Team:XMU-China/background

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Background and Approach

1. Logic Gates
A logic gate is an idealized or physical device implementing a Boolean function, that is, it performs a logical operation on one or more logic inputs and produces a single logic output^[1] .

 

AND gate INPUT OUTPUT A B 0 0 0 0 1 0 1 0 0 1 1 1

OR gate INPUT OUTPUT A B 0 0 0 0 1 1 1 0 1 1 1 1

The AIM of our project is to construct a fluorescent digital display device of genetic circuits with synthetic logic gates. The finished device will display and switch numbers. Similar to electronic circuits, logic regulation operation in cells integrates the extracellular and intracellular signals. We assembled several pairs of promoters and their activators or repressors into computing building block of the circuit: P_BAD-Arabinose, P_cI-CI, and P_tet-TetR. These computation units act as genetic logic gates perform AND, OR and NOR gate functions. 

(Alvin Tamsir , et al. Nature, 2011)

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2. Green Fluorescent Protein
The green fluorescent protein (GFP) is a protein composed of 238 amino acid residues (26.9kDa) that exhibits bright green fluorescence when exposed to light in the blue light wavelength range^[2]. Considering that the common types of GFP usually tend to be very stable and hard to "quench", we chose unstable GFP to make our device reusable and convert in a fast speed. This kind of GFP is tagged with a C-terminal extension that will be recognized and degraded by tail-specific proteases, leading to a short half-life and fast degradation of the protein. Degradation rate should be confined at a suitable range; otherwise the device would either have no light or take a long time to change numbers.

GFP crystal structure (Ormo, M., Cubitt, AB, Kallio, K., Gross, LA, Tsien, RY, Remington, SJ (1996) Science 273, 1392–1395)

(K., Gross, et al. Science, 1996)

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3. Cell Immobilization
It is a technique to fix cells in a suitable matrix. Immobilized cells have been employed to perform biotransformation and reported to have higher production rates than freely suspended cells. In our project, we have selected three methods to immobilize our engineering bacteria:

(1)Entrapping cells in calcium alginate beads;
(2)Entrapping cells in intra-hollow Ca-alginate capsules;
(3)Embedding cells in PDMDAAC-NaCS microcapsules.

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4. Quorum Sensing
It is a method of communication between bacteria that enables the coordination of group-based behavior based on population density^[3].

(Melissa B. Miller, et al. Annual Review of Microbiology, 2001)

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5. Time Delay
Constructing a unique display device which can let each unit light up one after another is beneficial for our team to accomplish the aim of time delay, which means that it is necessary to construct a series of cells those can express the characteristic of different time delay. As a result, we take advantage of quorum-sensing system to change the time delay of GFP expression via different intensity of RBS.

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6. Original Design
In reality, we selected this project from the first brainstorm which was put forward by the members of Group 2 and its previous name was Bioscreen, as you see, which turned to be E.lumoli nowadays. To gain more details about the original task, please link to:

Brainstorm: Contents: Group 2: Bioscreen

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7. Project Overview
Under usual condition, traditional seven-segment displays acted as a number “8” need 7 tubes. Through different combinations of the tubes, the device can display different numbers or characters. Actually most modern displays are controlled by electric signals which are converted by chemical or biological signals. But in several situations, especially in detect of trace amounts or minute amounts of biological signals, we found that it is difficult to transfer and display the results. Besides, the relevant parameters which are controlled in real time in some fields of industrial application are hard to be the same as the reality. The aim of our project is to design a shining synthetic device for digit or time-course display which can replace the traditional methods.

Fortunately, 2012 XMU iGEM team has constructed a fluorescent digital display device with synthetic logic gates, which is able to respond to signals by displaying and switching numbers. We put GFP equipped with degradation tags in downstream to illuminate our numbers and change them quickly as well. Considering our engineering background, we accordingly employ cell immobilization to build our device. Engineering bacteria have been embedded in intra-hallow calcium alginate microcapsules and in PDMDAAC-NaCS microcapsules respectively. In addition, 3D CAD design is performed for a perfect device.

Our genetic circuits vary in length and RBS strength, leading to different durations of time delay for GFP expression which inspired us to extend our work of 2011 XMU iGEM team. By altering the strength of RBS at five grades, another five circuits have been built.Once induced by arabinose, the duration of response time for GFP expression increases as the strength of RBS declines, bringing about a time-course display.

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8. References
[1] http://en.wikipedia.org/wiki/Logic_gate

[2] http://en.wikipedia.org/wiki/Green_fluorescent_protein

[3] Eberhard A, Burlingame A. L, Eberhard C, Kenyon G. L, Nealson K. H, Oppenheimer N. J. Structural identification of autoinducer of Photobacterium fischeri luciferase[J]. Biochemistry, 1981, 20 (9): 2444–2449.