Team:ZJU-China/Experiments
Light Control
To achieve the function of the first generation of our cipher machine, our bacteria should be sensitive enough to the light signal around specific wavelength long and output a fluorescent signal which should be strong enough to be detected. So we designed several experiments to prove the sensitivity and reliability of the two TCSs.
Firstly, we chose sfGFP as the reporter and exposed our bacteria to red or green light when culturing. After some time, we harvested all the culture and measured the fluorescent intensity .
What’s more , to make our cipher machine more complicated and hard to be cracked, we induced the oscillation circuit. But the key to couple these parts was to prove that the time our “light-sensors” need between sensing the light and output a signal was exactly shorter than the half-period of oscillation. So we tried to measure this delay time of the two TCSs. We also tried to stimulate this process by modeling.
Here we want to show our sincere appreciation to Team HZAU who had provided us with original plasmids the paper used, also to Team XJTLU who had designed the device for culturing the bacteria and measuring the fluorescent intensity.
In the green-light system, ccaS protein is fixed on the cell membrane. After combined with chromophore ho1 and pcyA, ccaS could absorb green light around 520nm wavelength long, and thus, its own phosphate group will transfer, resulting in the phosphorylation of ccaR, an intracellular protein. The ccaR then could activate the expression of PcpcG2 promoter. Using this system, optical signal can be used as input, and can be received by engineered E.coli. However, after being irradiated by red light around 650nm wavelength long, the phosphorylation transfer process (from ccaS to ccaR) will be inhibited.
After a discussion about our literature, we settled the size of each trap and the length, width, and height are 100 um, 85um and 5um, respectively, which is the most favorable one for oscillation experiment and dynamic response model. Two traps stand 150um apart at the same side, while for the opposite side, the groove is 200um wide and 100um deep which is convenient for fluid to pass. Also, according to our calculation, we are quite sure that this design can increase the flow of AHL quorum sensing substance, and can let the cells at the edge of expansion colonies be watered away, making it possible for bacteria in the traps maintaining a continuous exponential growth.For observing convenience, we divide the traps into 4 groups, and each has 30 traps. The distance between two groups is sixty thousands um.
Others
For overall experiment, we also designed a device to help us using the microfludic chip. As for the body part, the upper platform has four through-holes, for putting in the LEDs. The lower part has the two slots, size of each slot are the same as the microfluidic chip. And we have a shading baffle whose size is appropriate with slots. Devices and shading baffle are produced by 3D printing, the material we chose is resin. It has smooth surface, high precision, and good hardness. Taking into account that the device will be placed in the thermostat, we used a kind of resin that has small thermal deformation coefficient, this will ensure that assembling won’t be a problem. In order to ensure that the experiment will not be interfered by the stray light, all surfaces are sprayed with black matte paint.
The LED we used is a common cathode four-pin light-emitting diodes, we only use 1 (red), 2 (negative), 3 (green) three pins. The wavelengths were 630 ~ 640 nm and 515 ~ 525 nm, respectively. The brightness of the lights is around gigahertz candela. All of them can meet our experimental requirements
