Team:XMU-China/Content mechanism

Three Parts in Circuit Designation

In a synchronized oscillatory system, three important parts should be included: the oscillator, which is the biochemical machinery that generate the oscillatory output; the coupling pathway that ensure the connection among cells; and output pathway, which is also known as a reporter that reflect the state of the oscillator to downstream targets (Fig. 2-1).

Fig. 2-1 Circuit working mechanism

Oscillator

Quorum sensing (QS) is a cell-to-cell signaling mechanism that refers to the ability of bacteria to respond to chemical hormone-like molecules called autoinducers. When an autoinducer reaches a critical threshold, the bacteria detect and respond to this signal by altering their gene expression. In our circuit, QS (from Vibrio fischeri) is installed as a positive feedback while aiiA (from Bacillus Thurigensis) acts as a negative one to compose an oscillator together. In the quorum sensing part, the luxI gene is at low expression level and produces LuxI protein that synthesize a kind of acyl-homoserine lactone (AHL), which is a small molecule that can diffuse across the cell membrane and mediate intercellular coupling when it reaches the threshold as enough biomass accumulated. AHL will bind intracellular protein LuxR, which is also consecutively produced by luxR gene. The LuxR-AHL complex can activate the luxI promoter, and the positive feedback loop is built. At the same time, the aiiA gene, which is under control of the same promoter is expressed and produce a lactonase enzyme known as aiiA that hydrolyzes the lactone ring of AHL. (Fig. 2-1) In this system, the activator enhances the expression of both activator and repressor, which shares the common motif of many synthetic oscillators.

Coupling

The communication range of quorum sensing, however, is limited by the diffusion rate of AHL, could only reach cells over tens of micrometers. So, we introduced the coding gene for NADH dehydrogenase II (ndh) and put it under the control of the same luxIpromoter, which means ndh also has a periodical expression in accordance with the oscillator. NDH-2 is a membrane-bound respiratory enzyme that generates low level H₂O₂ and superoxide (O₂^-) and H₂O₂ will permeate to neighboring colonies. Periodic production of H₂O₂ changes the redox state of a cell immediately and interacts with the synthetic circuit through the native aerobic response control systems, including ArcAB, which has a binding site in the lux promoter region. Before the oxidizing condition is triggered, ArcAB is partially expressed, so lux is partially repressed. When the concentration of H₂O₂ is increasing, ArcAB is gradually inactivated, and the repression of lux is relieved. With the periodically produced vapor phase H₂O₂ that can diffuse quickly among colonies, the oscillation is not only strengthened but also expanded to millimeter scales(Fig. 2-2).

Fig. 2-2 H2O2 functions as an activator to the quorum sensing promoter

When compared with the strong but short ranged coupling by QS, H₂O₂ might be weaker but long ranged because its disperse characteristic. These two levels of communication between cells formed the basic oscillatory system inside our host.

Reporter

A good reporter in an oscillatory circuit should be steadily expressed and can be easily detected by regular equipment in laboratory, so broadly used reporter green fluorescent protein (GFP) came into our mind.

First, we built regular gfp gene into our circuit and it worked well. Its fluorescence can be observed by fluorescence microscopy under 1s of exposure time on the microfluidic array 1(Fig. 2-3).

Fig. 2-3 GFP under fluorescence microscopy on a microfluidic array

However, there is a newly found GFP variant Superfolder GFP, which has shown outstanding performance in many ways. It has two advantages in observing:
1) Fold 3.5 to 4 times faster than regular GFP;
2) Yield up to four times more total Fluorescence than regular GFP.
The greater the fluorescence strength is, the shorter exposure time will be needed, thus can decrease the photobleaching (GFP will be eventually destroyed by the light exposure that tries to stimulate it into fluorescing) that will lead into lapses in data processing.
Besides, we also want to make a comparison between two different GFPs' performances in our oscillatory circuit. For example, peaks, troughs and periods in oscillatory curves. The following picture shows the difference between GFP and sfGFP both on colE1 backbone.(Fig. 2-4)

Fig.2-4 Difference between GFP and sfGFP both on colE1 backbone (P. S. Thanks the Peking iGEM team 2013 for providing us the sfGFP part.)

Please come with us to see how we built our magic circuit in E. coli in Construction!