This summer, we collaborated with six 2019 iGEM teams for greater progress in the project and hosted 2019 Southwestern iGEM Exchange Conference with UESTC-Software.

We provided CAU_China with a plasmid and their simultaneous validation experiments. We discussed with SZU-China how to improve the feasibility of the project and the expression of toxic protein. We designed a questionnaire analysis method together with CSU_CHINA. ZJUT-China provided us with lysin. We offered a trial of our part of the last year to XMU-China. And we shared the experience of team building with USTC. A total of five teams from Southwest China participated in the meetup we held.
Host the meetup
On 3rd May 2019---4th May 2019, UESTC-Softerware and we (UESTC-China) hosted 2019 Southwestern iGEM Exchange Conference in order to encourage the development of iGEM in the west of China. BM-AMU, SCU-China, SICAU-iGEM, XJTU-China, SCU-China participated in this meeting. We also invited Asian Ambassador Dorothy Zhang. She anylysed the difference of the rules for new medal criteria between this year and before, and introduce the After iGEM for us in the meetup. She told us human practices should follow social science. Each team made a presentation to show their amazing ideas.
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We have collaborated with team CAU_China and help each other mutually. Their project this year is to degrade the straw to produce astaxanthin, and we have the same place in last year's project (degrading straw to generate clean energy butanol and hydrogen), so we put our piGEM2018 -Module001 gave them to detect their new part INP-N. In the process of improving part (BBa_J364000), we encountered the problem of not being able to successfully build the carrier INPNC. We asked them about the experience of building INP-N. They suggested that we add a linker. If it is not added, it will easily lead to the frameshift of the subsequent reading frame. They also provided us with their INP-N. At their suggestion, we sequenced the entire plasmid.
SZU-China tested the suicide mechanism of the toxic protein expressed by the T7 promoter this year, but they encountered many problems. For example, the endogenous proteolytic enzyme of E.coli will hydrolyze the protein after the expression of the toxic protein, resulting in a low expression of the detected protein. This year our project also designed a suicide switch to lyse E.coli. Therefore, we recommend that they use BL21 strain which was modified from DE3 to reduce the hydrolysis loss of protein. We advised them to increase the induced concentration of IPTG. We also offered new ideas. Another possible reason is that IPTG is a strong inducer, which probably makes proteins incorrectly folded because of proteins expressing too quickly, resulting in inclusion bodies and reduced protein activity. So we suggested that they could try to induce expression between 16 degrees Celsius to 30 degrees Celsius, prolong expression time, and reduce the production of inclusion bodies.

After hearing our thoughts on degrading antibiotics in sewage, they asked us what we planned to do to achieve it. We realized that we have not considered the application environment or going out of the laboratory. Based on their suggestions and our visits to the sewage plants afterwards, we decided to make an expired drug recycling bin.
When we encountered difficulties in the questionnaire analysis, we decided to conduct a video discussion with the students of CSU_CHINA to analyze the statistics in the questionnaire we posted. They suggested that we use SPSS analysis, and analyze multiple factors together in the analysis process. We also discussed with them how to carry out public participation on the elderly. They told us about their square dance activities in 2018. They said that although they did a good job, they did not do well in the news. As a result, they were not recognized by the judges during the presentation, and they suggested that we do more in the news promotion.
This year we intend to design a suicide switch. So we found ZJUT-China with rich experience, they provided us with an arabinose-induced cell autolysin gene (BBa_K2556051), one of the components created in 2018. After the experiment, we characterized the expression of the cleavage gene in different host cells, and plotted the corresponding growth curve (Figures 2, 4), suggesting that there is a problem of cleavage gene leakage expression in some competent states. We provided them with suggestions, and they also performed repeated experiments (Figures 1, 3) to verify the leaked expression of the lytic gene.

Figure 1 ZJUT-China DH5ɑ
Figure 2 UESTC-China DH5ɑ
Figure 3 ZJUT-China BL21
Figure 4 UESTC-China BL21
Our experimental data showed that the growth of both host bacteria (E.coli DH5α and E.coli BL21) was strongly inhibited when the inducer arabinose (final concentration 10mM) was added to induce lysin gene expression. The cell density (OD600 value) of E.coli DH5α was changed from 0.38 at 5 h to 0.14 at 8 h, a decrease of 0.24. The cell density (OD600 value) of E.coli BL21 (DE3) decreased from 0.32 at 4 h to 0.06 at 7 h, a decrease of 0.26. At the same time, our data also showed that the lysin protein cleaves the E.coli BL21(DE3) for about 5h. When the expression is more than 5h, E.coli BL21(DE3) re-grows at a certain rate. This phenomenon suggests that when using lysin protein to control the growth of BL21, special attention should be paid to the processing time and additional treatments should be added. We recommend that ZJUT-China pay more attention to the time period during which the lytic gene is functional and to discard the lytic gene during this time period. By changing the growth environment, the acid is used and the overheating condition makes the strain no longer have the ability to reproduce.
In this year's project, XMU-China used two parts of our 2018 BBa_K118022 (Cex) and BBa_K118023 (CenA), but encountered difficulties in the expression of these two parts. At CCiC, we communicated on how these two parts expressed. And we gave them a reference of the enzyme activity detection for two cellulase, Cex and CenA. They performed a Congo red test to determine the enzymatic activity of CenA and performed a MUC enzyme activity test under a modified protocol to determine the enzyme activity of Cex.
Our project this year wants to degrade antibiotics. In order to understand the source of antibiotic pollution, we went to the fourth water purification plant in Chengdu. At CCIC, we learned that USTC's project this year is Azo Dye Degradation. We shared the information we found in the water purification plant. Currently, the water purification plant in Chengdu uses the MP-MBR system, in the MP-MBR system, activated sludge is used, and microorganisms in the activated sludge are used for degradation, and the MP-MBR membrane system filters out pollutants larger than 0.1 μm and most bacteria. And ask them to help us investigate the treatment technology of the local water purification plant. They told us after the investigation: The water treatment plant which they visited used microorganisms to degrade the chemicals, and ozone to oxidize the azo dye molecules.

And we shared the experience of team building with them, including project selection , division of labor, etc. We decided to make more exchanges on Giant Jamboree to lay the foundation for the next year's project.
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