Overview

In wetlab, as the real part of MR-Cell ,we construct two genetic oscillator circuits in E.coli. The one is the quorum sensing oscillator based on quorum sensing, the other is the dual feedback oscillator based a hybrid promoter. They have different character and application for our project to couple with the virtual part. To connect with the oscillator in computer, we adopted a light control system associated with the oscillator in E.coli, so we can regulate the oscillator through light controlled by computer and the two parts can couple with each other eventually.

Furthermore, in order to further achieve our goal, we did some verification and test about the real part. Such as the verification of oscillation, the effect of light on the expression of genes associated with the oscillator, and the test of the related promoter : para/lacI, pluxpR.

Besides when doing that work, we have done some communications and collaborations with other teams. For example we helped HSTU-CHINA team to construct a essential part , and WHU-CHINA team helped us to construct a section of light control system.

Finally we wetlab group is full of love and happiness. Even we must work very hard, but everyone of us enjoyed the process and tried their best to achieve our goal . Because we are firmly convinced that what we are doing is worthwhile and great.

Design

The quorum sensing oscillator

The genetic oscillator based on quorum sensing. The luxI protein generates AHL , it’s a signal molecule. In the presence of luxR ,the complex can activate the promoter .When promoter activated ,the LuxI and AiiA express and accumulate. Because the AiiA protein can degrade the AHL and repress the promoter indirect, the expression of LuxI and AiiA are depressed, Thus forming an oscillation by the negative feedback circuit. (Danino T, et al. 2010)

Fig 1. The quorum sensing oscillator

The key point of this oscillator is that the promotor LuxpR is induced by quorum sensing molecular AHL, so the state of cells can be synchronous by communicating with each other, being convenient for us to observe and regulate the oscillator in the population level.

The dual feedback oscillator

The genetic oscillator is based on a negative feedback loop and a positive feedback loop. The hybrid promoter (Plac/ara-1) is composed of an activation operator site and a repression operator site .It is activated by the AraC protein in the presence of arabinose and repressed by LacI protein in the absence of IPTG. The araC, lacI, and GFP genes are under the control of three identical copies of the hybrid promoter thus formed three co-regulated transcriptional modules.

Fig 2. The dual feedback oscillator

The addition arabinose and IPTG will activate the promoter and result in transcription of each component of the circuit , and increased production of AraC in the presence of arabinose results in a positsive feedback loop that increase promoter activity. However the concurrent increase in production of LacI results in a linked negative feedback loop decreases promoter activity. So the concentration of the GFP would change with the variation of promoter activity.（Hasty, Jeff. 2008.）

The key point of this oscillator is that the hybrid promotor can be effected by chemical revulsive IPTG/Arabinose, so we can make the oscillator more tunable and robust by adding the chemical revulsive.

Regulation to the oscillator--the light control system

In this system , the three genes can generate a lightsensitive complex, which can phosphorylate ompR protein in dark, and the phosphorylated ompR protein will active the ompC promoter and the downstream gene can express. But in the presence of red light, the kinase activity is inhibited, resulting in repressing the promoter and inhibiting the expression of related genes.（Anselm, Levskaya, et al. 2005.）

Fig 3. The light control system

Fig 4. The genetic circuit of light control system

If the downstream genes is lacI or Arac , luxI or AiiA ,like this ,we can regulate the oscillator by the red light.

For the quorum sensing oscillator:

Fig 5.

For the dual feedback oscillator:

Fig 6.

Characterization

To ensure that our system of the real part can work as we expect and we can achieve our goal, we did some verifications and tests.

The verification of oscillator

When the genetic oscillator was completed, we verified its function by using GFP as a reporter indicating the state of oscillation. We gained some pictures and videos by observing the GFP in E.coli through fluorescence microscope.

Fig 1. The E.coli with fluorescence through fluorescence microscope.

video:The state of E.coli cultured in microfluidic chip through fluorescence microscope.

Furthermore, due to the GFP protein as the reporter has delay and accumulation character in our experiment, we also tried to verify the oscillator in transcriptional level using a RNA aptamer as the reporter, which is sensitive and can real-time reflect the state of oscillation.

We adopted a new generation RNA aptamer named dBroccoli. (Filonov G S, et al. JACS, 2014) It can activate the fluorescence of DFHBI-1T when binding with DFHBI-1T and shows green fluorescence in cells. ex = 465nm,em = 535nm. When the related gene added the RNA aptamer sequence, we can observe the state of oscillator in E.coli through fluorescence microscope. Meanwhile the function of the RNA aptamer was verified in our experiment showing as the following pictures.

Fig 2. The fluorescence in E.coli through fluorescence microscope.

Ps:

1) The Molecular Formula of DFHBI-1T is

            (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1Himidazol-5(4H)-one

2) The Chemical synthesis process of DFHBI-1T

Fig 3.

3) Excitation peak: 465 nm Emission peak: 535 nm

The effect of light on the expression of genes

We verified the effect of light on the expression of genes by controlling the light condition of E.coli transformed with the genetic circuit of light control system.

Two sample owning the same genetic circuit were set in our test experiment, the one in dark, the other in red light, and each sample had three biologic repeats. Besides we designed a simple and practical device to control the light condition of E.coli as the following picture shown.

Fig 4. A red LED powered by battery is set at the bottom of a cup to provide red light.

Fig 5. Two light conditions in our test device.

After some times gropes and tests, we gained related data and did correlation analysis as showing from the following table. As we can see from it, the presence of red light can inhibit the expression of target genes.(In the test, we used GFP as the target gene).

Fig 6. The effect of red light on the expression of genes

The test of the related promoter

In order to fully understand the features of the oscillator, we specifically ran some related tests on the hybrid promoter(Plac/ara-1). We put a mRFP behind the promoter as a reporter in our experiment. We had used two different chemical inducer(Arabinose/IPTG) in our test to run respective tests on the promoter. A series of different inducer's concentrations are set to test amples, and the expressions level of mRFP is linked with the influence that different concentration of inducer had on the promoter.

We got and analysed related datas showing as the following charts.

Fig 7. The effect on the promoter of Arabinose.

Fig 8. The effect on the promoter of IPTG.

Reference

1. Danino T, Mondragón-Palomino O, Tsimring L, et al. A synchronized quorum of genetic clocks. [J]. Nature, 2010, 463(7279):326-330.

2. Hasty J. A fast, robust and tunable synthetic gene oscillator. [J]. Nature, 2008, 456(7221):516-519.

3. Anselm L, Chevalier A A, Tabor J J, et al. Synthetic biology: Engineering Escherichia coli to see light [J]. Nature, 2005, 438(7067):441-442.

4.Filonov G S, Moon J D, Nina S, et al. Broccoli: Rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution.[J]. Journal of the American Chemical Society, 2014, 136.