Team:BIT-China/Collaborations
Collaboration with Jilin-China
[debugging a construct]
◎ BIT-China for Jilin-China
This year, team Jilin_China aimed to propose a new way to cure the solid tumor by expressing the TAT-Apoptin. The chassis they chose is Bifidobacteria, which can target the anaerobic region of the solid tumors since the microorganism is also anaerobic.
However, the functional device should firstly be transformed into E.coli to operate the methylation process in case that the devices were digested by the restriction enzymes inside the Bifidobacterium longum. They tried several times, but the results were not ideal. Since our team BIT-China is good at genetic operation on E.coli, we helped them realize the expression of TAT-Apoptin in E.coli.
We transformed four kinds of plasmid provided by Jilin_China into E.coli DH5α. The vector is pUC18, and the fragments are separately listed below:
| num | Fragment kinds |
|---|---|
| 1 | positive control |
| 2 | TAT-Linker-apoptin |
| 3 | apoptin and Tmp1 signal peptide |
| 4 | apoptin and Sec2 signal peptide |
After four kinds of positive clones separately containing these fragments were selected, we activated cells in LB culture medium overnight. We inoculated bacteria into new LB culture medium the second day and used IPTG for induction when the OD600 is 0.6. The induction was processed under 16℃ and 37℃ overnight.
After that, we treated the protein samples. We break cells after enrichment. We obtained the separation products of precipitation and supernatant. Separately, we ran those samples.
We have run the SDS-PAGE twice, here are the results:
Fig.1 Different temperature induced with supernatant
Fig.2 Different temperature induced with precipitation
◎ Jilin-China for BIT-China
This year, Jilin_China mainly helped us improve our model.
After constructing the primary model, we met some difficulties in proving the accuracy of these equations, as well as simulating these equations by software. Some members of Jilin_China majored in physics, math, computer science and chemistry, they were familiar with solving these problems and kindly provided us with much useful advice.
We provided them with the condition and equations of each stage of chemical reactions. They added some chemical reactions to improve our model and got the analytic solutions of the linearized equations.
Fig.1 The experiment circuit
Table.1 The description of variables used in differential reaction equations
| Variable1 | Variable2 | Description |
|---|---|---|
| Dt | C3 | Number of plasmids |
| [Sinductor] | y1 | Concentration of Arabinose |
| [XAraC] | y2 | Concentration of Arac protein |
| [DpBAD] | y3 | Number of pBAD promoter without inductor |
| [XAraCSinductor] | y4 | Concentration of combination of AraC protein and arabinose |
| [DpBADXAraCSinductor] | y5 | Concentration of combination of pBAD promoter, AraC and arabinose |
| [mRNAlacIq] | y6 | Concentration of LacIq mRNA |
| [proteinlacIq] | y7 | Concentration of LacIq protein |
| [DpTac] | y8 | Number of pTac promoter without inductor |
| [DpTacproteinlacIq] | y9 | Concentration of combination of LacIq protein and pTac promoter |
| [mRNARFP] | y10 | Concentration of RFP mRNA |
| [proteinRFP] | y11 | Concentration of RFP |
Based on the equations we provided, they added some modification.
Fig.2 The equations improved by JLU
After discussion, we found it’s hard to measure some parameters. They suggested us to simplify the equations, making it more close to the linear relationship.
The picture has two parts, one is about the equations replaced by other symbols (Fig.3), the other is about the linear equations to avoid measuring the parameter (Fig. 4).
Fig.3 Linear equations made by JLU
Fig.4 Equations replaced by other symbols
They also helped us improve another model (Fig. 5), they also rely on some equations provide by us to simplify the equations, making it more close to the linear relationship.
Fig.5 The circuit constructed by BIT-CHINA
Table.2 The description of variables used in differential reaction equations
| Variable1 | Variable2 |
|---|---|
| [mRNAlacIq] | y1 |
| [proteinlacIq] | y2 |
| [DpTacproteinlacIq] | y3 |
| [DpTac] | y4 |
| [mRNAkiller] | y5 |
| [proteinkiller] | y6 |
| [DT] | D |
They also helped us replace equations by other symbols.
Fig.6 The equations replaced by other symbols
Fig.7 The linear equations made by JLU
Collaboration with Lanzhou
[confirming the mercury concentration]
◎ BIT-China for Lanzhou
This year, team Lanzhou aimed to employ the gut flora to treat the mercury pollution inside fish body. To transform the gut flora, they planned to feed the fish with genetic engineered bacteria.
Above is the gene circuit they designed, INP (Ice Nucleation Protein) is an extrinsic membrane protein of E.coli and acts as an anchor. gfp acts as a reporter gene while metal catcher can bind heavy-metal ions.
We helped them designed and operated the pre-experiment to identify the maximum concentration of mercury ions which can keep the fish alive. From literature research, we chose the following concentration: 0.3mg/L, 0.2mg/L, 0.15mg/L, 0.1mg/L, 0.05mg/L, 0.025mg/L.
Then we raised 10 fishes and measured the number at the same time in fifteen days.
At last, we found that the concentration of mercury ions should be maintained at 0.1 milligrams per liter. When the concentration is higher than this value, the fish will die. When the concentration is lower than this value, the difference will not be obvious.
◎ Lanzhou for BIT-China
On September 18th, 2016, we met and exchanged ideas with members of teams from Lanzhou by the Beijing Institute of Technology. After the meeting, we confirmed our collaboration relationship. To further extend our system, we asked them for helping us constructing one of our gene circuits.
They constructed one of our testing circuits.
The "inhibitor" system is very important for our project, we use the concentration of inhibitor proteins as a signal indicating the plasmid numbers, and it can trigger the downstream reaction by activating the killer gene while the plasmid concentration reduce the threshold.
3. Collaboration with CIEI-BJ
[mentoring a new high school team]
◎ BIT-China for CIEI-BJ
CIEI-BJ is a high school team and it’s their first time to participate in iGEM competition this year. In the middle of July, team CIEI-BJ came to visit Beijing Institute of Technology and communicated with our team BIT-China.
As an undergraduate team, we shared the core concept of our project this year and explained a little bit about how we have exchanged ideas among the team members and how to assess the feasibility of our design as a reference for high school students.
Later, team members in high school also shared their fascinating ideas. However, some plans are not that practical due to the objective reasons. A lot of questioned were raised by members of BIT-China to help shape the project of team CIEI-BJ this year. The considerations refer to the practical meaning and the possibility of conduct for an appropriate project. What’s more, some suggestions are given to optimize the details like designing an experiment to prove a concept.
After the discussion, we showed high school students around our lab and explained the brief function of some experimental instruments. Besides, we introduced them to the previous projects of our team with the aid of posters preserved.
After they designed the basic gene circuit, we gave some advice on how to improve it. In the next few months, the guidance of BIT-China to CIEI-BJ are not only limited to the project itself, but also extended to the details about the competition procedures. We have been in touch with CIEI-BJ through the Internet and give advice on many aspects, like how to present our iGEM program to potential sponsors and how to achieve the requirements for deliverables.
In September 17th, 2016, some members of BIT-China are invited to share their experience on how to design the wiki page. A few examples were given to explain the good elements in introducing the project in a simple way. What should be put on the wiki were also discussed during this meeting. Considering the web fluency and in order to design a clear page, notes are listed for high school members to pay attention when they are working on their wiki.
In the process of mentoring a high school team, we also benefited a lot. Not only did we engage in the competition, but also we had a better understanding of the community connected by iGEM.
