Team:HFLS ZhejiangUnited/design.html
According to our previous study, interview and social investigation results, we planned to develop an efficient and affordable method that monitors the concentration of dissolved oxygen, total nitrogen and phosphorus. It would be used by publics, especially for farmers and aquarists. After collaborating a great number of related researches and reading a plenty of documents, we eventually discovered the VGB promoter, which has good sensitivity of oxygen. The VGB promoter happened to be one of the provided resources from the kit plates. Based on this discovery, our minds came up with a practical idea ——whether it could be possible for us to use the synthetic biological methods to construct an E. coli strain into a detector. With putting a huge amount effort on researching and instructed by professors, we got an answer: Yes! This will be possible!
In order to avoid the unnecessary problems that we might meet, the final products will be three individual strains of E. coli, with three different circuit for oxygen, nitrogen and phosphorus(figure1).
figure1.our design
The oxygen detector was designed to include the Pvgb promoter as a sensor and a GFP gene as a reporter. Different concentration of dissolved oxygen will be reflected in different amount of GFP in a single cell. For us, it will be different fluorescence intensity(figure2).
figure2. the oxygen detector
The nitrogen detector and phosphorus detector are basically the same as the oxygen one. The nitrogen detector uses the PyeaR promoter as a sensor and a BFP gene as a reporter, in the meantime, the phosphorus detector uses the external phosphate sensing promoter as a sensor and a RFP gene as a reporter(figure3,figure4).
figure3. the nitrogen detector figure4. the phosphorus detector
Despite all the tedious constructing work, the latter two designs have the same pattern “Sensor-Reporter” as the former one. Not only does it help when new detectors are designed and engineered, it also turn the constructing work into a modularity one(figure4).
As experiments of constructing these three plasmids were carried forward, we meet a problem that can’t avoid: the ordinary expression efficiency of GFP promoted by Pvgb would take up to 8-10 hours, when it is enough for people to see the fluorescence without using a fluorescence microscope. Fortunately, with great help from Dr. Zhu, we improved our circuit using T7 RNA polymerase gene and PT7 strong promoter to enhance the expression of GFP. The artificial gene cluster now contains a Pvgb promoter, a T7 RNA polymerase gene, a PT7 strong promoter and a GFP coding gene. The Pvgb promoter will not directly control the expression of GFP, but the T7 RNA polymerase instead. The latter fits with PT7 promoter and has a much higher expression efficiency that other combinations cannot match. We expected that the GFP, which is indirectly regulated by Pvgb promoter, will have a much higher expression efficiency.
To be differentiated from the others, we call this improved circuit ‘Oxygen 2.0’, and the previous circuit is called ‘Oxygen 1.0’(figure5).
figure5. “oxy2.0”
While the test results for Oxygen 1.0 and Oxygen 2.0 did not satisfy us, we designed the circuit ‘Oxygen 3.0’. The first half part is totally the same as the ‘Oxygen 2.0’, while we add another half part, including a tetR coding gene, a Ptet promoter and an amilCP coding gene. tetR protein is specific to Ptet promoter and will combine on it, preventing the polymerase from transcribing the downstream gene. And the amilCP is a dark-blue protein which is rather easy to observe. We hope that, when in a mionectic environment, the expression of BFP and tetR will be strong which will cause the inhibition of Ptet promoter activity and weaken the expression of amilCP, and when in a hyperoxia environment, the expression of BFP and tetR will be weak and the one of amilCP will be strong. Different levels of dissolved oxygen is expected to be reflected in the color and fluorescence intensity(figure6).
figure6. “Oxygen 3.0”Design
