Difference between revisions of "Team:ZJU-China/Experiments"

    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 order to validate the feasibility of the logic gate part, we designed experiment from the following aspects.
             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.

             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.

             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.

             In order to understand and describe the state of the single period oscillation system better, we need to know the relationship between the pluxR response strength and the AHL molecular concentration. To this end, we designed the AHL concentration gradient from 10^-11 to 10^-6, and used it for the induction of bacteria that contain pLuxR-sfGFP report loop. After fully cultured, the fluorescence intensity was measured, and we can get the concentration of AHL -- fluorescence intensity diagram.
              In order to detect single-period oscillation system composed of pTD103aiiA and pTD103luxI-sfGFP, these two plasmids were co-transfected into MG1655.The selected positive clones were cultured overnight. We inoculated them in 96-well plates in 1:1000, culture under 37°C and check the fluorescence intensity of GFP, to monitor the occurrence of oscillation.
              By analyzing the data, we found that, because of the increase in the amount of bacteria as well as the accumulation of AHL, the oscillation will become unstable, and the whole system will collapse, too. In order to eliminate the influence of all above, we designed a microfluidic device. Microfluidic chip can provide sufficient nutrition for the growth of the bacteria and wash away excess bacteria and AHL, so that the system can run stably. Under the fluorescence microscope, we captured pictures every 5min.
             In order to realize the double periodic oscillation, we also need to introduce another kind of auto-inducer into the oscillation system, we chose DPD, another auto-inducer in E.coli. We connected RBS with luxS to aiiA, which allows them to co-express. LuxS catalyzes the formation of DPD, so that the concentration of DPD in theory will be a half cycle difference with the peak of AHL, we named it as pTD103aiiA-luxS. PLsr is regulated by DPD promoter, it is followed by YFP as a reporter gene, so that we can monitor the change of the concentration of DPD, we named it as pTD103YFP. pTD103aiiA-luxS, pTD103luxI-sfGFP and pTD103YFP three plasmids constitute a double cycle system, sfGFP and YFP as reporter gene. We hope to observe the green and yellow light stable appear alternately, and each cycle length is fixed.
              As the method we used for the detection of single period oscillation, we put the treated bacteria solutio into the microfluidic device, and inject the fresh LB with proper flow rate, in order to stabilize the system. Under the fluorescence microscope, we captured pictures every 5min.