iGEM 2018 — Team CSU_CHINA

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# Team:CSU CHINA/Notebook

* ![](https://static.igem.org/mediawiki/2018/thumb/b/b2/T--CSU_CHINA--TeamLogo.png/320px-T--CSU_CHINA--TeamLogo.png)
* [HOME](https://2018.igem.org/Team%3ACSU_CHINA)
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  + [Description](https://2018.igem.org/Team%3ACSU_CHINA/Description)
  + [Design](https://2018.igem.org/Team%3ACSU_CHINA/Design)
  + [Experiments](https://2018.igem.org/Team%3ACSU_CHINA/Experiments)
  + [Notebook](https://2018.igem.org/Team%3ACSU_CHINA/Notebook)
* [Results](https://2018.igem.org/Team%3ACSU_CHINA/Demonstrate)
  + [Demonstrate](https://2018.igem.org/Team%3ACSU_CHINA/Demonstrate)
  + [Parts Overview](https://2018.igem.org/Team%3ACSU_CHINA/Parts)
  + [Basic Part](https://2018.igem.org/Team%3ACSU_CHINA/Basic_Part)
  + [Composite Part](https://2018.igem.org/Team%3ACSU_CHINA/Composite_Part)
* [Lab](https://2018.igem.org/Team%3ACSU_CHINA/InterLab)
  + [Safety](https://2018.igem.org/Team%3ACSU_CHINA/Safety)
  + [InterLab](https://2018.igem.org/Team%3ACSU_CHINA/InterLab)
* [Human Practices](https://2018.igem.org/Team%3ACSU_CHINA/Human_Practices)
  + [Human Practices](https://2018.igem.org/Team%3ACSU_CHINA/Human_Practices)
  + [Education & Engagement](https://2018.igem.org/Team%3ACSU_CHINA/Public_Engagement)
  + [Collaborations](https://2018.igem.org/Team%3ACSU_CHINA/Collaborations)
* [Team](https://2018.igem.org/Team%3ACSU_CHINA/Team)
  + [Team Members](https://2018.igem.org/Team%3ACSU_CHINA/Team)
  + [Attributions](https://2018.igem.org/Team%3ACSU_CHINA/Attributions)

# Notebook

---

You know what we did from last winter to autumn! In this Notebook you’ll be able to follow what and when we did it. We documented the experiment protocols and chronological important events, including how we started, how we determined our project, weekly meeting, experiment development, human practice project and many more! Thanks to this Notebook you can travel back in time and see how we managed to get big things done in such a small time frame. Once you get to know the past, you learn to appreciate the present day and the hard work done by CSU\_CHINA team.

Recruiting team member(January-Feburary)

This is the first time for our school to participate in IGEM competition. Since we were financed and sponsored by our school, we had begun to recruit our team member using various ways. We spread out the message on the Internet and made a detailed self-reporting questionnaire, including major, personal strength and so on.

![](https://static.igem.org/mediawiki/2018/5/51/T--CSU_CHINA--NB1.png)
Brain storm and discussing(March-April)

Then we welcomed all primary participaters coming together to think of an available project, which begins with the innovation ideas, gene circuit design and applications.

![](https://static.igem.org/mediawiki/2018/e/eb/T--CSU_CHINA--NB6.jpg)
Preparing preliminary experiment and make final project design (May-June)

After several meetings and preliminary lab experiment, we determined an available and innovative project targeted therapeutics of HCC using a way of synthetic biology.

![](https://static.igem.org/mediawiki/2018/7/78/T--CSU_CHINA--NB2.png)
Lab Experiment(June-October)

Seeing those plates for Interlab study and tubes for luciferase assay, you can imagine how much time and energy our IGEMers have spent. During the hot summer vacation, our lab members in turns came to the lab to finish the huge task.

![](https://static.igem.org/mediawiki/2018/9/93/T--CSU_CHINA--NB3.png)

![](https://static.igem.org/mediawiki/2018/9/9d/T--CSU_CHINA--NB4.png)
Human practice(August-Sepetember)

Working molecule iGEM. The inspiration for "Working molecule" comes from a Japanese anime called "はたらく細胞". The difference is that this animation anthropomorphizes all kinds of cells, but we regard people as molecules instead. Because everyone is similar to a molecule, which has its own use and value, even if this value has not been discovered. Everybody has his or her own duties and steps, and there will be a “mutation” and create many beautiful accidents and progress. Since liver cancer is caused by a variety of molecular combination mutations, we do can use this method to explain and promote our own concept of biosynthesis better. In this spirit, we did many things, and tried our best to perform it in a “Working molecule” way.

![](https://static.igem.org/mediawiki/2018/8/8d/T--CSU_CHINA--NB5.png)

THE TEAM includes a mix of complementary expertise. In the end the work was naturally split between the members. Below each member of the efficient team is linked to their identifying tasks.
Xiangyu Li was responsible for the whole experiment. He performed cloning, BioBrick construction and validation of the constructed parts. Xiangyu also coordinated our collaboration with team Nanjing-China and CPU\_CHINA.
Qingxin Shi was responsible for the construction of the first plasmid. He assembled the reporter plasmid and the corresponding biobricks. Qingxin is also in charge of handing all files submission online.
Yunfeiyang Li did all work on coding wiki design into HTML and CSS. Throughout the project he provided supportive models for us.
Zhaoyang Xiang is a student at the Faculty of Medicine. He brought to the group a wealth of information regarding medical practice. In the lab he started off with the Interlab study and then played an integral role in Human Practice having an interesting science class.
Jiaqi Xiao is a student at the Faculty of Medicine. Early on she helped with the Interlab study. Subsequently she played a supportive roles in Human Practice.
Jiamiao Liu was responsible for Human Practice . She organized several fantastic activities to expand the influence of our project to societies.
Jiayi Li studies life science at the Central South University in Changsha. She is a versatile teammate. In Human Practice, she was responsible for the Flash Mob. In our lab, she played a supportive roles in different modules as needed. She is also a supportive role in graphic design.
Ying Yang performed experiments to ensure the construction of our plasmids and assisted with other experiments. She was also responsible for the Flash Mob in Human Practice.
Shangcheng Yu is a bachelor student at the Faculty of Life Science at Central South University in Changsha. Shangcheng provided valuable knowledge and ideas during the development of the concept of our project and the design. He also assisted with our experiments.
Xiandong Peng is responsible for all work on graphic design. He made the standard results image of our experiments.
Keyu Yao is responsible for our graphic design. She beautified our wiki.

THE ADVISORS kept the work on track and helped answer the tough questions. They also checked wiki pages related to their field of expertise.
Weiqian Zeng was the main advisor of the team. She taught the students basics of synthetic biology and showed them how to use specialized equipment. She supervised the Biobrick construction and submission.
Yuelin Chen made sure all the deadlines are met and the loose ends are closed. In addition, she provided relevant literature and advice.
Shuai Guo provided valuable knowledge and ideas in modeling. His experience with data processing also proved useful in modeling our project.

THE INSTRUCTORS were closely involved keeping the message clear and the team on track to reaching the goals.
Kun Xia  as our first PI keeps the great mood in the lab and his accurate questions leads everyone to the result without any need for explanation.
Jiada Li is the PI of Li lab on which we depend and so gave us a number of advices of our experiments.
Shanni Li provided tremendous organizational support for our team throughout the duration.

Project Development and Important Meeting Documentation

[Click Here](https://static.igem.org/mediawiki/2018/2/27/T--CSU_CHINA--project_documentation.pdf)
for more information.

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[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:CSU CHINA/Experiments

* ![](https://static.igem.org/mediawiki/2018/thumb/b/b2/T--CSU_CHINA--TeamLogo.png/320px-T--CSU_CHINA--TeamLogo.png)
* [HOME](https://2018.igem.org/Team%3ACSU_CHINA)
* [Project](https://2018.igem.org/Team%3ACSU_CHINA/Description)
  + [Description](https://2018.igem.org/Team%3ACSU_CHINA/Description)
  + [Design](https://2018.igem.org/Team%3ACSU_CHINA/Design)
  + [Experiments](https://2018.igem.org/Team%3ACSU_CHINA/Experiments)
  + [Notebook](https://2018.igem.org/Team%3ACSU_CHINA/Notebook)
* [Results](https://2018.igem.org/Team%3ACSU_CHINA/Demonstrate)
  + [Demonstrate](https://2018.igem.org/Team%3ACSU_CHINA/Demonstrate)
  + [Parts Overview](https://2018.igem.org/Team%3ACSU_CHINA/Parts)
  + [Basic Part](https://2018.igem.org/Team%3ACSU_CHINA/Basic_Part)
  + [Composite Part](https://2018.igem.org/Team%3ACSU_CHINA/Composite_Part)
* [Lab](https://2018.igem.org/Team%3ACSU_CHINA/InterLab)
  + [Safety](https://2018.igem.org/Team%3ACSU_CHINA/Safety)
  + [InterLab](https://2018.igem.org/Team%3ACSU_CHINA/InterLab)
* [Human Practices](https://2018.igem.org/Team%3ACSU_CHINA/Human_Practices)
  + [Human Practices](https://2018.igem.org/Team%3ACSU_CHINA/Human_Practices)
  + [Education & Engagement](https://2018.igem.org/Team%3ACSU_CHINA/Public_Engagement)
  + [Collaborations](https://2018.igem.org/Team%3ACSU_CHINA/Collaborations)
* [Team](https://2018.igem.org/Team%3ACSU_CHINA/Team)
  + [Team Members](https://2018.igem.org/Team%3ACSU_CHINA/Team)
  + [Attributions](https://2018.igem.org/Team%3ACSU_CHINA/Attributions)

# Design

---

Protocols
Chemical transformation

Thaw 50µL DH10B competent E. coli cells on ice.
Add 5 µl DNA from a ligation reaction mix or 10-100ng DNA of a plasmid.
Place the mixture on ice for 20-30 minutes.
Heat shock at 42°C for 90 seconds.
Place on ice for 5 minutes.
Pipette 200 µl of room temperature LB media into the mixture.
Incubate at 37°C and 220 rpm for 60 minutes.
Add 50-100 µl of the transformed cells to the selection plate.

Growing overnight cultures

Overnight cultures were prepared under sterile conditions using a Bunsen burner
Add 3 mL liquid LB media into test tubes
Add 3 μL of appropriate antibiotic（1000x） into the broth
Using the pipette tips, pick a single colony and inoculate the cultures by dipping the tip into the LB broth or by adding 50 μL stored cells
Put caps on the tubes and incubate overnight at 37°C shaking at 200-250 rpm

PCR From Plasmid DNA Template

In a PCR tube on ice, combine 1-10 ng of plasmid DNA or just 1 µL template, 2 µL of 10 µM forward primer, 2 µL of 10 µM reverse primer to a PCR tube on ice, 25 µL of 2x Phanta Mastermix, and sterile water up to 50 µL.
Gently mix the reaction
If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly
Transfer the PCR tube from ice to a PCR machine preheated to 98°C to begin thermocycling

![](https://static.igem.org/mediawiki/2018/5/5f/T--CSU_CHINA--Exp1.png)
Preparation of LB Broth

Add 10 g Tryptone, 5 g Yeast Extract and 10 g Sodium Chloride to 1 litre purified water
Autoclave

Preparation of LB Agar

Add 10 g Tryptone, 5 g Yeast Extract, 10 g Sodium Chloride and 20 g Agar to 1 litre purified water
Autoclave

Ligation

Add vector plasmid, insert DNA, T4 DNA ligase and T4 DNA ligase buffer to the microcentrifuge tube
Make reaction up to 10 µL using sterile water
Incubate at room temperature for 30 - 60 min

Restriction Digestion

Set up the reaction following the instruction below, depending on whether test digest or assembly digest is being performed.
For a Test digest (10 µL):
5 µL Plasmid DNA
0.5 µL Restriction Enzyme 1
0.5 µL Restriction Enzyme 2
1 µL 10× buffer
Add sterile water to 10 µL
For gene assembly(30 µL):
15 µL Plasmid DNA
1.2 µL Restriction Enzyme 1
1.2 µL Restriction Enzyme 2
3 µL 10X buffer
Add sterile water to 30 µL
Incubate digestion reaction at 37°C for 30 minutes-1 hour
Perform heat deactivation at 80°C for 20 minutes, if not running on a gel at the end of incubation.

Gibson assembly

1.Keep 15µL Gibson mix on ice.
2.Add 5µL DNA fragment (Vector fragment: less than 15ng; small fragment: 20-50ng).
3. Incubate at 50℃ to 60min.

![](https://static.igem.org/mediawiki/2018/3/39/T--CSU_CHINA--Exp2.png)

Store in -20℃, aliquot 15µL

![](https://static.igem.org/mediawiki/2018/0/07/T--CSU_CHINA--Exp3.png)

Add ddH2O to 6mL, store in -20℃,Aliquot 100µL

DNA Gel Electrophoresis

Prepare 1% w/v solution of agarose powder in 1×TAE buffer (e.g. 1g agarose powder in 100 mL buffer) using a conical flask.
Heat the mixture until agarose is completely dissolved.
Add 5 µL EB to the solution.
Pour the solution into a gel mould.
Allows the solution to set (approx 15-20 minutes)
Transfer the agarose gel to a tank, remove the comb and apply:
3-5 µL of the DNA ladder
DNA samples with the corresponding amount of DNA loading buffer (6X)
Run the gel for 20 minutes at 120V.
PCR Purification, Gel Extraction & Miniprep
PCR purification was performed according to the Generay PCR Purification Kit
Gel extraction was performed according to the Generay Gel Extraction Kit
Plasmid extraction were carried out according to the Magen Miniprep Plasmid Kit
1.Prepare 3mL bacteria solution grown overnight in LB broth, centrifuge at 10,000×g for 1min. Discard the supernatant.
2.Add 250µL Buffer P1/RNase A to resuspend bacteria cells and transfer to a 1.5mL centrifuge tube.
3.Add 250µL Buffer P2 and gently invert the tube 8-10 times to mix.
4.Add 350µL Buffer P3 and gently invert the tube immediately but gently 8-10 times.
5.Centrifuge for 10min at 13,000×g in a microcentrifuge.
6.Apply the supernatants from step 5 to the HiPure DNA Mini Column Ⅱspin column by pipetting.
7.Centrifuge for 1min at 13,000×g. Discard the flow-through.
8.Wash the HiPure DNA Mini Column Ⅱspin column by adding 500µL Buffer PW1, and centrifuging for 1min at 13,000×g. Discard the flow-through.
9.Wash the HiPure DNA Mini Column Ⅱ by adding 600µL Buffer PW2 (diluted with absolute ethanol), and centrifuging for 1min at 13,000×g. Discard the flow-through.
10.Repeat the step 9.
11.Centrifuging for an additional 1min at 13,000×g to remove residual wash buffer.
12.Place the HiPure DNA Mini Column Ⅱ in a clean 1.5mL microcentrifuge tube. Add 50µL Elution Buffer (65°C) to the center of the HiPure DNA Mini Column Ⅱ, let stand for 1min, and centrifuge for 1min at 13,000×g. For better production, repeat Step12 for a second time.
13.Discard the column，measure the concentration of plasmid by Nanodrop2000，then preserve it at -20°C.

Cell subculturing

1.Remove culture medium from the dish. Gently add 1 ml of phosphate buffered saline (PBS) to the dish, and shake it several times.
2.Remove PBS from the dish. Add 1 ml of pre-warmed 0.1% Trypsin to the dish. Gently shake it to get complete coverage of the cell layer. Incubate the cells at room temperature for 40-60 seconds. (Note: The actual incubation time varies with the cell line used.)
3.Add 2ml of pre-warmed complete growth medium (DMEM with 10% FBS). Disperse the cells by pipetting over the cell layer surface several times.
4.Transfer the cells to 15 ml centrifuge tubes and centrifuge at 1200 ×g for 3 minutes.
5.Resuspend the cell pellet in 1-2 ml of pre-warmed medium and take 10 μl of medium for cell counting.
6.Cells were seeded in plates at the recommended density, and pipe the rest of the cells into new cell culture dishes, and put dishes back to the incubator (37°C, 5% CO2).

Plasmid transfection

1. Thaw FBS. Make sufficient 10% FBS in Opti-MEM media (w/o antibiotics) sterile filter
2. Aspirate medium off cells
3. Add 2-3 ml PBS to wash
4. Aspirate off PBS
5. Add 8 ml of 10% FBS/ Opti-MEM media /plate
6. Incubate 30min at 37°C while mixing following components in hood (see spreadsheet
for amounts, this example is for one 10cm plate):
a. A Tubes: Add selected DNA, and Q.S. w/ Opti-MEM to 100µl
b. B Tubes: Add LF2000 and Q.S. with Opti-MEM to 100µl
c. Incubate in hood for 5 min
7. Add Component B to A by gently bubble mixing
8. Incubate A/B complexes at RT for 30 min
9. Drip 200µl A/B complex mixture onto cells respectively
10. Gently mix media on cells via figure 8 pattern and back and forth motions
11. Incubated 37° for 48 hours
12. Remove Opti-MEM media containing A/B complexes and discard
13. Add fresh 8ml/ plate of 10% FBS/DMEM with antibiotics

Cell lysis

1. Remove growth medium from cultured cells.
2. Rinse cells in 1X PBS. Do not dislodge cells. Remove as much of the final wash as possible.
3. Dispense a minimal volume of 1X lysis reagent (CCLR, RLB or PLB) into each culture vessel (e.g.,400µl/60mm culture dish, 900µl/100mm culture dish or 20µl/well for a 96 well plate).
4. For culture dishes, scrape attached cells from the dish, and transfer the cells and solution to a microcentrifuge tube. Pellet debris by brief centrifugation, and transfer the supernatant to a new tube.
5. Mix 20µl of cell lysate with 100µl of Luciferase Assay Reagent and measure the light produced.

## Single-tube Luciferase reporter assay

![](https://static.igem.org/mediawiki/2018/9/9f/T--CSU_CHINA--Exp4.png)

Experiments
Cell culture and transfection

HepG2, LO2, Huh7 and MCF7 cells were cultured in DMEM (GIBCO) supplemented with 10% FBS. All cells were cultured at 37°C and 5% CO2. For transfection, HepG2, LO2, Huh7 and MCF7 cells were seeded at 4 ×  cells/well in 24‐well plates and transiently transfected by the lipo2000-mediated method. Briefly, each well is transfected with 15-30ng of constructed plasmid on two consecutive days.

Luciferase reporter assay

Luciferase assays were performed as described previously. Briefly, the cells were transfected with 100 ng of the luciferase (firefly) reporter plasmid as designed containing different promoter into 24-well plate. After 48h transfection, cells were collected and lysed. Firefly luciferase activities were measured with the single-tube luciferase Reporter mechine and β‐galactosidase luciferase activity was used to normalize for potential differences in transfection efficiency.

Molecule cloning and mutation

We use a series of methods above and fusion enzyme to construct targeted and mutated plasmid.

QUANTIFICATION AND STATISTICAL ANALYSIS

Results are expressed as mean ± SE. Differences between two groups were assessed using multiple two‐tailed Student's t‐tests（\*p<0.05, \*\*p<0.01, \*\*\*p<0.001, \*\*\*\*p<0.0001）. Statistical calculations were performed using Graphpad Prism 6.0.

EXPERIMENT DEVELOPMENT

\*For experiment development, you can turn to [notebook](https://static.igem.org/mediawiki/2018/c/c1/T--Edinburgh_OG--2018--EducationOwen2.pdf) to get the detail information

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[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:CSU CHINA/Results

* ![](https://static.igem.org/mediawiki/2018/thumb/b/b2/T--CSU_CHINA--TeamLogo.png/320px-T--CSU_CHINA--TeamLogo.png)
* [HOME](https://2018.igem.org/Team%3ACSU_CHINA)
* [Project](https://2018.igem.org/Team%3ACSU_CHINA/Description)
  + [Description](https://2018.igem.org/Team%3ACSU_CHINA/Description)
  + [Design](https://2018.igem.org/Team%3ACSU_CHINA/Design)
  + [Experiments](https://2018.igem.org/Team%3ACSU_CHINA/Experiments)
  + [Notebook](https://2018.igem.org/Team%3ACSU_CHINA/Notebook)
* [Results](https://2018.igem.org/Team%3ACSU_CHINA/Demonstrate)
  + [Demonstrate](https://2018.igem.org/Team%3ACSU_CHINA/Demonstrate)
  + [Parts Overview](https://2018.igem.org/Team%3ACSU_CHINA/Parts)
  + [Basic Part](https://2018.igem.org/Team%3ACSU_CHINA/Basic_Part)
  + [Composite Part](https://2018.igem.org/Team%3ACSU_CHINA/Composite_Part)
* [Lab](https://2018.igem.org/Team%3ACSU_CHINA/InterLab)
  + [Safety](https://2018.igem.org/Team%3ACSU_CHINA/Safety)
  + [InterLab](https://2018.igem.org/Team%3ACSU_CHINA/InterLab)
* [Human Practices](https://2018.igem.org/Team%3ACSU_CHINA/Human_Practices)
  + [Human Practices](https://2018.igem.org/Team%3ACSU_CHINA/Human_Practices)
  + [Education & Engagement](https://2018.igem.org/Team%3ACSU_CHINA/Public_Engagement)
  + [Collaborations](https://2018.igem.org/Team%3ACSU_CHINA/Collaborations)
* [Team](https://2018.igem.org/Team%3ACSU_CHINA/Team)
  + [Team Members](https://2018.igem.org/Team%3ACSU_CHINA/Team)
  + [Attributions](https://2018.igem.org/Team%3ACSU_CHINA/Attributions)

# Results

Here you can describe the results of your project and your future plans.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember all measurement and characterization data must be on part pages in the Registry.
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)