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 W1.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.

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Fig W2. 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.）

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Fig W3:the light control system

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Fig W4: 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:

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Fig W5

For the dual feedback oscillator:

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Fig W6

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.