This year, we not only learned from our instructors the biotech skills of molecular biology such as gene cloning, protein analysis, spectrophotometry, also we were sponsored and helped from a local biotech company, BIONIME Corp (and its new spin-off company, Zensor R&D Corp.) who is an expert and innovator of blood glucose meters and electrochemical analyzers.
In addition, we sought NCTU-FORMOSA as our mentor and advisors. They gave the BioBrick [BBa_K1694002: Lpp-OmpA-NcoI/pSB1C3] to us for designing and displaying our enzymes on the E. coli cell surface by Lpp-OmpA outer membrane proteins (They used it and the design for their iGEM project in 2015). We confirmed the plasmid by DNA sequencing and improved the existing BioBrick part by replacing NcoI site (There’s an extra NcoI site present on Cm resistance gene) with BamHI site to facilitate creating a fusion protein linked with Lpp-OmpA [BBa_K1991004: Lpp-OmpA-BamHI/pSB1C3].
On the other hand, we used the skills learned in the lab in Mingdao High School and have helped NCTU-FORMOSA 2016 to confirm a function of their Biobrick in protein analysis experiment.
By the way, NCTU-FORMOSA 2016 also helped us to examine our AOX enzyme activity. They proved the function of our Biobrick. Based on the results from us, NCTU-FORMOSA and BIONIME Corp., we firmly believe that our product will be applied in the real world in the near future.
On august 23rd, some of our members (Jiun-Jie Huang, Hsuan-Yi Lin, Shih-Chen Chen, Ming-Jui Tseng) went to the lab of NCTU-FORMOSA in National Chiao Tung University in Hsinchu and helped them to confirm a protein expression of one of their BioBrick.
We learned some experiment skills in molecular biology at Mingdao’s lab from our instructors. After discussion with NCTU-FORMOSA, we went to their place not only for learning more especially from an experienced iGEM team, but for doing them a favor by experiment skills we knew so far.
The following data was performed by us for a part of NCTU-FORMOSA’s project. We prepared E. coli BL21 with a plasmid of pSB1A3 carrying T7 promoter-driven Hv1a-lectin gene. When bacteria grew to OD600 around 0.6, 500uM of IPTG was added for induction. After incubation for 4hr, the lysates were collected and subject to SDS-PAGE with a 12% gel. And the proteins were observed by Coomassie Blue staining. (Lanes 1&2: samples without induction; 3: a sample with induction; 4: wild-type bacteria as control lysates. M refers to a protein marker).
Collaborating with another team definitely helped us improve our skills of doing experiments, especially when working with college students. By helping NCTU-FORMOSA team to do a part of an iGEM project, we once again proved ourselves that we are capable of doing lab experiments well although we are high school students. By working with NCTU-FORMOSA, we learned details of the experiment and got to know more about their project this year.
The following is the message from iGEM team NCTU-FORMOSA:
“To test the enzyme activity of AOX protein, which catalyzes the oxidation of alcohol and generates H2O2 products, produced by iGEM Team Mingdao, we performed HRP-TMB assay. TMB (3,3',5,5'-Tetramethylbenzidine) is oxidized and changed to a deep blue color during the enzymatic degradation of H2O2 by horse radish peroxidase (HRP). The concentration of H2O2 can be determined by checking the color change, indicating TMB assay can be used to examine the enzyme activity of AOX with the substrate of alcohol.
We cultivated 3ml of the E.coli DH5α carrying the Mingdao iGEM team’s biobrick (i.e., Pcons-RBS-LO-AOX/pSB1C3, BBa_K1991009) in the LB media supplemented with Chloramphenicol. And we prepared another one as the control which didn’t have any plasmid. The next day, we centrifuged 1mL of bacteria expressing LO-AOX enzyme followed by being suspended in PBS. The control group was adjusted to the OD values at the same level of the LO-AOX group. After mixing bacterial samples with ethanol, TMB was added as a substrate of HRP and incubated in the dark for 3 min. The color of solution was significantly changed to blue at an OD650 of 1.51 compared to 1.21 in the control group, clearly demonstrating the ethanol was oxidized by AOX. As a result, we proved the function of their BioBrick by testing the AOX enzyme activity.”
This year, our project was inspired by the Blood Glucose Meter (BGM). To develop a Blood Alcohol Meter (BAM) used in the sobriety test, we looked for a help from a biotech company, BIONIME corp. and its spin-off Zensor R&D corp, who are experts in designing and creating electrochemical analyzers.
On august 25th, the executive vice president of Bionime, Dr. Wiley Chung (Shie-Shiun Jung), came to our lab and taught us the electrochemistry and principles behind BGM. He also shared his successful story of creating an innovative BGM and gave valuable advices for us to design a blood alcohol meter.
Zensor Simulator & Zensor test strips
The data of cyclic voltammetry and amperometry
On that day, Dr. Wiley brought an electrochemical simulator designed and created by his research team and himself in Zensor R&D corp. He helped us to examine the chemical properties of AOX enzyme we prepared in our lab. By doing experiments in cyclic voltammetry and amperometry, we obtained data for a calibration curve based on alcohol concentrations. In the end, according to the experimental results, he told us that our products (enzymes) are functional and ready to apply in blood alcohol meter.
It’s definitely a big hit for us to collaborate with a biotech company, BIONIME. By the modeling experiments, we proved that the Alcohol Oxidase we created is functional and the design is practical. With the help of Dr. Wiley, we witnessed the whole process of developing a brand new product. The valuable collaboration not only gave us directions to improve our product, but also let us believe our design may become reality.
