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.
Logic gate
In order to validate the feasibility of the logic gate part, we designed experiment from the following aspects.
1.Qualitative experiment
We set 4 experimental groups and 1 control group to validate the logic gate qualitatively. The control group is MG1655 without plasmid that contains the logic gate circuit transformation. The 4 experimental groups are MG1655 with target plasmid transformation. And the treatments are: 1) adding nothing, 2) adding IPTG, 3) adding ARA, 4) adding both IPTG and ARA.
By comparing the data from these 5 groups, we can find if there is any leakage of the two promoter, and validate the circuit.
2.Quantitative experiment
We set a series of concentration gradient for ARA and IPTG from 10-7M/L to 10-3M/L. The specific setting is as follows.
The group that neither ARA nor IPTG is added is set as the control group, in order to proof the rigor of the logic gate, and explore the minimum concentration to trigger the expression of the circuit.
3. The exploration of the response time
In order to explore the response time of the logic gate circuit, we change the time of adding ARA and IPTG. We set several experimental groups at one-hour intervals, and measure the fluorescence intensity to find the response time.
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
