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.

Notes
    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.


Logic Gate


Overview
    We simplified the three-plasmid AND gate system based on our reference by constructing all the segments in one plasmid. And we tested it in E.coli MG1655. We used arabinose promoter (PBAD) and lactose promoter (Plac) as two inputs and we have verified the corresponding response concentration and time.

    
1. Construction of Plasmid：

                The plasmids:

Input
Output
Intput+Output

        The construction:

        
2.Precision of the Logic Gate

        We did four parallel experiments: the bacteria solutions were added by IPTG of 10^-4mol/L, arabinose of 10^-3mol/L, both of them, and neither of them. The following picture is our result.
        As is shown in the figure, solutions with no inducers or only one inducer can produce little GFP fluorescence which verify the precision and feasibility of the AND gate. But, GFP fluorescence is high when only adding IPTG. This may because the leak of Pbad. Considering the principle of AND gate, it is reasonable. Only the protein T7ptag can activate the gene controlled by T7 promoter, though T7ptag has two amber mutations, some E.coli endogenous tRNA may also identify the stop codon, which causes the leaking of Pbad seemingly.

        
3.Response Concentration：

        Next, we designed experiments to verify the response concentration. Through these experiments, we can also conclude under which concentration can the AND gate have the best effect. We designed a series of gradient about how many inducers should be added. The concentration of IPTG and arabinose varied from 10^-3, 10^-4, 10^-5, 10^-6, to 0. Following is the result.
        Seeing from the right figure, with the increase of the inducers’ concentration, the fluorescence intensity also increases. This figure has met our expectation. As the absorbance value (shown in the left figure) indicates the amount of bacteria, we can make sure that the increasing of fluorescence intensity is not caused by the bacteria growth.

        
4.Response Time：

        We measured the sample every certain period after adding inducers, and make the response curves in the figure. We found that after adding arabinose and IPTG, the fluorescence intensity satisfies a linear growth curve. However, the fluorescence intensity of the group with no inducers flattens after 2 hours. The difference of fluorescence intensity between two groups becomes apparent after 2 hours. Thus, we estimate that the response time of logic gate is around 2 hours.

        
5.Analysis of Result

        In general, we have verified the feasibility of logic gate. We successfully simplify the three-plasmid system based on our reference, and obtain a good result. After adding two inducers, fluorescence value changed apparently. Its response time is around 2 hours. After coupling with the light-induced system, the overall response time can be matched up with the oscillation period.
        Meanwhile, logic gate system has some problems. Firstly, T7ptag has a high possibility of leak, possibly caused by pBAD, or by E.coil’s endogenous tRNA.
        Secondly, we integrate the logic gate input and output parts together in a single plasmid. And these two systems may have mutual negative effect.
        Thirdly, the logic gate plasmid is relatively large and according to the references, the tRNA we used is toxic and may cause damage to the cell. That makes our bacteria grow relatively slow.

        
6.Future Plan

        (1)Change Promoters. Due to the inequality of these two circuits, we have constructed a plasmid (Bba_K1886016) to change the position of plac and pbad promoters. After verifying the changed logic gate, we could learn more about its precision. And thus, we can explore the influence caused by input circuits towards the whole logic gate.
        (2)Integrate with the light-induced system to verify the cipher machine’s function (first generation).We have already constructed a plasmid using light-induced promoter to replace an existent one. Next, after co-transformation with bacteria, we can verify the response time of the integrated system (light-induced + logic gate), and thus, verify the cipher machine’s basic function in its first generation.
        (3)Integrate with the light-induced system, and the oscillation system, to verify the function of Enigma.We have already constructed a plasmid (Bba_K1886010，Bba_K1886011，Bba_K1886012，Bba_K1886013) for Enigma. Our next step is to verify its function in the microfluidics device.

        
Notes:

        1.We use MG1655 as the chassis, which contains the Lac operon system and Arabinose operon system. If you use a strain does not contain these systems, you need to add the corresponding repressor protein into the circuit when constructing plasmid .
        2.Make sure A600nm is more than 0.3 before you add sugar into the bacteria solution.
        3.Note that LB has very high absorbance, when you detect the fluorescence intensity you’d better use PBS to suspend bacteria.
Oscillation


        To describe the single-period oscillation system in a better and more precise way, we measured pluxR promoter’s response states towards AHL of different concentration. By using PCR and one step cloning, we removed luxl gene (fig1a, fig1b), and thus the plasmid, pTD103luxI-sfGFP, was mutant. In this way, we excluded its interference to our experiment.
figure 1a
figure 1b


        After removing luxl gene, we combined the plasmid with its specific binding inducer and conducted PCR. The results verified that we had successfully removed luxl gene in structure. After comparing the results being induced by no AHL and AHL of 10^-6M, we verified we had successfully removed luxl gene in function. Also, it had the corresponding AHL and the ability to express sfGFP.
        We did inducement experiment after constructing pluxR-sfGFP circuit. We designed a gradient from 10^-6 to 10^-11M. After a night of inducement, we harvested the cell and resuspended in PBS (pH=7.2). Also, we did two parallel experiments for each gradient, and measured the fluorescence intensity. Following is the relational graph of fluorescence intensity and the concentration of AHL (fig2).

Figure 2


        We used hyperbolic curve to analyze the result. We found that pluxR began to be activated when the concentration of AHL was about 10^-9M, and began to saturate when AHL was about 10^-6M.

        We have figured out the relationship between pluxR and AHL, and our next step was inserting single-period oscillation system into MG1655, verifying the oscillation system. After being cultivated for a whole night, the co-transformation bacteria was transferred into the 100 ml LB medium with the proportion of 1:1000. When reaching OD0.1, we took out 1ml sample every 10 minutes, diluting to the same OD value and measuring the fluorescence intensify. Following is the result (fig3).

Figure 3


        Although we observed periodic oscillation, it became unstable gradually due to the increase of bacteria quantity and the accumulation of AHL.
        We designed the microfluidics device to solve this problem and repeated our experiment. We took pictures every 5 minutes and supervised the change of fluorescence intensity in each chamber. Following is the result (fig4a).


Figure 4a


        We then could observe a stable oscillation, and the period was about 3h. We used image to measure the fluorescence intensity and drew the oscillation curve (fig4b). Seeing from the figure, we could find the oscillation was stable enough.


Figure 4b


        After the single-period oscillation was proved, we tried to test the double-period oscillation system. We inserted double-period oscillation system into MG1655. After being cultivated for a whole night, the co-transformation bacteria was transferred into the 100 ml LB medium with the proportion of 1:1000. When reaching OD0.1, we took out 1ml sample every 10 minutes, diluting to the same OD value and measuring the fluorescence intensify. Following is the result (fig5).

Figure 5


        Due to the limited time, we did not realize double-period oscillation. However, after comparing our data of these two oscillation systems without microfluidics condition, we can speculate that in the microfluidics chip, the stable double-period oscillation can be realized.         
Future work:

        1.Realize the double-period oscillation system in the microfluidics chip. And then couple these systems together: the light-induced system, the logic gate system, and the oscillation system.
        2.Decrease the device’s scale in order to get a more stable oscillation. Also, find solutions to solve the accumulation of bacteria and other unexpected problems.
        3.Explore the relationship between velocity of flow and the length of each period.
        
Notes:

        1.When measuring fluorescence intensity, the first step is to do re-suspension. Otherwise, LB will affect our results.
        2.AHL uses DMSO as solvent which is poisonous. We should improve the concentration of AHL and decrease the quantity of DMSO. When the volume fraction falls below 10%, we did not observe obvious differences.
        3.When using microfluidics device, don’t forget to add Tween20 of 0.075% in the medium to avoid bacteria being stuck in the chip.
        4.We recommend to use MG1655 because it grows fast and is the suitable engineering bacteria for this experiment.

Contact Us
Room 413,Biology lab center, Zijingang Campus
Zhejiang University, YuHangTang Road NO.866
Hangzhou, China
iGEM ZJU-China 2016 Team
igem_zjuchina_2016@outlook.com