In June, we communicated with our instructor and got in touch with another 2 high-school IGEM teams. We, LZU-HS-China, Think-Edu, XHD-Wuhan-Pro-China, are in the same track and we have similar ideas about our projects. We formed a high-school league to promote further collaborations. We also invited LZU, SCU, and SJTU to be our undergraduate and graduate mentor. We invited other school teams and bioers to join our league and held forums online twice a month. We discussed potential problems within capacious domains under tight collaborations throughout the whole iGEM season.
P1: Group photo at Bio-X Academy in SJTU
After we determined our project, which is the probiotics synthesis of SeNPs, we were impeded by choosing an appropriate cell carrier. XHD-Wuhan-Pro-China's project, the degradation of alcohol, also concentrated on the goal to construct an intestinal probiotic. We talked about our problem with them, and with the help of the instructor in LZU and their experimental results, they suggested us to use Nissle1917 (EcN), a good probiotic which has been used in the treatment of a variety of intestinal diseases because it does not secrete exotoxin, as our cell expression vector.
P2. Experiment Data from XHD
P3. A scheme of the implementation of EcN
We were ready to adopt their advice to do further experiments, but there were still some problems remain.
Another problem did existed. Instead of expressing our enzymes and proteins within the cell membrane, we wanted to figure out another feasible way to express our foreign functional protein. We talked about this issue on the third conference. Think-Edu members raised an eminent proposal, which was to apply the Microbial Cell-surface display system to make our project more economically and effectively advantageous. We discovered that this system can also enhance the stability and efficiency of reactive enzymes as well as prevent the degradation of foreign proteins by intracellular enzymes produced by the host. This system, allowing the protein to bind to the cell wall and do its job, is significantly compatible with our idea. We worked with them at SJTU for two weeks to do the wet-lab experiments together.
P4. Think-Edu-China（Debiotics）
P5. Schematic diagram of plasmid construction and surface display.
We adopted two teams' suggestions and managed to express our protein in vitro in further experiments. Finally, It was observed that the reduction efficiency of selenites to nano-selenium was significantly improved in vitro environments.
However, after a long period of test and examination, our group discovered that because EcN contains secret plasmids, it can interfere with the expression of foreign genes at specific locations to some extent and disturbed the efficacy of the gene and protein. Thus, we ameliorated our project by adopting a new high copy plasmid-pSB1A3. Moreover, the strong promoter before the expressive sequence and INP, an appropraite and stable protein transporter binding on cell membrane, were added in front of the target gene to promote the efficiency and stability.
We shared our achievements and helped other league members to avoid potential deficiencies in their experiments. Our contributions are definitely an anesis of our common difficulties.
P6. Cell surface display systems constructed by different transporters and their application directions
P7. Heterologous expression diagram of probiotic Escherichia coli Nissle1917 in our mode
P8. The map of recombinant plasmid