
2. Grouped online, with each group choosing their own direction for a project and there's an advisor in each group to help the new members.

### Week 5

### Project

1. Each group was very motivated during the iGEM journey and successively carried out their own project direction.

### Week 6

### Project

1. The five groups began to make presentation for their projects.
2. The advisors of each group supported and guided their presentation of the project.

### Week 7

### Project

1. Celebrated Chinese New Year.

### Week 8

### Project

1. Interpreted previous years' projects.
2. We brainstormed HP's activities.

### Week 9

### Project

1. The presentation of each group was completed, and the advisors gave suggestions for us on March 4th.

### Week 10

### Project

1. Added *Synthetic Biology* to our coursework and began to study.
2. Team members revised the presentation and began to prepare for the offline presentation.

## Week 11-20

### Week 11

### Project

1. Determined the list of team members, and made an outline of HP part on March 16th .
2. The tasks of posting tweets were determined at the meeting on March 18th.

### Week 12

### Project

1. Determined the direction of the questionnaire on March 21st.
2. Posted "Synthetic Biology" on the public account on March 25th.
3. A meeting was held, the project interpretation report was conducted at the meeting on March 26th.

### Week 13

### Project

1. "Project Interpretation Series ①" was posted on the public account on March 30th.
2. Summarized the problems of tweets on April 3rd.
3. Discussed making a vocabulary list about synthetic biology.

### Week 14

### Project

1. "No distance to synthetic biology" was posted on the public account on April 4th.
2. "Project Interpretation Series ②" was posted on the public account on April 6th.
3. "The Beautiful World of Synthetic Biology" was posted on the public account on April 8th.
4. We watched the live broadcast of the synthetic biology competition which named "Symposium on Intersection of Structure, Cell and Synthetic Biology".

### Week 15

### Project

1. "Project Interpretation Series ③" was posted on the public account on April 13th
2. "what can we learn from synthetic biology" was posted on the public account on April 15th.
3. The seventh meeting was held on April 17th. At the meeting, we planned the activities of HP, shared the notebook and reviewed the work content; student leader emphasized the importance of team integration; members reported on project interpretation.

### Week 16

### Project

1. "Project Interpretation Series ④" was posted on the public account on April 20th.
2. "Synthetic Biology Is Changing the World" was posted on the public account on April 22nd.
3. Communicated with SZU and SUSTech April 22nd.

### HP

1. Our team came to Watsons to interview salesmen on April 18th.

### Week 17

### Project

1. The project interpretation tweet was posted on the public account on April 27th.
2. The tweet of the Skin Care English Class was published on the public account on April 29th.
3. A meeting was held on April 30th.

### Week 18

### Project

1. The topic and content of the lecture was discussed on May 2nd.
2. "The Synthetic Biology Popular Science" was posted on the public account on May 6th.
3. We discussed the topic of the questionnaire on May 8th.

### HP

1.We went to Winkey Pharmaceutical R&D Co., Ltd. for an interview on May 2nd.

### Week 19

### Project

1. We had the second meeting with SZU and SUSTech on May 10th. At the meeting, students from SUSTech put forward the requirements for the declaration structure and declaration title.
2. The project interpretation was posted on the public account on May 11th.
3. The first tweet of "reader" (audiobook) was posted on the public account on May 12th.
4. "Skin Care English Class" was posted on the public account on May 13th.
5. Carried out the project report of each group's winter vacation project offline on May 15th.

### Week 20

### Project

1. The second tweet of ""reader"" was posted on the public account on May 16th.
2. The students of the EXP group went to the Shenzhen Institute of Advanced Technology, Chinese Academy of Csiences for training on Laboratory Safety Regulations on May 18th.
3. The third tweet of "reader" was posted on the public account on May 19th.
4. "No distance with synthetic biology" was posted on the public account on May 20th.

### HP

1. Interviewed cosmetic consumers on May 18th.

## Week 21 - 30

### Week 21

### Project

1. The fourth tweet of "reader" was posted on the public account on May 23rd.
2. The fifth tweet of "reader" was posted on the public account on May 26th.
3. "English class" was posted on the public account on May 27th.
4. An offline meeting was held on May 29th.

### HP

1. Interviewed dermatologists Jingkai Huang and Jun Huang, plastic surgeons Yuru Shang at Southern University of Science and Technology hospital on May 24th as well as interviewed Zhijun Tan of a plastic surgeon at Shenzhen University General hospital.

### Week 22

### Project

1. The students of the EXP group went to Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences for safety training and laboratory safety examination on June 1st.
2. Participated in the Southern China iGEM meetup on June 3rd.
3. Held an offline meeting on June 5th.

### Week 23

### Project

1. The eighth "reader" was posted on the public account on June 6th.
2. An offline meeting was held on June 12th.
3. The ninth tweet of "reader" was posted on the public account on June 9th.

### HP

1. We cooperated with the Japanese Club of our school, saw the film *Escape from Clone Island*, which is about synthetic biology to the members online to promote synthetic biology.

### Week 24

### Project

1. The tenth tweet of "reader" was posted on the public account on June 13th.
2. The eleventh tweet of "reader" was posted on the public account on June 16th.
3. We watched the seminar online on the topic of "Innovative Application of Synthetic Biology in the Field of New Materials for Personal Care" on June 17th.
4. "Chinese version of synthetic biology" was posted on our public account on June 17th.

### HP

1. Conducted a popular science activity of synthetic biology with members of Inspiration Creativity Club online.
2. interviewed Dr. Mingxing Tang on June 16th.

### Week 25

### Project

1. The twelfth tweet of "reader" was posted on the public account on June 20th.
2. The thirteenth tweet of "reader" was posted on the public account on June 23rd.
3. The EXP group reported and summarized their experiment on June 26th.

### HP

1. Held an activity in Nanshui Kindergarten to on June 22nd.
2. Reinterviewed Dr. Mingxing Tang on June 25th.

### Week 26

### Project

1. The fourteenth of "reader" was posted on the public account on June 27th.
2. The eighth of project interpretation was posted on the public account on June 29th.
3. The fifteenth tweet of "reader" was posted on the public account on June 30th.
4. "English class" was posted on the public account on July 1st.

### Week 27

### Project

1. The sixteenth tweet of "reader" was posted on the public account on July 4th.
2. Summarized last week's problems and assigned tasks in an online meeting on July 6th.
3. Posted the seventeenth of "reader" tweets on the public account.
4. Held an online meetup with NAU-China，NJTech\_China，UCAS-China，UCAC-China，HBUT-China on July 8th.
5. Participated in the "2022 Synthetic Biology Competition-Innovation Competition".

### Week 28

### Project

1. Posted the eighteenth tweet of "reader" on the public account on July 11st.
2. A questionnaire was posted on July 13th.
3. The first meeting was held with BNUZH-China on July 14th.
4. The first meeting with HBUT-China was held on July 14th.
5. A tweet about skin care was posted on the public account on July 14th.
6. Posted a tweet about participating in the "Synthetic Biology Competition-Innovation Competition" on the official account on July 15th.
7. Participated in the 2022 China iGEM Online Meetup hosted by GXU-China, Jilin China 2022, Jiangnan\_China, and NPU\_CHINA on July 16th
8. The first meeting with NEFU\_China was held on July 17th.

### Lab

1. The J23102-RBS003422-gshF-T0-pSEVA331(JRgTp) plasmid was constructed and transformed into *E. coli* TOP10.
2. Built the plasmids about the lysis and safety module.

### Week 29

### Project

1. The project interpretation series tweets were posted on the public account on July 18th.
2. Held an online meeting with NWU-CHINA-A on July 19th.
3. A summary tweet was posted on the public account on July 19th.
4. "skin care class" was posted on the public account on July 20th.
5. "English class" was posted on the public account on July 23rd.
6. Participated in the 4th Northwestern China iGEM Meetup hosted by NWU-CHINA-A on July 23rd.
7. Summarized the HP work we've done as well as the feedback from the questionnaire on July 24th.

### Lab

1. Verified the experssion of GshF, and used The GSH and GSSG Assay Kit of Beyotime to test the production of GSH in *E. coli* TOP10.

### Week 30

### Project

1. Held an online meeting on July 26th.
2. Poseted "Skin care class" was posted on the public account on July 27th.
3. Held an online meeting on July 27th.
4. The third online meeting was held with HBUT-China on July 29th.
5. Had an online meeting On July 29th.
6. "English class" was posted on the public account on July 29th.
7. Held an online meeting with the BNUZH team on July 31st.

### HP

1. An activity called "A Midsummer 366 Appointment" was held.
2. The offline event "A Midsummer 366 Covenant" was held on July 31st.

### Lab

1. Shook strains that verified to be correct ,and extracted plasmid as well as transferred into *G. hansenii* ATCC53582 with electrotransformation.
2. Added LVA hydrolysis label on strains that grew in dark and didn't grow in light.
3. Conducted the preliminary exploration of emulsion.

## Week 31 - 40

### Week 31

### Project

1. "English class" was posted on the public account on August 5th.
2. Had an online communication with BNUZH-China at the Tencent Conference on August 7th.

### HP

1. Visited the Yakult factory with the CUHKSZ team and non-competition members on August 3rd.
2. Held a lecture named "Enter the World of Synthetic Biology", which was participated by nearly 300 college students.

### Lab

1. Verified whether J23102-RBS003422-gshF-T0-pSEVA331(JRgTp) plasmid exist into *G. hansenii* ATCC53582.
2. Used The GSH and GSSG Assay Kit of Beyotime to test the production of GSH in *G. hansenii* ATCC53582.
3. Optimised blue light responsive system of strains that lysedwell last week: LVA hydrolysis label was knocked out.
4. Determined the exploration conditions of live bacterial emulsion/gel.

### Week 32

### Project

1. "Skin care class" was posted on our public account on August 10th.
 2. Conducted an online interview with Lifegen Co., Ltd on August 10th.
 3. Had an online meeting with the HBUT-China at the Tencent Conference on August 11th.
4. Held an online meeting on August 11th.
5. "English class" was posted on our public account on August 12th.
6. Had an online meeting with the NWU team at the Tencent conference on August 12th.
7. Had an online meeting with the BNUZH at the Tencent conference on August 14th.

### HP

1. Conducted an activity in Wulonggang Village on August 12th.
2. Went to Guangzhou Question Mark Art Institute to conduct science popularization of synthetic biology on August 15th.
3. Interviewed the experimenters of Bode Chuangyan (Guangzhou) Biotechnology Co., Ltd. on August 14th.

### Lab

1. Plotted growth curve of *G. hansenii* ATCC53582 which contains J23102-RBS003422-gshF-T0-pSEVA331(JRgTp) plasmid.
2. Repeated emulsion experiment.

### Week 33

### Project

1. Participated in the meeting of CCiC.
2. "English class" was posted on our public account on August 19th.

### HP

1. Interviewed Ms.Lee, a skin health practitioner at Naturade Beauty Salon on August 22nd.

### Lab

1. Plotted the growth curve of *G. hansenii ATCC 53582* that containes J23102-B0034-gshF-pSEVA331 plasimds at different concentrations of chloramphenicol.
2. Extracted plasimds that lysed well in the *E. coli* and transferred into *G. hansenii* ATCC53582 with electrotransformation.
3. Explored the conditions of live bacterial gel.

### Week 34

### Project

1. We had an online meeting with HBUT-China at the Tencent Conference on August 25th.
2. We had an regular meeting on August 26th.
3. We had an online meeting with BNUZH-China at the Tencent Conference on August 28th.
4. We had an weekly meeting on August 28th.

### HP

1. Went to Liyayuan West Community for science popularization on August 23rd.

### Lab

1. Plaques of *G. hansenii* ATCC53582 that contains lysis gene were pointed two plates: some plates put on dark place, others put on light place.
2. Determined the condition of live bacterial gel.

### Week 35

### Project

1. We had an online meeting with NWU-CHINA-A at the Tencent Conference on September 2nd.
2. We had an online meeting with CUHKSZ at the Tencent Conference on September 3rd.
3. We had an weekly meeting on September 4th.

### Week 36

### Project

1. We had an offline meeting on September 9th.
2. We had an online meeting with BNUZH-China at the Tencent Conference on September 11th.

### HP

1. We presented our project and synthetic biology to freshmen on September 5th.
2. We brought the abiotics students into the lab to experience the product form of our project on September 10th.

### Week 37

### Project

1. We had an weekly meeting on September 13th.

### HP

1. We took the school's minority students into the laboratory on September 17th.

### Week 38

### Project

1. Edited wiki pages.
2. Prepared the final presentation.

### Week 39

### Project

1. Edited wiki pages.
2. Prepared the final presentation.

### Week 40

### Project

1. Edited wiki pages.
2. Prepared the final presentation.

### HP

1. We performed Chinese shadow puppetry on the spotground on October 6th.
2. Held the activity of biological Chinese paper-cutting on October 8th.

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block bgimg %}engineering.jpg{% endblock %}
{% block page\_content %}

{% include 'pages/wikimenu.html' %}

## Overview

![...](https://static.igem.wiki/teams/4325/wiki/page/engineering/engineering1.png)

Figure 1: Summary of the GshF expression and GSH production module

The engineering of "*Beauty G. hansenii*" is the central goal of our project, which involves the construction and assembly of the three different modules (See [Design](https://2022.igem.wiki/szpt-china/design) for details). Hereby, we demonstrate the engineering success in the construction of the GshF expression and GSH production module with two “DBTL” cycles.

## Engineering Success

### Cycle 1: Construction of GF22 in *E. coli* TOP10

### Design

![...](https://static.igem.wiki/teams/4325/wiki/page/engineering/engineering2.png)

Figure 2: The genetic circuit of the GshF expression and GSH production module

The biosynthetic pathway of GSH usually contains two ATP-dependent consecutive reactions that are catalyzed by γ-glutamylcysteine (γ-GC) synthetase (γ-GCS or GSH I) and GSH synthetase (GS or GSH II). However, it is difficult to accumulate high levels of intracellular GSH since the enzymatic activity of the native GSH synthetases (the above mentioned GSH I and GSH II) would be strongly inhibited by the GSH. To overcome this inhibition effect of the end-product, the gene encoding a bifunctional glutathione synthetase GshF originated from *Streptococcus thermophilus*, which is less sensitive to GSH, is selected and codon optimized for expressing in *G. hansenii*.

### Build

![...](https://static.igem.wiki/teams/4325/wiki/page/engineering/engineering3.png)

Figure 3: The agarose gel electrophoresis image showed the PCR products that were amplified using the constructed plasmid GF22 as templates (In *E. coli* TOP10).

In this section, our goal is to construct the gshF expression system using the vector pSEVA331. The recombinant plasmid pSEVA331-J23102-RBS003422-gshF-T0 is referred to herein as "GF22". Firstly, the CDS region of gshF was amplified by high-fidelity PCR, purified and ligated to the linearized vector pSEVA331. Next, competent cells of *E. coli* TOP10 were transformed with the newly constructed plasmid and spread on selective agar plates. Finally, a few of monoclonal colonies formed by the *E. coli* TOP10 transformants were individually selected for examining the presence of the inserted gshF sequence using PCR. The size of PCR products (2306 bp) was determined using agarose gel electrophoresis. In this fashion, we screened for correct builds and obtained the expected plasmid.

### Test

**Characterization of GshF****:** **Western blot**

![...](https://static.igem.wiki/teams/4325/wiki/page/engineering/engineering4.png)

Figure 4: Western blot analysis of GshF in *E. coli* TOP10

Constitutive expression of our protein of interest, GshF labelled with His-Tag, was enabled by the expression system. Then the bacterial growth was launched in liquid broth to ensure to have sufficient biomass, and cell pellets were collected by centrifugation and further disrupted by ultrasonics. Raw protein matrix was then obtained from the supernatant after centrifugation. The protein was purified by the immobilized metal affinity chromatography (IMAC) loaded with high affinity Ni-NTA Resins. The molecular weight (approximately 85 kDa) of the purified protein products was finally determined using immunoblotting, as shown in Figure 4.

**Detection****of****GSH****production****content****:** **C****olorimetry**

![...](https://static.igem.wiki/teams/4325/wiki/page/engineering/engineering5.png)

Figure 5: GSH yield of wild type *E. coli* TOP10 and *E. coli* containing GF22

The GSH content in the cell lysate of wild type*E. coli* TOP10 and *E. coli* TOP10 containing GF22 were determined using Glutathione Colorimetric Detection Kit (Beyotime). The results shows that the GSH production of *E. coli* TOP10 containing GF22 is much higher (~92fold) than that of wild-type *E. coli* TOP10.

### Learn

Our experimental results were within our expectation, indicating that our design is feasible in *E. coli*. This promotes us to expect that a high level ofGSH in *G. hansenii* ATCC53582 can also be achieved if this plasmid was introduced into our final chassis.

### Cycle 2: Introduction of GF22 into *G. hansenii* ATCC53582

### Build

![...](https://static.igem.wiki/teams/4325/wiki/page/engineering/engineering6.png)

Figure 6: The agarose gel electrophoresis image showed the PCR products that were amplified using the constructed plasmid GF22 as templates (In *G. hansenii* ATCC53582).

The recombinant plasmid GF22 was firstly extracted from *E. coli*, and then introduced into our bacterial chassis using electroporation. Figure 6 showed that the size of the DNA fragments amplified from *G. hansenii*, thus confirming the successful incorporation of the plasmid.

### Test

![...](https://static.igem.wiki/teams/4325/wiki/page/engineering/engineering7.png)

Figure 7: GSH yield of wild type *G. hansenii* and *G. hansenii* containing GF22

*G. hansenii* containing the plasmid GF22 exhibited enhanced GSH biosynthsis, as evidenced by colorimetric analysis of glutathione (Figure 7).

### Learn

To summarize, the GshF expression and GSH production module [(BBa\_K4325015)](http://parts.igem.org/Part%3ABBa_K4325015) functions well in our final chassis *G. hansenii*. We plan to improve the system in the future to achieve higher GSH production.

## References

*[1] Pophaly, S. D. et al. Glutathione biosynthesis and activity of dependent enzymes in food-grade lactic acid bacteria harbouring multidomain bifunctional fusion gene (gshF). J. Appl. Microbiol. 123, 194-203 (2017).
[2] Li, Wei , et al. "Production of glutathione using a bifunctional enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli." journal of biotechnology 154.4(2011):261-268.
[3] Dezheng, et al. "Glutathione production by recombinant Escherichia coli expressing bifunctional glutathione synthetase." Journal of Industrial Microbiology & Biotechnology (2016).
[4] Xiong, Z.-Q. et al. Functional analysis and heterologous expression of bifunctional glutathione synthetase from Lactobacillus. J. Dairy Sci. 101, 6937-6945 (2018).
[5] Cui, X. et al. Efficient glutathione production in metabolically engineered Escherichia coli strains using constitutive promoters. J. Biotechnol. 289, 39-45 (2019).
[6] [https://2018.igem.org/Team:H14Z1\_Hangzhou/Team](https://2018.igem.org/Team%3AH14Z1_Hangzhou/Team)*

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block bgimg %}experiment.jpg{% endblock %}
{% block page\_content %}

{% include 'pages/wikimenu.html' %}

## Medium and Antibiotic

### Materials

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

### Procedure

### Medium

| Medium | Materials | Mass/g |
| --- | --- | --- |
| LB Medium (1000mL) | Tryptone | 10 |
| NaCl | 10 |
| Yeast Extract | 5 |
| LB Agar (1000mL) | Tryptone | 10 |
| NaCl | 10 |
| Yeast Extract | 5 |
| Agar | 15 |
| HS Medium (500mL) | Tryptone | 2.5 |
| Yeast Extract | 2.5 |
| Glucose | 10 |
| H25Na2O16P | 3.4 |
| C6H10O8 | 0.75 |
| HS Agar (500mL) | Tryptone | 2.5 |
| Yeast Extract | 2.5 |
| Glucose | 10 |
| H25Na2O16P | 3.4 |
| C6H10O8 | 0.75 |
| Agar | 7.5 |

### Antibiotic

1. Set antibiotics until room temperature from -20℃ refrigerator, label the centrifuge tubes, microfiltration membranes and syringes, calculate and record the mass of the antibiotics;
\* Note: Calculation method: Preparation Volume (mL) \* Concentration (mg/mL) = Mass (mg)
2. When antibiotics thaw to room temperature, start weighing the antibiotics with analytical balance and dissolve it with pure water;
3. After dissolving, filter the solution through 0.22μm millipore filter and store it at 4℃.
\* Note: Chloramphenicol should be dissolved in absolute ethanol.

## Preparation of Cellulose: 40mg/mL

### Materials

* Cellulose powder
* ddH2O

### Procedure

1. Weigh 1g Cellulose powder into a 50mL centrifuge tube and add 20mL ddH2O to dissolve it. Ultrasonic can accelerate dissolution. After dissolution, there will be a lot of foam in the centrifuge tube, which affects our final volume calibration.
2. Centrifuge at 8000rpm for 10min. The centrifugal time depends on the foam volume so that it does not affect volume calibration. You can take out the centrifuge tube to observe every 10min.
3. After there is not much foam, calibrate the volume to 25mL and then pass through the membrane.

## Plasmid Construction

### Materials

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

### Procedure

1. Add 25μL 2 × Phanta Max Buffer, 18μL ddH2O, 1μL dNTP Mix (10 mM each), 1μL Phanta Max Super-Fidelity DNA Polymerase, 1μL DNA templates (need to be diluted to 30ng/μL) 1μL upper primer and 1μL downstream primer, mix and put them into the PCR thermal cycler (ProFlex 3x32-Well).
2. The amplified products were verified by 1% agarose gel electrophoresis, compared with bp and cut their correct range. Then carry out gel recovery:
2.1. Add 300μL Binding Buffer, centrifuge at 12000rpm for 1min, discard waste liquid——repeat twice;
2.2. Add 700μL SPW Wash Buffer, centrifuge at 12000rpm for 1min, discard waste liquid——repeat twice;
2.3. Centrifuge at 12000rpm for 2min;
2.4. Add 30μL ddH2O and stand for 2min, centrifuge at 12000rpm for 1min;
2.5. Measure the DNA concentration.
3. Ligation:
Add 2μL 5×CE Multis Buffer, 1μL enzyme (Exnase Multis), appropriate volume of fragments, vectors and ddH2O. And put them into the PCR thermal cycler.
\* Calculation:
(1) The volume of fragments and vectors=(0.02×bp)/Concentration of PCR;
(2) The volume of ddH2O=10μL-the volumes of all solutions expected ddH2O.
4. Transformation:
4.1. Thaw the competent cells (on ice), add the ligated products and apply ice for 30min. Heat it for 90sec at 42℃ in a water bath, then put in a ice bath again for 2min;
4.2. Add 700μL LB medium to bacterium liquid, shake it at 37℃ and 220rpm for 1h in a shaker, then centrifuge at 10000rpm for 1min;
4.3. Discard 700μL supernatant solution, resuspend the bacteria with pipetting gun, take 100μL to the plate (LB agar and antibiotic), evenly spread with a cell spreader and culture it overnight at 37℃;
5. The PCR verification:
5.1. Bacterial colonies were picked, to lysed in a water bath at 95℃ for 10min and centrifuge at 5000rpm for 30sec;
5.2. Take out 1μL supernatant and add 5μL buffer, 2μL ddH2O,1μL dNTP, 1μL upper primer and 1μL downstream primer, mix and put it into the PCR thermal cycler.
5.3. The amplified products were verified by 1% agarose gel electrophoresis to compare bp and record the corresponding bacteria. Then select and inoculate bacteria into medium (antibiotic included) and shake them at 37℃ and 220rpm for 12-16h in a shaker.
6. Plasmid extraction:
6.1. Take 1.5mL bacterial solution and centrifuge at 12000rpm for 1min, discard the waste solution ——repeat twice;
6.2. Add 250μL Solution I and resuspend; add 250μL Solution II and shake gently; add 250μL Solution III and shake rapidly, then centrifuge at 12000rpm for 10min;
6.3. Take the supernatant into the absorbent tube, and centrifuge at 12000rpm for 1min, and discard the waste solution;
6.4. Add 500μL HBC Buffer and centrifuge at 12000rpm for 1min, and discard the waste solution;
6.5. Add 700μL DNA Wash Buffer and centrifuge at 12000rpm for 1min, discard the waste solution——repeat twice;
6.6. Centrifuged at 12000rpm for 2min;
6.7. Add 35μL ddH2O, stand for 2min and centrifuge at 12000rpm for 1min;
6.8. Measure the concentration.

## Electro-transformation

### Materials

* HS medium + 0.2% Cellulose
* 1mM HEPES buffer
* 15% glycerol
* HS medium
* HS Agar plate (antibiotic included)

### Procedure

6.1. Inoculate *Gluconacetobacter hansenii* ATCC53582 in 50mL centrifuge tube with 5mL HS medium and 0.2%(v/v) Cellulose, and shake it at 30℃ and 220rpm for 24-72h until OD600>0.7.
6.2. Take 1mL bacterial solution and inoculate to 10mL HS medium and 0.2% Cellulose in a 250mL conical flask, shake it at 220rpm and 30℃ for overnight until OD600=0.4-0.7.
6.3. Pre-cool centrifuge to 4℃, prepare ice bucket and ice, and place 1mM HEPES buffer and 15% glycerol on the ice.
6.4. Bacteria that reached OD600 =0.4-0.7 are put on ice for 10min.
6.5. Centrifuge at 4100rpm and 4℃ for 5min, and discard the supernatant solution.
\* Note: Pour out the supernatant solution carefully, but not the pellet. *Gluconacetobacter hansenii* ATCC53582 doesn’t form precipitates as readily as Escherichia coli (*E. coli*), most likely due to the buffering effect of Cellulose. If the pellet doesn’t adhere to the tube wall after centrifugation, apply the pellet to the tube wall and centrifuge again for a longer period of time.
6.6. Resuspend with 10mL HEPES buffer.
6.7. Centrifuge at 4100rpm and 4℃ for 5min, discard the supernatant solution. Repeat steps 6.5. to 6.7.
6.8. After resuspending with 5mL of 15% glycerol, place the bacterial solution in a centrifuge at 4℃ for 5min at 4100rpm. Discard the supernatant solution.
6.9. After resuspending the bacteria with 1mL of 15% pre-cooled glycerol, pack each 100μL of it individually into 1.5mL centrifuge tubes.
\* Note: After freezing, the transform efficiency of the competent cell may decrease, so immediately use may yield the highest efficiency.
6.10. Add 600ng plasmid to competent cells and flick gently, then transfer the bacterial solution into a (0.1cm) electroporation cup and set 3000V for 5-8ms. After completion, immediately take the bacteria to 800mL HS medium with 0.2%(v/v) Cellulose, and culture it at 220rpm and 30℃ for 16h.
Calculation: the volume of plasmid=600ng/Concentration (no more than 10μL.)
6.11. After centrifuging for 10min, discard 700μL surpernatant solution resuspend with 100μL of HS medium and spread the bacterial solution on the plate with the HS agar (including antibiotic), the colonies will grow for 24-72h. It needs to be verified by PCR to ensure the transform results.

## GSH concentration test module

### Materials

Total glutathione assay buffer
Glutathione reductase
Oxidized glutathione (GSSG)

* DTNB

Protein removal reagent M

* DMSO
* GSH Scavenging Adjuvant
* GSH Scavenging Reagent

### Procedure

1.Preparation of the kit
1.1. Preparation of GSSG stock solution: 816μL pure water is added to 5mg GSSG provided in this kit, dissolve well and mix well, that is the GSSG stock solution with a concentration of 10mM. Except for the part to be used immediately, the rest of GSSG stock solution should be properly divided and stored at -20°C
1.2. Preparation of DTNB stock solution: 4.5mg DTNB is added to 1.5mL DMSO provided by this kit, dissolve and mix well, that is DTNB stock solution. Except for the immediately to be used part, the remaining DTNB stock solution should be stored at -20°C after proper aliquoting.
1.3. Preparation of protein removal reagent M solution: Weigh 0.2g protein removal reagent M, add 4mL total glutathione detection buffer, and prepare 4mL of 5% aqueous solution, protein removal reagent M solution must be freshly prepared and limited to be used on the same day.
1.4. Preparation of NADPH stock solution (40mg/mL): Add 100μL pure water to the 4mg NADPH provided in this kit, dissolve and mix well, that is NADPH stock solution. Except for the immediately available part, the rest of the NADPH stock solution should be stored at -70°C after proper divided solution.
1.5. Preparation of 5x diluted glutathione reductase solution: Take 50μL glutathione reductase, add 200μL total glutathione detection buffer, mix well, that is 5x dilution of total glutathione reductase.
1.6. Glutathione detection solution:

|  |  |  |  |
| --- | --- | --- | --- |
| Reagent name | One sample | Ten samples | Twenty samples |
| Five-fold dilution glutathione reductase | 6.6μL | 66μL | 132μL |
| NADPH Stock solution | 6.6μL | 66μL | 132μL |
| Total glutathione detection buffer | 150μL | 1.5mL | 3mL |

1.7. Preparation of 0.5mg/mL NADPH: Take 10μL NADPH stock solution, add 790μL total glutathione detection buffer, and mix well.
1.8. Preparation of diluted GSH removal auxiliary solution: Add 53μL GSH removal auxiliary solution to 47μl grade pure water and mix immediately.
1.9. Preparation of GSH removal reagent working solution: 89.2μL absolute ethanol is added to 10.8μL GSH scavenger reagent and mixed immediately. The GSH scavenger working solution must also be freshly prepared each time.
2. Preparation of samples
2.1. Take 1.5mL of bacterial liquid, centrifuge at 12000rpm for 1min, discard the waste liquid - repeat 3 times;
2.2. PBS washes bacteria collect bacteria by centrifugation at one time discard the supernatant;
2.3. Add three times of the bacteria volume of protein removal reagent M solution and fully vortex;
2.4. With liquid nitrogen and a 37 degree water bath, the samples are subjected to two rapid freeze-thaws, four degree or an ice bath for five minutes.
2.5. Centrifuge at 10,000rpm for 10min at 4°C and take the supernatant for the determination of total glutathione.
3. Preparation of samples with GSSG content to be detected:
3.1. Take the part of the sample prepared above with the total glutamine content to be detected, add 20μl diluted GSH scavenging auxiliary solution in proportion to each 100μM sample, add diluted GSH scavenging auxiliary solution, and immediately vortex;
3.2. Then add 4μL GSH removal reagent working solution per 100μL sample add GSH clearance working solution and mix well immediately. React for 60min at 25℃;
4. Preparation of the standard samples: Dilute the 10mM GSSG stock solution with protein removal reagent M solution to 15μM GSSG solution, anD then dilute it to 10, 5, 2, 1, 0.5 μM GSG solution sequentially, measure the absorbance of 15, 10, 5, 2, 1, 0.5μM solution and plot the standard curve;
5. Determination of GSSG content in standards:
5.1. Dilute the 10mM GSSG stock solution with protein removal reagent M solution into 15μM GSSG solution, and then dilute it into 10, 5, 2, 1, 0.5μ MGSSG solution sequentially, and take six points of 15, 10, 5, 2, 1, 0.5μM solution as the standard curve.
5.2. Add diluted GSH removal auxiliary solution according to the proportion of adding 20μL diluted GSH removal auxiliary solution per 100μL standard samples and immediately vortex;
5.3. Then add GSH scavenger working solution in the proportion of adding 4μL GSH scavenger working solution per 100μL samples, immediately vortex. React at 25℃ for 60min;
6. Determination of samples and standards samples:
6.1. Refer to the table below, use a 96-well plate, add samples and standards samples in turn, and mix well. Add 150μL total glutathione detection working solution, mix well and incubate at 25°C or room temperature for 5min;

|  |  |  |  |
| --- | --- | --- | --- |
| Reagent name | Blank control | Standard curve | Sample |
| Samples or standards | 0μL | 10μL | xμL |
| Protein removal reagent M | 10μL | 0μL | 10－xμL |
| Total glutathione detection working fluid | 150μL | 150μL | 150μL |
| 0.5mg/mL NADPH | 50μL | 50μL | 50μL |

6.2. Add 50μL 0.5mg/mL NADPH solution and mix well, incubate at 25°C or room temperature for 20min;
6.3. Immediately determine A412 with enzyme calibration.

## Preparation of emulsion

### Materials

* Tween 80
* EHP (Ethylhexyl Palmitate)
* 1%Carbormer
* 5 times diluted TEA
* HS liquid medium
* Steriled water

### Procedure

1. Culture the bacteria in 100mL conical flask with 50mL HS liquid medium and 0.2%(v/v) cellulase at 220rpm, 30℃ in shaker untill its OD600 reaches 1;
2. Transfer the bacterial solution to 50mL centrifuge tube and centrifuge at 5000rpm for 10 min, discard the supernatant liquid;
3. Resuspend and wash the bacteria with 10mL pure water, centrifuge at 5000rpm for 10min, discard the supernatant liquid. ——repeat twice;
4. Resuspend the bacteria with HS medium to achieve the bacterial solution which final OD600=3 by calculating according to the CfinalVfinal=CtargetVtarget.
5. Add the components from the fomula below orderly to test tube, vortex till the componets are emulsified homogeneously. Take out 2g emulsion on plate and observe the results of the BC film production.

|  |  |
| --- | --- |
| Compose | Mass/g |
| 1%Carbomer | 0.4 |
| ddH2O | 1.6 |
| 5 times diluted TEA | 0.01 |
| Bacterial solution | 2 |
| Tween 80 | 0.1 |

## References

[1] Green, M. R. Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, 2014).

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## Engineering of the GshF expression and GSH production module

The goal of this module is to construct an expression system of the bifunctional glutathione synthetase GshF, which can be employed to enhance glutathione production in *G. hansenii*. To this end, the gene *gshF* was firstly optimized regarding the codon sequence, and then ligated to pSEVA331, which was finally introduced into *E. coli* TOP10 to obtain the expression system. To the DNA constructs, specific primers were designed to amplify a partial region of *gshF*, and the size of the PCR product met with our expectation (Figure 1a). In addition, to examine the efficacy of the expression system, the molecular weight of the HiS-tag-labelled GshF protein was checked using western blot assay (Figure 1b). Interestingly, even in the genetic background of *E. coli* TOP10, GshF is active and functional since the GSH production has been significantly increased in TOP10 following transformation with the pSEVA311-based *gshF* expression system (Figure 1c).

![...](https://static.igem.wiki/teams/4325/wiki/page/results/figure1.png)

Figure 1: (a) PCR identification of *gshF* in *E. coli* TOP10.
(b) Confirmation of GshF protein expression in *E. coli* TOP10 by Western Blot.
(c) GSH yield of wild type *E. coli* TOP10 and *E. coli* TOP10 carrying GF22.

More importantly, this *gshF* expression system also functions well in *G. hansenii*, after introduction into this strain. PCR ascertained the presence of *gshF* (Figure 2a) and GSH production massively improved in our engineered strain (Figure 2b).

![...](https://static.igem.wiki/teams/4325/wiki/page/results/figure2.png)

Figure 2: (a) PCR identification of *gshF* in *G. hansenii*.
(b) GSH yield of the wild type *G. hansenii* and its derivative strain carrying GF22.

## Engineering of the lysis and safety module

This module was slightly modified from the module from [our last year project](https://2021.igem.org/Team%3ASZPT-CHINA), the objective of which is to construct a light-inducible system for cell lysis. To this end, the lysis genes originated from bacteriophages were combined with the blue light responsive system pDawn, so that illumination with blue light could induce the lysis gene expression and, in turn the bacterial cell death. In this way, the glutathione produced by the GshF expression system as well as cell lysate can also be released to exert cosmetic effect as active ingredients. Moreover, this could also act as a kill switch which enables confinement of an engineered bacterium to an environment without blue light.

Table 1: The library of the constructed kill switches

|  |  |
| --- | --- |
| Kill Switch | Part Component |
| BX01 | pDawn(cI-LVA)-X174E-T1 |
| BX02 | pDawn(cI-LVA)-RBS070-X174E-T1 |
| BX03 | pDawn(cI)-X174E-T1 |
| BX04 | pDawn(cI-LVA)-X174E-LVA-T1 |
| BL01 | pDawn(cI-LVA)-LKD-T1 |
| BL02 | pDawn(cI-LVA)-RBS070-LKD-T1 |
| BL03 | pDawn(cI)-RBS070-LKD-T1 |
| BL04 | pDawn(cI-LVA)-RBS070-LKD-LVA-T1 |

A small library of potential constructs were built to screen for effective kill switches (Table1). The agarose gel electrophoresis verified that the four genetic builds (BX01, BX02, BL01, BL02) in *E. coli* TOP10 were correct (Figure 3).

![...](https://static.igem.wiki/teams/4325/wiki/page/results/figure3.png)

Figure 3: Verification of the plasmids containing lysis genes in *E. coli*.
Lane1: BX01; Lane2: BX02; Lane 3: BL01; Lane 4: BL02.

Then, the strains that grew in the dark but failed to grow under blue light (red squares in Figure 4) were selected and cultured in fresh LB medium and the growth curves of these strains were monitored under the two different illumination conditions (Figure 5). The discrepancy in OD600 value in the dark and that under blue light was used to evaluate the efficacy of the blue light inducible system. We found that BX01 and BL02 were the optimal candidates. We found that BX01 and BL02 were the optimal candidates.

![...](https://static.igem.wiki/teams/4325/wiki/page/results/figure4-wjj.png)

Figure 4: (a) Colony growth of *E. coli* TOP10 containing BX01 (a1) in the dark, (a2) under blue light;
(b) Colony growth of *E. coli* TOP10 containing BX02 (b1) in the dark, (b2) under blue light;
(c) Colony growth of *E. coli* TOP10 containing BL01 (c1) in the dark, (c2) under blue light;
(d) Colony growth of *E. coli* TOP10 containing BL02 (d1) in the dark, (d2) under blue light.

![...](https://static.igem.wiki/teams/4325/wiki/page/results/figure5.png)

Figure 5: Growth curve of *E. coli* TOP10 containing (a) BX01, (b) BX02, (c) BL01, (d) BL02 respectively.

However, even for the most effective system BL02, the growth-limiting effect under blue light could only be maintained for several hours. Moreover, following growth in the dark until the logarithmic phase, blue light illumination did not even inhibit bacterial growth. These results indicated that this blue light-responsive suicide system become dysfunctional over a period of growth. We speculated that excessive expression of lysis protein under blue light and leakage expression of lysis protein in the dark might cause a survival pressure. Consequently, favorable mutants outcompeted the parental strains and thrived in the culture broth. Therefore, we intended to optimize both BX01 and BL02.

![...](https://static.igem.wiki/teams/4325/wiki/page/results/figure6.png)

Figure 6: Verification of the plasmids containing lysis genes in *E. coli*.
Lane1: BX03; Lane2: BX04; Lane 3: BL03; Lane 4: BL04.

![...](https://static.igem.wiki/teams/4325/wiki/page/results/figure7-wjj.png)

Figure 7: (a) Colony growth of *E. coli* TOP10 containing BX03 (a1) in the dark, (a2) under blue light;
(b) Colony growth of *E. coli* TOP10 containing BX04 (b1) in the dark, (b2) under blue light;
(c) Colony growth of *E. coli* TOP10 containing BL03 (c1) in the dark, (c2) under blue light;
(d) Colony growth of *E. coli* TOP10 containing BL04 (d1) in the dark, (d2) under blue light.

The kill switch variants BX03, BX04, BL03 and BL04 were therefore generated either by deleting the degradation tag LVA in the original repressor cI of pDawn or adding the degradation tag LVA downstream the lysis gene. The plasmids were introduced into *E. coli* TOP10 by electroporation to obtain the transformants (Table 1). Then, the *E. coli* strains that grew in the dark but not under blue light were selected (BX03, BL03, BL04 in Figure 7) and cultured in LB medium in the dark until the OD600 reached around 0.6. Finally, the bacterial growth was further monitored under blue light. We found that those three new engineered strains showed decreased OD600 values in response to blue light (Figure 8), indicating the effective refinement of the inducible suicide plasmid in rapid growing cells.

![...](https://static.igem.wiki/teams/4325/wiki/page/results/figure8.png)

Figure 8: Growth curves of *E. coli* containing different kill switches
 with (a) X174 lysis gene; (b) LKD lysis cassette.

To sum up, we optimized the efficacy of the kill switches and obtained three variants BX03, BL03 and BL04 for the following assembly experiments.

## Assembly of GshF expression module and lysis module

Table 2: The library of the Recombinant Plasmid for GSH production and release

|  |  |
| --- | --- |
| Recombinant Plasmid | Part Component |
| GX01 | pSEVA331-pDawn(cI-LVA)-X174E-*gshF*-T1 |
| GX03 | pSEVA331-pDawn(cI)-X174E-*gshF*-T1 |
| GL02 | pSEVA331-pDawn(cI-LVA)-RBS070-LKD-*gshF*-T1 |
| GL03 | pSEVA331-pDawn(cI)-RBS070-LKD-*gshF*-T1 |
| GL04 | pSEVA331-pDawn(cI-LVA)-RBS070-LKD-LVA-*gshF*-T1 |

The objective was to incorporate the lysis-induction circuit into the pSEVA311-based *gshF* expression system. To this end, the inducible suicide genetic circuit covering the pDawn promoter, the lysis gene and the associated regulatory region was amplified by high-fidelity PCR. Then the PCR product was purified and ligated to linearized the *gshF* expression system vector to generate a set of assembled systems (Table 2). Those plasmids were individually introduced into *E. coli* TOP10 competent cells, which were further spread on selective plates to obtain transformants. The assembly of two modules in each type of transformant was verified by PCR (Figure 9).

![...](https://static.igem.wiki/teams/4325/wiki/page/results/figure9.png)

Figure 9: Verification of the assembly plasmids in E. coli TOP10. Lane1~5 are the verification of gshF fragments in GL02, GX01, GL04, GL03, GX03.

To examine the efficacy of the inserted lysis modules in *E. coli,*the growth of those strains on agar surface under either the dark or the blue light condition was compared, and the result allowed us to confirm the insertion of optogenetic circuit for lysis in the colonies of those strains (Figure 10 and 11). Among them, the plasmid, GL03 was optimal since the frequency of *E. coli* colonies harboring this plasmid that can lyse under blue light was the highest.

![...](https://static.igem.wiki/teams/4325/wiki/page/results/results10-w.png)

Figure 10: (a) Colony growth of *E. coli* TOP10 containing GX01 (a1) in the dark, (a2) under blue light;
(b) Colony growth of *E. coli* TOP10 containing GX03 (b1) in the dark, (b2) under blue light.

![...](https://static.igem.wiki/teams/4325/wiki/page/results/results-11w.png)

Figure 11: (a) Colony growth of *E. coli* TOP10 containing GL02 (a1) in the dark, (a2) under blue light;
(b) Colony growth of *E. coli* TOP10 containing GL03 (b1) in the dark, (b2) under blue light;
(c) Colony growth of *E. coli* TOP10 containing GL04 (c1) in the dark, (c2) under blue light.

Finally, the GL03 plasmid was extracted and introduced into *G. hansenii* through electroporation. This plasmid can cause limited bacterial growth on agar plates, but not in the dark (Figure 12), indicative of a partial success in engineering the *G. hansenii* strains into the cosmetics-related bio-materials.

![...](https://static.igem.wiki/teams/4325/wiki/page/results/figure12.png)

Figure 12: Colony growth of *G. hansenii* containing GL03 (a) in the dark, (b) under blue light.

## Prototype

See [proof of concept](https://2022.igem.wiki/szpt-china/proof-of-concept) for details.

## References

[1] [https://2021.igem.org/Team:SZPT-CHINA/Design](https://2021.igem.org/Team%3ASZPT-CHINA/Design)
[2] Li, W., Li, Z., Yang, J. & Ye, Q. Production of glutathione using a bifunctional enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli. *J. Biotechnol.***154**, 261–268 (2011).
[3] Teh, M. Y. *et al.*An Expanded Synthetic Biology Toolkit for Gene Expression Control in Acetobacteraceae. *Acs Synth. Biol.***8**, 708–723 (2019).
[4] Pophaly, S. D. *et al.*Glutathione biosynthesis and activity of dependent enzymes in food-grade lactic acid bacteria harbouring multidomain bifunctional fusion gene (gshF). *J. Appl. Microbiol.***123**, 194–203 (2017).
[5] Ryu, M.-H. & Gomelsky, M. Near-infrared Light Responsive Synthetic c-di-GMP Module for Optogenetic Applications. *ACS Synth. Biol.***3**, 802–810 (2014).
[6] Florea, M. *et al.*Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain. *Proc. Natl. Acad. Sci. U. S. A.***113**, E3431–E3440 (2016).
[7] Ohlendorf, R., Vidavski, R. R., Eldar, A., Moffat, K. & Möglich, A. From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression. *J. Mol. Biol.***416**, 534–542 (2012).
[8] Witte, A., Wanner, G., Sulzner, M. & Lubitz, W. Dynamics of PhiX174 protein E-mediated lysis of Escherichia coli. *Arch. Microbiol.***157**, 381–8 (1992).
[9] Ceyssens, P.-J. *et al.*Genomic analysis of Pseudomonas aeruginosa phages LKD16 and LKA1: establishment of the phiKMV subgroup within the T7 supergroup. *J. Bacteriol.***188**, 6924–31 (2006).
[10] Guiziou, S. *et al.*A part toolbox to tune genetic expression in Bacillus subtilis. *Nucleic Acids Res.***44**, 7495–7508 (2016).
[11] Stirling, F. *et al.*Rational Design of Evolutionarily Stable Microbial Kill Switches. *Mol. Cell***68**, 686-697.e3 (2017).

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