Working together to make progress is always one of the most important spirit of Tsinghua iGEM team. We actively participated in meet-ups, providing resources for other teams, sharing ideas on project design. Throughout this process, we improved our project, offered help for others, and most importantly created bonds with a lot of highly motivated students.
In June 2019, we got two basic parts, SIM and SUMO, from Peking iGEM 2019. Thanks for their help, we successfully realized phase separation of (SIM-SUMO)3 in E.coli, which behave like a droplet as expected. Since Peking achieved (SIM)3 and (SUMO)3 phase separation in S.cerevisiae, we observed the same effect smoothly.
In addition, we discussed on how to determine the attributions of phase separation. During the meet-up, we learnt that three assays should be carried out to characterize phase separation. The first one is FRAP assay, exploring the fluidity of phase. Another way to validate it is to observe the fusion process of to droplet. Furthermore, the circularity or sphericity of phase can be calculated and compared to well-known liquid droplet.
Through our discussion, in fact, we realized it might be hard to observe fusion process of two droplet in bacteria who had rather narrow inner space for phase to move. Therefore, in reality, we focused on trying to elucidate phase separation in E.coli by FRAP assay and circularity calculation.
Collaboration with Peking University on modeling
On July 13th, we had another fruitful discussion with friends from Peking iGEM 2019 and exchanged our views on modeling and simulation. For using artificial organelle as their project theme last year, Peking iGEM have some experience and worth communicating about phase system modeling.
We'd already completed some preliminary learning about the mechanism of LLPS, numerical solution for Cahn-Hilliard equation, which is one of the most important equation describing the phase mechanism and established a effective simulation system for our LLPS system before July. For this discussion, we had prepared some questions in advance. We were really interested in whethter the conformation changes of the light-control elements caused by light intensity adjustment can be regarded as a fast reaction process relative to phase separaton.
The discussion lasted for about two hours, and our warm friends also led us to visit their igem lab and the newly completed CQB (Center for Quantitative Biology) at Peking University.We were happy to learned that, according to their experience last year, the comformation changes of light-control elements is orders of magnitude faster than phase separation process, which meant that we can use a simple first-oder kinetic equation to integrate and describe the relationship between unit impulse light signal and distribution of target enzymes.
Furthermore, They also gave us some constructive suggestions on our simulation, which they didn't have chance to try last year. The first suggestion is the 3D simulation and the implementation of fluid mechanism for lipid phase. The suggestions on 3D simulation was just in line with our plan. We try to use movements of particles in a cuboid cell model and try to simulate fluorescence quenching in our model. The second suggestion was about guiding significance and user interactivity of our mathematical model. We can use our model to estimate the minimum of element expression and the correct proportion between light-control elements and polyphase separation elements. We also design a user interaction interface for our simulation model.
In September 2019, we held two meet-ups with Peking iGEM 2019 and harvested great insights. Cheng Li, an advisor from Peking, introduced a very interesting paper to us, talking about asymmetric division of E.coli in creating multi-cell-type system. Combining the observation by us that some specific phase separation protein could form aggregates unequally in E.coli formed by, we discussed the potentiality of using phase separating system to control the asymmetric division of E.coli. What’s more, upon knowing that Peking could observe cell division using Microfluidic-control system, also called “mother machine”, we held another meet-up with them to discuss whether it could be used to observe asymmetric division of cells. Since the uneven distribution had been observed already, if they observed that one of the filial E.coli did not contain obvious phase while another did, this idea might be applicable for inducing asymmetric division in E.coli.
We were very glad that we could collaborate with iGEMers from BIT-China 2019. Thanks for the development of Safety-catch by Tsinghua iGEM 2018, the leakage of many systems can be improved. Upon knowing that, BIT-China 2019 discussed the possibility of utilizing it in their system with us. We explained our system in detail for them, and pointed out that the potential difficulty for applying it in their project might be the metabolic pressure of expressing this rather large controlling system. Then, we held a meet-up in September 28th, during which we offered Safety-catch for them. Members from both team designed the strategy of assembling lacI into their system. Moreover, advisors of Tsinghua iGEM 2019 shared experience on introducing point mutation on promoter, which could also benefit the program of BIT 2019.
IGEM team SJTU-BioX-Shanghai (https://2019.igem.org/Team:SJTU-BioX-Shanghai) contacted us. They need our Luxpr-HS promoter to perfect their information storage device. We wasted no time to prepare relevant samples overnight and sent them by SF express. We communicated with them online and patiently explained the principle to them. Fortunately, our samples passed through typhoon Lekima and reached Shanghai. The team, SJTU-BioX-Shanghai, carried out a successful experiment.
iGEM Meetup in Beijing
On August 6, BIT-China 2019 hosted an iGEM meet-up in Beijing. BIT-China, UCAS-China, Peking, Tsinghua-A, Tsinghua, OUC-China, CAU-China and BNU-China participated in the meet-up. We shared our project design and problems we encountered. After that, we exchanged ideas and discussed about the possibility of deeper cooperation. That was an enjoyable morning for us! Thanks for the invitation of BIT-China and the participation of all other teams.