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Team:XMU-China/Content3
From 2013.igem.org
Hi, Oscillation!
Are you familiar with oscillation?
No? Oh, you must be kidding!
Let me present you its definition in Wikipedia to help you get a clue. Oscillation is the repetitive variation, typically in time, of some measure about a central value (often a point of equilibrium) or between two or more different states1.
It seems that you have met it somewhere, right?
Have you ever used a GPS? Or lasers? Alright, you must have used AC power that supports nearly all electrical appliances in your houses. These three are typical applications of oscillation and amplified that signals in the frequency domain have obvious advantages over those steady-state design in terms of information gathering and procession. Take lasers for example, which are known for their intensity and can be focused to a tight spot over long distance. These characteristics all owe to their spatial coherent in the frequency of the light source.
Oscillation in bacteria
Scientists have proved oscillations also pervade biological systems at all scales as well, from gene expression to cell cycle progression, and these oscillations can incorporate the periodic variation in a parameter over time to generate an oscillatory output2. As mentioned above, oscillations can lead to fantastic applications and benefit our everyday life. Since the output of bacteria oscillations can be detected as a frequency, people started their journey on designing a bacteria reporter.
The noise of gene expression in biological systems, however, slowed our pace in this process, because it will generate noisy or stochastic oscillation with varying amplitudes and frequencies. To deal with this problem, we have to unify the expression of the reporter gene in bacteria. And we found…
First generation of oscillation, the second and the third…Brief intro
Synchronized Oscillation
Yes, we found synchronized oscillation!
It was in 1670s that Christiaan Huygens first observed coupled oscillations: two of his pendulum clocks mounted next to each other on the same support often became synchronized3. To get cells communicate and oscillate in the same amplitude and frequency like the two pendulum clocks, we adopted quorum sensing, a cell-to-cell signaling mechanism that refers to the ability of bacteria to respond to chemical hormone-like molecules called autoinducers, from Vibrio fischeri into our host E.coli (MG1655) using our “hero” synthetic biology to realize synchronization among cells over tens of micrometers4.
QS now or later
As far as we concern, more consistent the oscillation can be if more colonies are synchronized. In order to enhance the communication range of bacteria, a gas-phase redox signal molecular H2O2 is introduced into our circuit. According to a published research5, through H2O2 a faster and long distance instantaneous communication can be achieved to strengthen the oscillation.
For more detailed information about our circuit, please refer to the Mechanism part of our project.
Introduction
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.
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 luxl 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 luxl 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. 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 luxl promoter, 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.
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. [the host choosing]
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 works well. Its fluorescence can be observed by fluorescence microscopy under 1s of exposure time on the microfluidic array 1.
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.
[Data/Pictures]
P. S. Thanks the Peking iGEM team for providing us the sfGFP part.
Host
After building the whole circuit in DH5α, we have to find a suitable host for it. It is as challenging as casting for a right leading actor for our blockbuster Synchronized Oscillatory.
At first, we chose BL21 (DE3) to be this lucky guy for the following reasons:
1) ndh gene is originally expressed in BL21 (DE3), and that’s where we get this part by PCR. So we think BL21 must have a full developed ndh expression system that can be a credit for the normal function our coupling part.
2) BL21 is always known for its competent in transformation and protein expression among a variety of E.coli cells.
However, BL21with three plasmids let us down after some characterizations.
The DNA gel electrophoresis for digestion confirmation results showed that there is something wrong with the gfp plasmid.
The single digestion by EcoR I (Band 2) should only generate a single band which runs a little faster than the plasmid itself (Band 1). From the picture above, however, an unknown band is found (Circled in red), and we couldn’t find a solution for it.
To be continued…
Note:
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.
The autoinducer in our circuit is acyl-homoserine lactone (AHL).
http://en.wikipedia.org/wiki/Photobleaching
Photobleaching is the photochemical destruction of a dye or a fluorophore. In microscopy, photobleaching may complicate the observation of fluorescent molecules, since they will eventually be destroyed by the light exposure necessary to stimulate them into fluorescing. This is especially problematic in time-lapse microscopy.
Loss of activity caused by photobleaching can be controlled by reducing the intensity or time-span of light exposure, by increasing the concentration of fluorophores, by reducing the frequency and thus the photon energy of the input light, or by employing more robust fluorophores that are less prone to bleaching (e.g. Alexa Fluors or DyLight Fluors). To a reasonable approximation, a given molecule will be destroyed after a constant exposure (intensity of emission X emission time X number of cycles) because, in a constant environment, each absorption-emission cycle has an equal probability of causing photobleaching.
First
By constructing robust circuits in E.coli, we want to build a gene network capable of synchronizing genetic oscillations in multiple levels. Cells can be synchronized at the colony level via quorum sensing, and a gas-phase redox will be signaling (mainly H2O2) between colonies simultaneously. Two scales of coupling ensured extremely consistent oscillations.
First
By constructing robust circuits in E.coli, we want to build a gene network capable of synchronizing genetic oscillations in multiple levels. Cells can be synchronized at the colony level via quorum sensing, and a gas-phase redox will be signaling (mainly H2O2) between colonies simultaneously. Two scales of coupling ensured extremely consistent oscillations.
