Team:ZJU-China/Proof

Proof

Light Control

    we did co-transformation for the red light and green light system separately. the bacteria after co-transformation were irradiated by red or green light in our device, and we use sfGFP as reporter gene. By monitoring relative intensity of fluorescence, we could prove the system’s feasibility. Following is the result observed by multiple plate reader.


（The device we used for testing--designed by TEAM_XJTLU）



    In addition, to realize the second generation of cipher machine, we need to ensure that the light-induced system could be coupled with the oscillation system. That is to say, the response time of the light-induced system must be shorter than half period of the oscillation. Thus, we measured the system’s response time. Following is the results:


    Conclusion: After a series of experiment, we are able to prove the feasibility of the light-induced system. While there is a possibility of leak, the differences between the two groups can be measured by devices. Also, after our measurement of the response time about 3 hours, it falls within the oscillation’s regulating range, which is consistent with our modeling results. So far, we can prove that the concept of the second generation of cipher machine is correct.


Oscillation

    In order to realize the single-period oscillation, we design a circuit based on negative feedback mechanism (fig1), which is composed of gene luxI，aiiA and sfGFP.

    To prove the effect of this circuit, we implanted it in E.coli MG1655 and raised them in test tubes. Meanwhile, we monitored the change of GFP fluorescence intensity. Although we could observe oscillation, amplitude rose higher and higher. After analysis, we think this happened due to the increased bacteria population and thus, the resulting increased AHL accumulated in the medium.
    To get a stable oscillation, we designed a microfluidics device based on our project. In practice, we add bacteria solution to microfluidics chips, so each chamber is distributed with bacteria. Then, we add fresh medium with an appropriate velocity continuously, to provide adequate nutrition and take away the excessive bacteria due to proliferation and the accumulated AHL. By now, we ensure the system’s stability. We use fluorescence microscope to take pictures every 5 minutes and observe the stable oscillation. (Shown in the figures below:)

Views of the device

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