Team:XMU-China/Future

Three Parts & Three Plasmids in a Glee

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.)

Three Plasmids in circuit construction

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 reporter 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 Exploration (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.