Team:XMU-China/Content3

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.

We designed three plasmids to function as the three important pA rts above according to a published research, and all of them all in the charge of the same promoter – lux promoter to make sure that all target genes share the same oscillatory period.

The first plasmid is the pSB1C3-gfp-luxI (pG-L) plasmid, which will express GFP reporter and LuxI proteins; the second plasmid is pSB3T5-aiiA (pA ), which will express protein aiiA to degrade AHL; the third plasmid is pSB4K5-ndh (pN ), which expresses ndh to generate H₂O₂ to communicate between colonies.

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 …. A 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.

At first, we assume that the relationship between pG-L, pA and pN should be high-middle-low. [reason] So we chose pMB1, p15A and pSC101 because they seem to meet the requirement of copy number. However, the oscillation we observed on our microfluidic array is not …, because the light of green fluorescence protein is not dim enough at the trough. (We also discussed this problem with the author of this article, and his suggestion was to lower the copy number of the pG-L). Then we thought the explanation for this phenomenon is there was too much GFP but not enough aiiA to degrade them all in time, so GFP remained longer than we expected and weak the oscillatory effect. So we changed the backbone of plasmid pSB1C3--gfp-luxI into … with a … replication origin, and the copy number is ….

Why M&M?

If we want to capture gene expression dynamics and distributions reported by fluorescence, we need to create a chemostat that provide a near-constant environment for the growth of bacteria. The near-constant environment mainly includes two aspects: continuous fresh media to ensure the optimum growth state and constant small populations of bacteria to ensure single-cell resolution in observation. Steady growth state is needed because the oscillation curve is expected to be a near simple harmonic one. Besides, bacteria tend to form biofilm which will lead to the overlapping of cells and create a false impression that the fluorescence increases, and that’s why we have to prevent this to maintain a constant bacteria population with single-cell resolution.

What we mentioned above might have covered all aspects we need to consider for communication in a single colony, but if we still want to observe the coupling between colonies we have to create an array that contains many colonies and allows H₂O₂ to pass through freely.

Among many different techniques, microfluidic array seems to be our best choice with following advantages: 1) Precisely control of flowing rate and cell populations when equipped with syringe pumps;

2) Cells behavior including fluorescence strength can be captured by high resolution microscopy;

3) Trap numbers and dimensions can be adapted to different experimental need;

4) Productive: fewer resources and less time are required when compared with macroscopic cultures.

Design of Microfluidic Array

According to what we have discussed above, the microfluidic device should be able to provide flowing fresh media and maintain a constant bacteria population with single-cell resolution. So we designed our device as shown below.

About devices:

a) Material: PDMS

b) Incubation temperature: around 30℃

c) *Tween20 in media: 0.075%

d) Trap size: ？μm height 100*100 μm space between traps was 25μm channel wide 100 μm

e) Flow rate: around 0.01 ml/hr, 3mm radius, for LB

will be increased according to the cell density

This device is composed by two parts: the flowing channels and near 3,000 square trapping chambers, and their layout is like there are many passages with symmetry rooms distributed on both sides, so we call this array “passage” model. Firstly, cells are loaded from the cell port. When the loading stops, most cells would remain in channels, but if the loading cell density is dense enough, many cells would be pushed into chambers and form a monolayer of Escherichia coli growing in it. This is the time for running LB to get in. Fresh LB is loaded from the media port with Tween 20* and appropriate antibiotics. Flowing LB does not only permit a constant nutrition and exponentially growing cells for more than two days (that’s the longest time we have tried), but also washes away newly generated cells to sustain a certain cell density, which is crucial for the determination of fluorescence strength.

This device is composed by two parts: the flowing channels and near 3,000 square trapping chambers, and their layout is like there are many passages with symmetry rooms distributed on both sides, so we call this array “passage” model. Firstly, cells are loaded from the cell port. When the loading stops, most cells would remain in channels, but if the loading cell density is dense enough, many cells would be pushed into chambers and form a monolayer of Escherichia coli growing in it. This is the time for running LB to get in. Fresh LB is loaded from the media port with Tween 20* and appropriate antibiotics. Flowing LB does not only permit a constant nutrition and exponentially growing cells for more than two days (that’s the longest time we have tried), but also washes away newly generated cells to sustain a certain cell density, which is crucial for the determination of fluorescence strength.

It has been stated before in the Mechanism part that most quorum sensing systems have a critical cell density requirement, so the trap size is designed neither too big nor too small to capture enough cells and allows for nutrients to permeate to every corner of the chamber.

You may also notice that the height of the chamber is extremely small, which is really demanding for the researcher’s skills, but it’s important for the observation. The oscillation could only be seen when the height of the chamber is in a particular range as our modeling predicted for many complicated reasons. For more details, please refer to the Modeling part.

Polydimethylsiloxane (PDMS), a transparent polymer material, is chosen to build the devise because of its excellent breathability, which is necessary for the permeation of H₂O₂. Once H₂O₂ diffused outside cells, it can travel from colony to colony and synchronize them.

Our device is not only environmental friendly, but also a reusable one. After the flushing of a lysis buffer, most cells can be washed away, even those attached to walls. Then the device will be washed for three times by sterilized water before reuse.

The green fluorescence of bacteria in chambers is captured every 5 minutes by an inverted microscope (Eclipse Ti, Nikon) with a standard CCD camera manually and usually last more than 7 hrs. (This is really a big challenge for our team and very physically demanding since this is the only available fluorescence we could find. However, we managed to finish this data collecting process anyway. Good jobs, guys！)

Data are transferred into oscillation curves by ImageJ and software designed by our teammate for digital image batch processing. (More detail for our self-designed software, please refer to protocol.)

Experiments with different models

Before our experiments on the microfluidic array we described before, a series of pretests were done on another kind of chip.

“Swimming pool” chip in black field, those white squares (100μm×100μm×100μm) are chambers for bacteria

From the sketch we can see that the chamber is like a swimming pool where bacteria swim happily in the fresh LB that running from up above, and newly generated cells would be washed away.

A “swimming pool” chamber model would have a lager AHL concentration when compared with a “passage” chamber because the dilution effect is minor. According to a published research that the oscillation period will be shorter as the AHL concentration increases. So we assume that it will be more time-consuming to use this “swimming pool” model in pretests.

However, this chamber is about 100 μm deep and could grow E. coli far more than monolayer. As the time goes by, the oscillation in a single colony signaling by AHL could never go back to the original baseline, and the oscillation curves have a tendency like the following graph.

In spite of the effect of growing bacteria, a slight oscillation was still observed. However, this data was not solid enough since we only captured one period of oscillation because of the limited microscope using time.

The oscillation period is about 120 minutes with a tendency of increasing fluorescence strength. However, it still proved that our circuit could function as an oscillation.

BL21 strain with three plasmids (pSB1C3-gfp-luxI & pSB3T5-aiiA & pSB4K5-ndh) has also been tried several times on this array, but no obvious phenomenon could be captured. There are many factors involved, for example cell density, trap size and flowing rate. The strict requirement for the observation of oscillation has already discussed before.

DH5α strains have been tested on this array, too. They didn’t work well, either. The reasons could be found in Host part of our project.

Mathematical model

We modeled the oscillate gene circuit (without ndh) based on the paper “A synchronized quorum of genetic clocks” ( Reference: Danino, T., Mondragon-Palomino, O., Tsimring, L., & Hasty, J., A synchronized quorum of genetic clocks. Nature 463, 326-330 (2010) ). In that, the author characterized the oscillator by using the four delay differential equations (DDEs), which are the basic of our modeling. DDE can perfectly simulate our circuit due to its powerful function about solving the dynamic change in biological processes.

In the first two equations, item is a Hill function equaling to , which describes the delayed reactions about the complex processes in the cells (transcription, translation, maturation, etc.). This Hill function takes the history of the system into account, i.e. the concentration of AHL at the time it binds to LuxR to form the activation complex.

This model dose not includes an equation for LuxR assuming that it is constitutively produced at a constant level. All parameters are normalized for the convenience of analysis.

Modeling in MATLAB

We used MATLAB to help us solve the DDEs and draw the graphs. In addition, we developed a GUI user interface after finishing the main DDE code. The user interface makes people easier to modify and adjust the parameters without recoding the .m files and any MATLAB language knowledge. We called this software “Gene OS” which is short for Gene oscillate simulator.

First

We designed three plasmids to function as the three important pA rts above according to a published research, and all of them all in the charge of the same promoter – lux promoter to make sure that all target genes share the same oscillatory period.

The first plasmid is the pSB1C3-gfp-luxI (pG-L) plasmid, which will express GFP reporter and LuxI proteins; the second plasmid is pSB3T5-aiiA (pA ), which will express protein aiiA to degrade AHL; the third plasmid is pSB4K5-ndh (pN ), which expresses ndh to generate H₂O₂ to communicate between colonies.

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 …. A 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.

At first, we assume that the relationship between pG-L, pA and pN should be high-middle-low. [reason] So we chose pMB1, p15A and pSC101 because they seem to meet the requirement of copy number. However, the oscillation we observed on our microfluidic array is not …, because the light of green fluorescence protein is not dim enough at the trough. (We also discussed this problem with the author of this article, and his suggestion was to lower the copy number of the pG-L). Then we thought the explanation for this phenomenon is there was too much GFP but not enough aiiA to degrade them all in time, so GFP remained longer than we expected and weak the oscillatory effect. So we changed the backbone of plasmid pSB1C3--gfp-luxI into … with a … replication origin, and the copy number is ….

First

We designed three plasmids to function as the three important pA rts above according to a published research, and all of them all in the charge of the same promoter – lux promoter to make sure that all target genes share the same oscillatory period.

The first plasmid is the pSB1C3-gfp-luxI (pG-L) plasmid, which will express GFP reporter and LuxI proteins; the second plasmid is pSB3T5-aiiA (pA ), which will express protein aiiA to degrade AHL; the third plasmid is pSB4K5-ndh (pN ), which expresses ndh to generate H₂O₂ to communicate between colonies.

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 …. A 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.

At first, we assume that the relationship between pG-L, pA and pN should be high-middle-low. [reason] So we chose pMB1, p15A and pSC101 because they seem to meet the requirement of copy number. However, the oscillation we observed on our microfluidic array is not …, because the light of green fluorescence protein is not dim enough at the trough. (We also discussed this problem with the author of this article, and his suggestion was to lower the copy number of the pG-L). Then we thought the explanation for this phenomenon is there was too much GFP but not enough aiiA to degrade them all in time, so GFP remained longer than we expected and weak the oscillatory effect. So we changed the backbone of plasmid pSB1C3--gfp-luxI into … with a … replication origin, and the copy number is ….