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.

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.

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.

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.

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.

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

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

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.

What should this page have?

Chronological notes of what your team is doing.
Brief descriptions of daily important events.
Pictures of your progress.
Mention who participated in what task.

Inspiration

You can see what others teams have done to organize their notes:

Team:CSU CHINA/Notebook