Team:XMU-China/digitaldisplay

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Contents[hide][show]
  • Inspiration
  • Basic principles of digital display
  • Luminescent method: GFP with LVA tag
  • Circuit construction
  • Results and analysis
  • References
  • digitaldisplay

    Digital Display

    1. Inspiration:

    Synthetic biology is expected to design and construct new biological systems not found in the nature. This nascence field of science makes it possible to "build" living machines from off-the-shelf chemical ingredients, employing many of the same strategies that electrical engineers use to make computer chips[1].
    By analogy with electric circuits, many synthetic circuits like toggle switch[2], oscillator and repressilator[3] have been constructed. XMU 2012iGEM team also intended to construct biological circuits based on electric ones. However, not like those basic parts, we concern more about a REAL MACHINE, just like the full name of iGEM. The genetic engineered machine we construct should function in a visualized manner. Yes, we intended to construct a biological display device.
    Compared with electric display, biological display shows great advantages in many aspects: 1) Input signals: Unlike the only signal electrons in electric circuits, there are various types of input signals, making it possible to perform parallel computation; 2) Inspired by traditional logic gates in electric circuits, we assembled existed biological logic gates into synthetic circuits working as a genetic Seven-Segment Display. As shown in Figure 1, a seven-segment display requires seven elements to arrange. Individually on or off, they can be combined to produce simplified representations of the Arabic numerals. Traditional displays usually convert chemical or biological signals into electric signals as outputs. However in many other situations, especially in trace analysis of biological signals at microscale level, research has consistently shown that signal converting and transmission is still a tough problem.

    Figure 1. A traditional electric seven-segment display



    2. Basic principles of digital display:
    In our project, we utilized tubes with different kinds of bacteria as seven segments. According to the theory of network design, genetic logic gates were assembled into circuits and then constructed using synthetic biology method. Based on the knowledge of bioinformatics and synthetic biology, there are only two results for a single logic gate: on or off. Here, instead of electric signals representing streams of binary ones and zeros, the chemical concentrations of specific DNA-binding proteins and inducer molecules act as the input and output signals of the genetic logic gates, i.e. present or absent of a specific signal molecule represents binary one or zero. The genetic circuits we constructed actually using Binary-Coded Decimal (or BCD, a device which converts the binary numerical value to decimal value) to compute signal molecules. To display two results (in this case, number one and zero) necessitates one kind of signal molecule, we called it a Single-input BCD Decoder (As shown in Table 1). The inducer is arabinose, which effect on promoter PBAD (an E.coli promoter, see more[4] ). In the absence of arabinose, cI regulated promoter(PcI) expresses GFP with LVA tag. In the presence of arabinose, PBAD starts transcription and then repressor CI protein is translated and represses PcI. The number of displaying results determines how many kinds of input signals we need, i.e. the kinds of inducers. More specifically, if there are N pairs of inducers and their corresponding regulators, we can get 2N kinds of results (As shown in Table 2).



    3. Luminescent method: GFP with LVA tag
    The device utilizes green fluorescent protein as luminescent source. The use of the fluorescence protein (GFP) from the jellyfish Aequorea Victoria is ubiquitous in the field of biological signal protein and become widely applied in situ monitoring and other researches. But traditional mutated GFP tends to be very stable, leading to a long degradation time, being unfavorable to the reuse of the device. A novel type of GFP solves the problem with its specific C-terminal oligopeptide extensions which can render stable proteins susceptible to degradation by protease. The project applies GFP with LVA tag, which is one of unstable types of GFP and presents fast degradation and relatively short half-life. Equipped this kind of GFP, the device could light up and die out in a fast speed, therefore becomes available to be repetitively used.



    4. Circuit construction:
    In order to display the number related to the environmental substances investigation, the device should contains different combinations of tandem and parallel logic gates. As mentioned in explanation of principles, input signals are inducer molecules, functioning as the switches of protein expression in circuit. Output of the circuit is GFP with LVA tag.
    We assembled several pairs of promoters and their activators (or repressors) into computing blocks of the circuit: PBAD -Arabinose, PcI -CI, and Ptet -TetR. These computation units act as genetic logic gates perform AND, OR and NOT gate functions. Table 3 presents the design of device displaying, in a way which we called Dual-input BCD Decoder. Once the construction is completed, Engineered E.coli with different circuits can be immobilized into microcapsules and then be put in seven transparent tubes which acting as segment of display.



    5. Results and analysis:
    5.1 Optimization of the inducers concentrations
    In order to achieve a better visual impact, first thing we concerned is to optimize conditions for the display device. Concentration of inducers is a crucial factor effecting on both fluorescent intensity and response time. Therefore, the first set of analysis examined the impact of the concentration of added additional inducers on circuit.

    Part PBADGLT (Part:BBa_K750000, short for PBAD-rbs-gfp(LVA)-tt)
    From Figure 2 we can see that fluorescent intensity increase with the concentration of arabinose increase. By comparison the three curves, we drew the conclusion that 1mM group shows the best fluorescent performance, for its significant up and down, high peak value and little fluctuations. The optimized concentration (1mM was applied in following experiments without additional explanations.

    [click to data]

    SDS-PAGE was also applied as another characterize method to verify the function of arabinose. As can be seen from Figure 3, the expression of GFP is significant at peak time(75 min after induction) and the degradation caused by unstable peptide tails also worked as desired. Fluorescence value of the same sample has also been analyzed and showed on the right, which presents a great correlation to the result got by SDS-PAGE.

    [click to data]

    Part PtetGLT (Part:BBa_K750114, short for Ptet-rbs-gfp(LVA)-tt)
    Similarly, another experiment has been conducted to optimize the concentration of adding anhydrotetracycline(aTc). As can been seen in Figure 4, the result after inducing are both significant, which 70 ng/mL shows better properties for the device: fast response as well as fast degradation. Therefore, 70 ng/mL became the optimize concentration for the device, in spite of the higher peak value reached using 140 ng/mL.
    Another thing worth to be mentioned is that one part in the Distribution Kit seem failing to work as expected. The trouble circuit is Part BBa_K145280, consisting of a TetR generator and a GFP with LVA tag under control of a TetR promoter. It is supposed to be able to response to the addition of (anhydro)tetracycline. However, almost no fluorescence can be observed or determined after the addition of aTc. At first, we suspected that aTc we use might be the cause for the problem, for its unsuitable concentration (140 ng/mL, as the designer mentioned on their wiki). But further experiment showed that all gradients of concentration also failed (250, 25, 0.25, 0.025 ng/mL). We even suspected that aTc itself might be ineffective due to inappropriate storage or preparation. Then we noticed that the strength of RBS in front of gfp is 0.3, a medium level, which may lead the low expression and finally counteracted by fast degradation. Consequently, we constructed a new circuit, changing the strength of RBS from 0.3 to 1.0 and successfully obtaining satisfying data as mentioned above.

    [click to data]



    5.2 Functional assay
    Once obtained the optimized conditions, our next step is to confirm the function of circuits we constructed. Figure 5 presents the result obtained from fluorescent test on two circuits used in Single-input BCD Decoder. As can be seen from the graph, both two circuits function as expected. PcIGLT (Part:BBa_K750103, short for PcI-rbs-gfp(LVA)-tt) continually expresses GFP while PBADGLT (Part:BBa_K750000) shows a significant up-and-down after adding arabinose.

    [click to data]

    But when moving on to other more complicate circuits in Dual-input BCD Decoder, we encountered a thorny problem. Synthetic NOT gate in PBADcIT-PcIGLT (Part:BBa_K750113, short for PBAD-rbs-cI-tt-PcI-rbs-gfp(LVA)-tt) and PtetcIT-PcIGLT (Part:BBa_K750114, short for Ptet-rbs-cI-tt-PcI-rbs-gfp(LVA)-tt) failed to function, which is supposed to express GFP without any inducer. However, no fluorescence can be observed or detected. This result may be explained by the possible leakage of expression of CI repressor protein. In order to verify our suspicion, we performed SDS-PAGE to detect the existent of CI protein. We use the front parts of the circuit as new parts, i.e. PBADcIT (Part:BBa_K750104, short for PBAD-rbs-cI-TT)
    But we got no result from the SDS-PAGE, it is possible to hypothesize that the SDS-PAGE is not so sensitive to detect the low concentration of CI protein, since we have no time to transfer the strain from DH5α to BL21. We did further experiment using an additional group of another part with lower strength of RBS: PBADcIT-0.07 (Part:BBa_K750107 , short for PBAD-rbs0.07-cI-tt). The aim of constructing this new part is to reduce the hypothetic leakage of CI expression. If succeed in this function, the new part could then be assembled with the downstream part, functioning as genetic circuits mentioned before. Additionally, the experiment conducted on strains that had been transferred into BL21 earlier, to prevent the low expression level due to the defection of DH5α. By comparing BL21 group and the group shown in Figure 6, no leakage of CI protein can be observed, though changes in the strength of RBS are effective.


    Another assumption is that the design of circuits might be unstable in E.coli, causing the totally failure of the function. Due to intrinsic uncertainties and extrinsic disturbance, this situation reported happening lot of times, much more than one can expect. To examine the circuit, a similar circuit has been constructed, where cI gene was changed into gfp gene at the downstream of PBAD. As shown in Figure 7, PcI is working and expresses GFP in the group without arabinose. For another group, after addition of arabinose, PBAD is activated and GFP in its downstream therefore start expression, resulting in a higher peak value of fluorescence. From the data, we got a preliminary result that the circuit is able to work, though without consideration about the individual difference. However the conclusion is too early to be drawn, further researches are badly needed. We intend to exchange the position of two subparts (i.e. PcIGLT-PBADcIT instead of PBADcIT-PcIGLT), but due to time limitation, this intension can only be placed in our future plan.

    [click to data][click to result]

    For our current achievement, we have great ambitions to construct Multi-input BCD Decoder and widely applied in many fields. See more on our future plan page.
    To solve the problem we encountered this summer, more research works are badly needed. As David Sprinzak and Michael B. Elowitz mentioned at the beginning of their paper [5], to answer fundamental questions about existed circuits, one could design and build new circuits using similar components. The main goal of this nascent field of synthetic biology is to design and to construct biological systems and study their dynamics in organism[6].



    6. Reference:
    [1] S. B. Ron Weiss, Sara Hooshangi, Natural Computing 2003, 2, 47-84.
    [2] T. S. Gardner, C. R. Cantor and J. J. Collins, Nature 2000, 403.
    [3] M. B. Elowitz and S. Leibler, Nature 2000, 403, 335-338.
    [4] R. Schleif, Trends in Genetics 2000, 16, 559-565.
    [5] A. Eldar and M. B. Elowitz, Nature 2010, 467, 167-173.
    [6] B.-S. Chen and C.-H. Wu, BMC Systems Biology 2009, 3, 66.
    [7] http://2008.igem.org/Team:KULeuven