Team:SJTang/Partnership
1. Partnership with Team SCAU-China
This year, Team SCAU-China is focusing on heavy metal pollution in water bodies. They plan to use Chlamydomonas reinhardtii to adsorb heavy metals within the water bodies and thereby purifying it. We have carried out in-depth cooperation with their team in various aspects. The specific cooperation content is as follows:
1.1 Biological Modeling for Rhodopseudomonas palustris
In the early stages of the project, we discovered that the hydrogen yield process occurs primarily under an anaerobic environment, which is not ideal for direct quantitative experiments, and it hindered our data-collecting efficiency. Hence, finding a feasible methodology for biological modeling for this project remains a challenge. We have had many discussions with SCAU-China in this regard. During the discussion, they suggested that we can begin by not considering the first principles, but viewing the entire hydrogen production process as a black box, only focusing on the relationship between the substrate as input and hydrogen as output. We found it to be a great idea, however, in the follow-up refinement, we realized that such a modeling method requires massive data on the relationship between the substrate and hydrogen. Nevertheless, due to the complexity of accurate gas collection and analysis, such high-throughput experiments seem unrealistic, so we end up giving up this idea.
As the discussion continues, SCAU-China noticed a new phenomenon observed in our experiments, that is, R. palustris, which normally requires anaerobic culture, does grow under aerobic conditions. Through research, we found that R. palustris was a facultative anaerobic bacteria, but most of the recent publications only focused on its hydrogen production ability, therefore only describing the anaerobic culture process. Based on this information, SCAU-China recommends that we carry out more in-depth studies, as it is generally believed that the aerobic growth of bacteria is faster than that under anaerobic conditions. After testing, we found that it is true that R. palustris can grow under aerobic conditions in YPMOPS medium.
Figure.1.1Aerobic growth rate of R. palustris. Sodium succinate used here is 10 mM.
Following SCAU's advice, we further tested the aerobic growth of R. palustris and found that it can reach OD660nm=0.8 in optimized YPMOPS within 2 days of culture, a speed that far exceeded the record in the previous articles. On this basis, our team held a more thorough discussion with SCAU-China, established a modeling method with R. palustris' aerobic growth as the core, and worked out an experiment plan together. After the experiment, SCAU-China also helped us to build the model.
In general, through a series of cooperation with SCAU-China, we have successfully modeled the aerobic growth process of R. palustris, which can provide much convenience for related research in the future.
1.2 Further steps about MESEG
While SCAU-China assisted our team with modeling, we also helped their project. The full name of SCAU-China's project is "Molecular magnEt Super Environment Guard" (MESEG). It utilizes Chlamydomonas reinhardtii to adsorb heavy metal ions in the water, focusing on the treatment of heavy metal pollution. We hope to look at the problem from a different angle and use this to assist their projects. We believe that it is important not only to consider how to deal with the pollution but also how to detect the pollution promptly. Researches are conducted from this standpoint, and it is found that a mean using DNAzyme that can be used to detect heavy metal ions. We conducted a preliminary investigation on this method and found that the method does not rely on enzymes that are difficult to preserve but uses DNA, a relatively easy-to-storage substance, for reaction, and can form visualized output. Moreover, the sensitivity of DNAzyme also lies within the desirable range of the MESEG project.
Based upon this, we have put forward our proposal to SCAU-China, which is to combine the processes of heavy metal ion detection and heavy metal ion adsorption, in order to achieve a complete solution from pollution detection to pollution control. This solution has been recognized by Team SCAU-China, but they prefer to put the detection stage after the treatment stage, using it as a tool to monitor the removal of heavy metal ions. That being so, SCAU-China is happy to integrate DNAzyme-based heavy metal ion detection into their project in the future in assisting heavy metal ion removal.
Figure.1.2Screenshot of discussion with SCAU.
2. Partnership with Tongji China
Our team and Team Tongji_China met during the 8th CCiC(Conference of China iGEMer Community). Their project focused on the odor produced during bacterial metabolism. They hope to use their modified bacteria to reduce the hydrogen sulfide and ammonia produced by other bacteria during the growth process. When Team Tongji_China heard from our team that the modified E. coli and R. palustris might be co-cultured for hydrogen production in the future, they showed great interest in our work, as their project will eventually be used in a scene where a variety of microorganisms are mixed and cultivated, and the modified strain will be used to remove the "exhaust gas" produced by other bacterias. Therefore, we reached a consensus and decided to conduct co-cultivation experiments with our modified hydrogen-producing bacteria and their deodorizing bacteria.
Considering that Tongji_China uses E. coli BL21 as the chassis organism, we also selected our modified E. coli with hydrogenase. To determine whether the two could be cultivated together, we conducted a preliminary test as below:
Figure.2.1Growth curve of 3 different strain. HoxG1 for Hydrogenase expression, T7-Sqr and SSAS for sulfide oxidization.
The test results suggest that the SSAS bacteria (which can degrade S2-) of the Tongji_China have similar growth rates to E. coli modified by our hydrogenase. Therefore, we chose SSAS and our hydrogenase strain for co-cultivation. So we further carried out the following experiments:
Figure.2.2Growth curve of different strains. Both SSAS and HoxG1 uses E. coli BL21 as chassis.
We mixed the two kinds of bacteria and measured the growth curve, and found that the culture conditions of them all are similar. After 6 hours of culture, when the OD 600nm reached about 0.7, the addition of IPTG began to induce the expression of sulfide-removing enzymes. After induction for a period of time, we followed the detection method provided by Tongji_China to detect the possible presence of sulfide ions in the culture medium. The results are as follows:
Figure.2.3S2- concentration test result. Use OD 665nm for concentration domination. The higher the OD 665nm value, the greater the S2- concentration.
The results indicate that after the hydrogenase was expressed in E. coli, the sulfide ion content in the solution increased. But the differences between the two are not as significant as anticipated, and the difference between the two kinds of mixed-cultured bacteria is not very big compared to the hydrogen-producing strains cultured separately. Therefore, we believe that from the results, SSAS has an unsatisfactory sulfide ion removal effect for the HoxG1 strain. However, the specific reason for this result requires the cooperation of the two teams to conduct a more in-depth exploration afterward.