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

It is 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 ,the aiia protein can degrade the AHL, repressing the promoter indirect, thus forming a oscillation.

Fig 1.The quorum sensing oscillator

As AiiA accumulating, it begins to degrade AHL, and after a sufficient time, the promoters return to their inactivated state. Because AHL is swept away by the fluid flow and is degraded by AiiA internally, the production of AiiA is then attenuated, which permits another round of AHL accumulation and another burst of the promoters.（Danino T, et al. 2010）

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 synthetic 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 3 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 and concentration of the GFP would change with the variation of promoter activity. The key design of the oscillator is a time delay in the negative feedback loop, the positive feedback loop increases the robustness of the oscillator.（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

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.

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.

1、 the verification of oscillator

2、 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.

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

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

3、 the test of the related promoter