Team:NEU China B/Collaborations

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Collaboration

Collaboration with Fujian normal university

1. Online meeting

In the process of online meeting (Figure 1), we initially introduced our projects and aimed to help each other. Then we talked about our problems that we meet during the experiments. For example, we often failed in the construction of plasmid. Through our communication, we hoped that we can have a further cooperation and help each other to overcome the current difficulties.

Figure 1. Online Meeting

Human practice collaboration

(1) Beer factory in FJNU（Beer Factory）

When we mentioned that the lactic acid biosensor can be used in fermentation factory, students from FJNU said that the beer factory in their school can produce beer by yeast fermentation. To better understand the production of lactic acid in fermentation, we visited the beer factory with the help of FJNU.

(2)Brochure of FJNU_China

In order to publicize the project of team from Fujian normal university, they hoped that we can help them to propagandize their notebook. After receiving the well-made brochure made by the team from Fujian normal university (Figure 2), we went to the campus, explained the project and IGEM competition to students from other majors.

Figure 2. Propaganda

Collaboration of experiments

After discussion, we found that the team from Fujian normal university constructed their plasmids in a different way. According to our protocol, when we construct our plasmid, we firstly used overlap to connect each gene, and then combined them with plasmid by enzymatic ligation. But the team from Fujian normal university used Gibson assembly master mix system to construct the composite plasmid. Therefore, we decided to help the team from Fujian normal university construct plasmid proU-GFP, They help us build plasmid proLsrA-GFP.

Finally, we helped them construct plasmid proU-GFP (Figure 3a) successfully. According to their verification, this plasmid can express GFP (Figure 3b).

Figure 3. Results of plasmids construction. (a) Colony PCR; (b) Characterization Result.

Collaboration with DLUT

Our two teams have held a meeting (Figure 3) to discuss some question about the whole project.
During the meeting, we solved some problems of our projects.

Figure 4. Meeting

Question from NEU

Quiz1：The idea of your team is very novel and has important practical value. But as a medicine that enters the human body, how to control it does not harm the intestinal flora?
Answer: We have reviewed a group quantity control system of Imperial College of Science and Technology in 2016. We have made improvements in light of our own situation. Simply put, the concentration of signal substances controlled by the bacteria itself controls the opening and closing of the suicide system, thereby regulating the number of bacteria.
Quiz2：Your final product is a bacterial preparation. There are great potential threats to the environment. Have you thought about any suicide mechanism? We are doing a cold lethal system, and if you are interested we can cooperate.
Answer：Thank you very much for your proposal. We also checked the information and found the Blu-ray lethal system, etc., but I am still very happy to work with you. You can provide our plasmids. We can help you to characterize and provide relevant data.

Question from DLUT

Quiz1: As a system for detecting lactic acid, what are the advantages of your products with lactic acid detection products on the market? Answer: Compared with the normal detection of lactic acid products, we have more use of biological related technologies. By referring to some of the design experience of the 2015-year team at the ETH Zurich, we used the group sensing system of E. coli and used fiber optic circuitry for detection,. It is low cost and ease of use.

Quiz2: How do you ensure that E. coli in your system does not pollute the fermentation industry? Answer: We intend to paste our product into a portable device, and the part that touches the fermentation liquid is only the probe. The E. coli inside the unit does not pollute the fermentation broth.

Collaboration with SCAU-China

Our team conducted an exchange discussion with NEU_China_B in August 2018. This meeting (Figure 5) is mainly to share the two team's projects main content and two teams each encountered in the experiment problem. The Q&A are as follow.

Figure 5. Meeting

NEU_China_B: How do you ensure that the integrative vector in transformation will not lose?
SCAU-China: The integrative vector we used is pFQ20 provided by the Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences. The homology arms in integrative vector for homologous recombination allows the target genes to be stably transferred into the genome of Microcoleus vaginatus. It has been verified through our experiment results.

SCAU-China: How to match the concentration of AI-2 expressed by the first plasmid pCFDuet-1 with the GFP concentration expressed by pet28a(+).

NEU_China_B: Our experiment was to release the AI-2 signal molecule after the pCFDuet-1 expression plasmid induction of lactic acid concentration, and AI-2 signal molecule can inhibit the lldr sequence on the second plasmid pet28a(+).

The lldr sequence is capable of producing GFP-deterring proteins. In the experiment, we verified that two plasmid copies were more similar, and that the O1O2 sequence could be started normally when the lactic acid concentration was lower (less than 1 mM), and then the GFP gene could be produced fluorescent signal.

Therefore, the expression of two plasmids is more matched, and the experimental results are not affected in this project.

Besides, as SCAU is famous for their tasty yogurt (Figure 6a), we detected, using our device, whether lactate is remained in their yogurt provided by them. We used their yogurt as inducer of our engineered E.coli, and green florescence could be observed under fluorescence microscope (Figure 6b).

Figure 6. Result of Yogurt Indution. (a). Yogurt; (b). Green Florescence Protein Expression.