Team:BS United China/Partnership

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Partnership

Partnership with Jilin_China

After knowing each other from the 2021 China iGEM Online Meetup in May, Jilin_China and we have gone into continuous partnership throughout the season. By communicating with each other through online meetings and WeChat, we helped each other in many different ways including organizing educational meetup and video together, offering project advice, etc. We also conducted experiments in our labs to improve projects for each of us. Meanwhile, we received encouragement from Jilin_China every now and then. The partnership with Jilin-China made us realize that collaboration can be so beneficial.

Click here to visit their PARTNERSHIP page

Our partnership timeline

Our communication

Jilin_China and we frequently communicated with each other online this year by using several tools, including WeChat and conducting online meetings on Tencent Meeting.

Fig.1 Screenshots during our several online meetings.

Fig.2 Screenshots of some of our chatting on WeChat.

Experiments

After online meetings between Jilin-China to exchange our ideas, we reached a consensus to send our existing constructs to each other to conduct certain experiments. Specifically, we sent our E. bsuahlscout to Jilin_China to test the validity of this construct for the detection of the AHL concentration in the environment. Mutually, Jilin_China sent their LL-37 peptide to us to verify its sterilization function combined with the PVDQ protein in our engineered bacterium part 2, E. bsuahlterminator, to sterilize P. aeruginosa in particular.

Experiments conducted by Jilin_China

In August, we sent our E. bsuahlscout to Jilin_China to test the validity of this construct for the detection of the AHL concentration in the environment. We initially offered Jilin_China a protocol and they modified the protocol during repeating this experiment for several times.

• Experimental purposes

- The first purpose of the experiments conducted by Jilin_China is to help us verify the validity of our construct part 1, E. bsuahlscout, to detect the presence of AHL in the environment, especially the low concentration of AHL. Our ideal experiment result is that the E. bsuahlscout can produce enough red color that is visible enough for naked eyes to observe. This is because the eventual purpose of our E. bsuahlscout is to warn people about the presence of AHL in the environment that they cannot notice with naked eyes.

- The second purpose is to prove that with the use our E. bsuahlscout, Jilin_China will be able to detect AHL in the environment. In other words, our E. bsuahlscout acted as an indicator of AHL for Jilin_China to better shape their projects.

• Results and Modifications

- By using the initial protocol we provided, Jilin_China first conducted an experiment. The result showed that the E. bsuahlscout successfully expressed mCherry to change the color to pink in high AHL concentration. Nevertheless, the expression of the color change is inconspicuous when the AHL concentration is low. Eventually, Jilin_China modified the protocol and conducted experiments that showed the more visible color expression by E. bsuahlscout in low AHL concentration. Several findings or modifications are listed below:

1. Since E. bsuahlscout includes an inducible promoter to express LuxR gene while Jilin_China uses a constitutive one, the insufficiency of LuxR protein expression might be the real culprit of the low expression of E. bsuahlscout regarding AHL.

2. Jilin_China found that the concentration of DMSO is excessively high in the culture medium and this could have an adverse effect on E. bsuahlscout. So they decided to expand the culture medium volume. Additionally, they changed the solvent of AHL they used from DMSO to LB culture medium to directly avoid the adverse effect DMSO could cause.

3. Our protocol standard the inducing temperature to be 25℃ because this low temperature can inhibit bacteria from producing cell walls and prompt more protein expression. Jilin_China verified this belief and incorporated the low inducing temperature into their project.

4. Since the quorum sensing signaling molecules of the Gram-negative bacteria (AHL) have various types, we realize that the types of AHL in Jilin_China’s lab and ours are different. Specifically, AHL in Jilin_China’s lab is N-(β-Ketocaproyl)-DL-homoserine lactone while ours is N-(3-Oxododecanoyl)-L-homoserine lactone. Therefore, we need to carefully convert the formula weights of the two types of AHL into the same concentration.

Fig.3 The pink color expression of E. bsuahlscout of the experiment by the initial protocol（Left）The more conspicuous pink color expression of E. bsuahlscout of the experiment by the modified protocol（Right）

Fig.4 After centrifugation. The more conspicuous pink color expression of E. bsuahlscout of the experiment by the modified protocol (Left)The more conspicuous pink color expression of E. bsuahlscout of a repeating experiment by the modified protocol(Right)

Experiments conducted by BS_United_China

• Experimental purpose

- The purpose of this experiment is to test the validity of the combination use of PVDQ from our E. bsuahlterminator and LL-37 peptide from Jilin_China to sterilize P. aeruginosa. We aim at showing that PVDQ is able to assist LL-37 peptide to function well.

• Results

- The results from the plates are obvious. By counting the numbers of bacterial colonies, we found that initially LL-37 peptide is able to sterilize part of the P. aeruginosa, but the combined use of PVDQ from E. bsuahlterminator and LL-37 peptide can sterilize P. aeruginosa more efficiently and validly.

Fig.5 3 groups in the experiment and each group contains 3 individuals. (The last individual of the last group that is with LL-37 + PVDQ was not evenly distributed, but the result can still be obvious.)

Fig.6 Numbers of bacterial colonies on average of plates of each group.

Part combination

We combined parts from each team to BBa_K3882003. Engineered bacterium Part 2 from BS_United_China provides the function to up-regulate the expression of PVDQ. PVDQ is able to convert N-Acyl Homoserine Lactone (AHL) molecule into L-Homoserine Lactone (LHL) that is able to generate QS signals. Lac operator is used for releasing LuxR. T7 promoter is served to start the whole synthetic gene. PVDQ protein functions as restraining P. aeruginosa from growing and activating GFP gene (GFP is a green fluorescence reporting gene). K3078107 is the LL-37 peptide fused to the secreted peptide SPusp45 from Jilin_China. The K3078107 uses a constitutive promoter 59 (BBa_K3078009), secretory signal peptide SPusp45 (BBa_K1830002), RBS from pVE5523 (BBa_K3078011), a double terminator (BBa_B0014), and LL-37 coding region (BBa_K3078005). The composite part makes LL-37 able to be secreted. We conducted an experiment to verify the validity of the combination of PVDQ from part 2 and LL-37 peptide, see the result above.

Fig.7 The diagram of BBa_K3882003.

With NEU_CHINA

We sent our bacteria construct Part 1 to NEU_China to do one experiment. Specifically, the experiment is about using the supernatant of engineering bacteria culture medium at different culture time to induce our bacteria construct Part 1 called E. bsuahlscout to produce red fluorescent protein. The result of the experiment on the whole has the trend that we expect. The relationship between the time cultivated and the productivity of the bacteria is shown in Figure 8 below.

The two teams gain mutual benefits from the experiment. In one way, by using our E. bsuahlscout to detect the AHL concentration in the environment, NEU_CHINA could better test their project. This is because AHL is critical for NEU_CHINA to manipulate its genetic elements in its project. In another, NEU_CHINA helps us to verify the validity of our construct Part 1 E. bsuahlscout as an indicator of AHL in the environment.

Click here to visit their PARTNERSHIP page

Fig.8 One of the team members of NEU_China is doing the experiment（Left）The relationship between the time cultivated and the productivity of the bacteria（Right）