Team:ZJU-China/Results

Result

Light Control

    We have successfully constructed plasmid used in the light-induced system and achieved co-transformation in E.coli JT2. After then, we have tested the light sensitivity and the response time.
    Construction of Plasmid
        (1) The construction of Part




        (2) The green light system


        (3) The red light system


        (4) The result of co-transformation


     We can conclude that under ultraviolet, bacteria would emit green fluorescence.
    About response


     We measured our system’s ability to produce fluorescence signal under different conditions of light. Due to time limit, we measured the green light system and the red light system without the NOT gate. It can be observed that these two systems had obvious leak. But after a series of experiments, we could observe stable differences both after irradiated by light and in the dark. Also, the green light system is more stable than the other one. However, compared with our reference, we did not test the obvious constraint on the red light.
    Delay


     We observed that the sharp increase of fluorescence mainly happens after 3 hours. And this sharp increase is really a helpful index for us to test the system. Thus, we think the response time of TCS is between 3 to 4 hours.     In general, we have verified that these two systems are feasible. However, they did not match up to our expectations, and the results seemed far different from our references. Following are the possible reasons:
    1、The results can be affected by the copy number of plasmid and the replicon’s intensity, especially for the red light TCS.
    2、Maybe this is because the LED intensity was not strong enough. Also, we used LB medium and it absorbed light of different wavelength, making light attenuate too much and could not activate TCS effectively.
    3、The antibiotics we used may affect the system’s expression.

Future plan
    1、Decrease the leak level
         In the light-induced system, the reporter gene’s leak will affect our measurement. What’s more, it may even lead to unexpected errors for users to read the information. In order to avoid such errors, we have several alternative plans:Use a new light-induced system - we have already found a system using blue light. Keep balance between every components by changing the intensity of rbs. Keep balance between every components by changing the copy number of plasmid and the replicon’s intensity.
    2、Future measurement of the red light system after inserting NOT gate.
    3、Mix bacteria of the green light and red light system together, and measure their response towards light.
    4、Analyze the response curve, to find the optimum light intensity.
    5、Replace LB medium as M9 medium and compare their results.
    6、Explore the optimum quantity of antibiotics.

The key points for repeating experiment
     1.When constructing plasmid, with the existence of double terminator, it is prone to appear disorder when using overlap pcr to join two segments together. Using seamless connection to construct plasmid is also prone to appear deletions of segments, and we can consider to use the method of enzyme-digestion and enzyme-connection.
    2.When measuring the fluorescence expression of bacteria after co-transformation, the results are affected by medium. Thus, we recommend to centrifuge first, then use PBS buffer solution to clear the remaining medium. These two steps can be repeated by 2 times in order to preclude the effects caused by medium.


    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.


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