Team:XMU-China/digitaldisplay

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Project

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Inspiration:

	Figure 1: A traditional electric seven-segment display (showing number "0")

Synthetic biology is expected to design and construct new biological systems not found in nature. It 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[4], oscillator and repressilator[5] 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 device, a device which can be observed not only by scientific equipment, but by our own eyes. 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.

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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 and 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 (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 (As shown in Table 1). 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).

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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.

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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: P_BAD - Arabinose, P_cI - CI, and P_tetR - TetR. These computation units act as genetic logic gates perform AND, OR and NOT gates function. 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.

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Future plan:
For our current achievement, ...
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[2], 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[3].

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