Team:XMU-China/Content3

Forming the Glee

To be a good glee, first it needs a good song. The most intriguing song on earth is called OSCILLATION. If you look about, you will find it ubiquitous and extremely useful. After we taught each member this song, we need to help them sing it together, so oscillation became SYNCHRONIZED OSCILLATION. Thus how we form a glee!

Oscillation: The Magic Song of World

Once upon the time, there was a magic song called oscillation, it permeated every corner of the world. It regulated nearly everything on the earth. The interaction of moon and ocean generated the magnificent tide oscillation, and the biological process that displays a 24-hour oscillation inside human body.

Fig. 1-1 The Tides depend on several factors, including where the sun and moon are relative to the Earth	Fig. 1-2 The circadian clock inside human body

Besides, oscillation also plays an important part in signal transition. 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.

Fig. 1-3 Helium-Neon laser demonstration at the Kastler-Brossel Laboratory at Paris VI: Pierre et Marie

Oscillation in Bacteria

Synthetic biologists have done a lot of work to teach bacteria to sing this oscillation song by building artificial genetic circuit inside them, since oscillations can lead to fantastic applications and benefit our everyday life. Besides regulation and signal transmission, oscillations in living organisms can also react to its growing environment, which probably will activate the trigger of oscillation inside cells and generate oscillation signals. From collected signals we can tell how an environment factor influences the cells behavior, and in turn the environment factor can be indicated by the signals. Furthermore, if we interpret these kinds of oscillation signals like 1010 in binary system, then we could even make an electronic environmental factor detector, using signals from environment as an input, and numbers on computer screen as output (Fig. 1-4).

Fig. 1-4 How to make a signal converter

Here is the progress of artificial synthetic biological oscillations have made.
In the first generation of oscillation, three transcriptional repressor systems that are not part of any natural biological clock are used to build an oscillating network, termed the repressilator, in Escherichia coli (Fig. 1-5). The network periodically induces the synthesis of green fluorescent protein as a readout of its state in individual cells. However, plasmids can hardly pass from generation to generation and this artificial clock is not robust enough and always displays noisy behavior.

Fig. 1-5 The first generation oscillator, known as repressor

Scientists constructed the second generation oscillator rapidly. This improved oscillator is fast, robust and persistent; with tunable oscillatory periods as fast as 13 min (Fig.1-6). The oscillator is designed using a previously modeled network architecture comprising linked positive and negative feedback loops. But this circuit could only monitor oscillations in individual cells through multiple cycles, which means the song is still limited to single individual.

Fig. 1-6 2nd generation of oscillation consists of positive and negative feedback loops

Thinking that singing alone is lonely, scientists introduce quorum sensing to connect isolated cells and let them sing together, and technically it's called synchronized oscillation. The synchronization effect of quorum sensing, however, is limited to over tens of micrometer (Fig.1-7). If the synchronized colony could be enlarged, then the oscillation signal will be much stronger and steadier. Longer ranged synchronized oscillation is realized by a gas-phase redox signal H2O2 to the third generation circuit, which could achieve the communication among colonies at over millimeter scales and became the fourth generation. Oh yeah, finally our bacteria can sing the same oscillation at the same time, so they decided to form their own glee!

Fig.1-7 3rd generation involved in quorum sensing

That's how our little E. coli friend formed their glee, a long but cheerful story. Thanks to hardworking synthetic biologists. Let's go and see how this fourth generation oscillation glee functions inside in THREE PLASMIDS!

Reference
1. http://en.wikipedia.org/wiki/Oscillation
2. Elowitz M. B. & Leibler S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335-338 (2000)
3. Stricker J. et al. A fast, robust and tunable synthetic gene oscillator. Nature 456, 516-519 (2008)
4. Danino T., Mondrago´n-Palomino O., Tsimring L. & Hasty J. A synchronized quorum of genetic clocks. Nature 463, 326-330 (2010)

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!

Overview

So we designed three plasmids to function as the three important parts in the oscillation circuit, and all of them are in the charge of the same quorum sensing promoter to make sure that all target genes share the same oscillatory period (Table 2-1).

In a synchronized oscillation circuit, each A, B and C plasmid is necessary. The plasmid A expresses GFP breporter and LuxI proteins, which is the positive feedback in oscillator; the plasmid B expresses protein aiiA to degrade AHL and acts as the negative feedback in oscillator; the plasmid C expresses NAD-2 to generate H2O2 to communicate between colonies, is the coupling part in circuit (Fig 2-5).

Fig. 2-5 Plasmids A, B and C in oscillation circuit

You may have noticed that these three plasmids have different resistant genes and replication origins on their backbone. Different resistant genes are used mainly for selection. And since different replication origin means a different copy number of target genes in this plasmid. Using different replication origins is to produce different target proteins in an appropriate ratio, and only in this way can oscillation be observed. Just like a glee needs Soprano, Tenor and Bass to cooperate to finish a song. Besides, three artificial plasmids with same copy numbers always have a competitive relation in one cell and cannot be well expressed.

Plasmid A has two different backbones with high and middle copy numbers respectively. We built them to see the copy numbers' effect on oscillation. Results can be seen in Improvement (For a better Glee).

All of the plasmids we constructed are confirmed by agarose gel electrophoresis, and result can be seen in Parts, where you can also get the link to parts we submitted.

After the onerous construction work, our glee is ready to perform the synchronized oscillation song, so we have to find them a stage. Let's go to see the beautiful opera in Microfluidics (Stages).

Reference
1. Prindle, A., et al., A sensing array of radically coupled genetic 'biopixels'. Nature 481, 39-44, 2012;
2. Pedelacq, J. et al., Engineering and characterization of a superfolder green fluorescent protein. Nature 24, 79-88, 2006;
3. Waters C. M. & Bassler B. L. Quorum Sensing: Cell-to-Cell Communication in Bacteria. Annu. Rev. Cell Dev. Biol 21, 319-46, 2005.