NEU_China_B & DLUT_China & DLUT_China_B 【Hosting Seminar and Meetup】
The NEU_China_A is proud to be able to host the first meetup of all of the iGEM Teams of Dalian University of Technology and Northeastern University this year at August, 27th 2018. By this great opportunity, we also collaborated with DLUT_China, DLUT_China_B, and NEU_China_B to hold a seminar. During this seminar, we had the chance to share each of team’s project, and discuss the specific schemes of the experiments. The seminar greatly promoted the smooth development of these four iGEM teams. At the end of the seminar, we had a discussion regarding the opportunity for collaboration in the next future and reached an experimental cooperation intention with the DLUT_China in kill switch section.
【Optimization of protein expression conditions and product purification for AHUT_China】
During the online communication with AHUT_China, we learned that they were distressed by the optimization of CSCA protein expression conditions. It happens that our team was also optimizing the expression conditions of the Myrosinase, which has contributed to our cooperation in the optimization of CSCA protein expression conditions and product purification for AHUT_China. We tested the expression efficacy of CSCA at different temperatures, different induction time, and different IPTG concentrations. By comparing the protein expression level, we determined that the best induction condition was induced at 37 °C, 0.5mM IPTG for 6 h. After induced by IPTG, CSCA protein was detected by SDS-PAGE and Western blot, which suggested that the CSCA protein was expressed with molecular weights of about 60 kD. Since the N-terminal of CCA carries a 6 x His tag, the expression product was purified by passing the Ni+ affinity chromatograph column, which ultimately helped AHUT_China to obtain 1 mg of purified CSCA protein with a purity greater than 85%.
【Data comparison of kill switch】
Security issues have always been a very important part of the iGEM competition. This year NEU_China_A and DLUT_China have worked together in the kill switch section. From the proposing the idea of a kill switch, the choice of lethal system, the construction of the plasmid vector to the characterization of the part, NEU_China_A and DLUT_China cooperate with each other. After discussion between two teams and with reference to the experience of the previous iGEM team, we choose the maz-EF system and Lysis two different systems. Two characterization methods were used to verify the function of the kill switch, one is plate induction and the other is a growth curve.
NEU_China_A and DLUT_China compared the lethal efficiencies of two different kill switch systems. The two teams verified the efficiency of cold shock expression of Lysis and mazF by measuring the growth curve under the same experimental conditions. Our data indicate that due to the high toxicity of mazF, even low expression of toxic proteins is sufficient for cell death. In contrast, the cold shock expression Lysis showed a better kill switch property. In vivo at 37℃, low expression of lysis is not enough to cause functional probiotics to die, while short-term (<4h) cold shock induction in vitro can fully inhibit cell growth. In short, cold shock expression of Lysis as a kill switch is a better choice.
AHUT_China & SDU-CHINA & NWU-China
Collaboration is always about exchanging ideas and we believe that collective thinking can do a lot of good for iGEM projects. NEU_China_A have participated in a virtual meet-up with AHUT_China, SDU-CHINA, and NWU-China. In the virtual meetup, our four iGEM teams mainly exchanged the progress of each of team’s project, especially the problems encountered in the experiment, the progress of the modeling work, the interpretation of the requirements of the awards, etc.
Jilin_China used sfGFP_optimism as reporter protein in their gene circuit this year. The existing sfGFP in the iGEM registry Kit has a BbsI restriction site and lacks ease of application. Therefore, this year Jilin_China optimized the sequence of sfGFP, and the optimized sfGFP no longer contains BbsI restriction sites, so sfGFP_optimism can be conveniently applied to GoldenGate assembly. To verify the necessity for sfGFP optimization, NEU_China_A sent experimental materials:superfolder GFP (BBa_I746916) in the Distribution Kit to them. NEU_China_A utilized blue pigment protein（amilCP）as reporter protein after detecting NO signal this year. In return, Jilin_China very kindly sent Nitrate reporter: PyeaR-GFP composite (BBa_K381001) and Test Device 4 for interlab (BBa_J364007) to our team. After constructing the plasmid, we verified by experiments that the blue protein can be expressed normally after exposure to NO.
This year our project requires the use of the intestinal probiotic, Escherichia coli Nissle 1917, as the chassis organism, but this strain is not a common strain in the laboratory. Knowing that JNFLS has relevant strain information, we turned to the JNFLS iGEM team for help. Fortunately, they sent it very generously to us with scribing plates and glycerol cryotubes of Escherichia coli Nissle 1917 so that we can use it in the future.
At 5th CCiC, we learned that SJTU-BioX-Shanghai intends to develop new methods for diagnosing colorectal cancer, which is closely related to our project. Our two teams conducted in-depth exchanges on our respective projects. Coincidentally, they also use NO as a signal molecule. In order to compare the efficiency of different NO sensors, we asked them to send us a plasmid which containing promoter PyeaR and GFP so that we can verify the ability of NO sensor to respond to NO. In addition, they mailed us the colorectal cancer cell line HT-29, which is used to characterize the anti-cancer function of Myrosinase.