During the summer, we worked hard to build our system, which consists of mainly three parts, and we got some quite promising results. Here we show a list of our major achievements, including experiments, modeling, hardware, human practice and collaborations.
- We cloned several potential antibiotic degrading enzymes, including TetX monoxygenase (TetX) and MnCcP, and successfully expressed and purified them.
- After enzymatic catalyzed reaction of tetracycline (Tet), it is necessary to enrich the remaining Tet before any further test. Working with our Advisor Cheng Hu, we successfully established a solid-phase extraction (SPE) method using SupelTM –Select SPE (Sigma-Aldrich). This work shall to some extend, if not definitely, provide some guidance for future teams.
- We qualitatively and quantitatively measured the antibiotic degrading abilities of four enzymes, and found out the most efficient one, the TetX.
- By in vitro experiments, we characterized TetX comprehensively, getting its turnover number and its reactive kinetic properties.
- As a proof-of-concept, we showed that TetX can degrade Tet in vivo. To measure the degrading rate, samples were taken for 30 minutes’ interval, and the amount of remaining Tet were measured by LC-MS or UV absorption.
- We established a method of utilizing LC-MS and UV spectrophotometer to measure the degradation of Tet in a QUANTITATIVE way.
- We demonstrated that Mn(III) can also degrade Tet, providing a basic idea for future work.
- We successfully cloned 11 putative toxins onto a cloning vector, induced by aTc (anhydrotetracycline). We further cloned four antitoxins induced by IPTG.
- Among the 11 hypothetical toxins, 7 showed different levels of toxicity. We measured the growth curve of bacteria expressing the toxins. By doing so, we can evaluate the toxicity of varies toxins.
- We demonstrated that out of the four antitoxins cloned, three effectively neutralized their corresponding toxins.
- We conducted an online survey concerning with antibiotic residues and resistance. We got more 1,200 responses, which provided sufficient data for us to analyze the knowledge and opinions of the public. Besides collecting information from the public, we also get to the next generation. We talked with high school students, introduced our project as well as iGEM Competition and SynBio to them. Many of them should impressing interests and enthusiasm in these topics.
- We met with iGEMers from several universities including Peking University, Tianjin University, Beijing Institute of Technology, and Xiamen University, during which we discussed each other’s projects and problems occurred in experiments.
- We also attended this year’s CCiC held during 2nd-4th Sept. at Sun Yet-Sen University in Guangzhou, where we lectured on our projects and took part in the poster session.
- As a collaboration, we measured the growth curve of E. coli harboring toxin MazF by 96-well microplate spectrophotometer for the iGEM team from Beijing Institute of Technology. They are also working on toxins to build a kill-switch, so our measurement can be quite important for them.
In previous years, dozens of teams have worked on tackling antibiotic resistant bacteria, while a small number of teams paid attention to degrading antibiotics, and few team showed solid data on how much antibiotic can be degraded in their projects. So based on the achievement above, we believe we deserve a gold medal.