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From 2011.igem.org

Introduction

        In previous pages, we have built the promoting cycle according to how the Wu xing system work. However, in Wu xing system, the restraining effect between each other is as important as the promoting one.
        In promoting cycle, some parts like AHL synthetase, activator protein, promoter that can be activate by the AHL-activator dimmer have palyed rather an important role in the signal communicating.
        In the restraining cycle, you will see them act as the heroes again. Besides, you will find how important the promoters are, they are like switches, can be turned on and off according to the change of outer physical and chemical environment.

The General Mechanism

        In restraining device, each one includes maily two production; the activator protein production, which is the same with the activator protein in the promoting device of the circuit before it, and the tetR, which will bind with the ptetR in promoting device and inhibit its downstream gene’s expression.




        In the promoting cycle, we use ptetR, which have a constitutive expression. So once the five kinds of bacteria with different promoting devices is added into the medium. their downstream activator gene will have an expression. When input AHLs accumulats to a certain desenty, these activators will bind with AHLs and activate the quorum sensing promoter’s downstream genes expression.
        In the restraining cycle, we will use plac, which is inhibited by the lacI in the lac operon in E.coli, so only when we add IPTG into the medium can the restraining device be activated. The plac will make its downstream gene, a AHL-related activator protein, express. This activator will only accept the AHLs produce by its corresponding AHL synthetase, then they form a dimmer, activate the quorum sensing promoter (the second promoter in this restraining circuit), and make the downstream gene tetR express. The tetR, in return, will inhibit the ptetR in promoting device.

The Specific Mechanism in Each Device

        In device I, the plac will express its downstream gene rhiR when IPTG is added, producing rhiR protein, then rhiR will bind with C6-HSL、C7-HSL、C8-HSL from the device Ⅳ, activating the promoter of rhiABC, express the tetR, inhibiting the ptetR in promoting circuit.

The Wu xing and Promoting Cycle

        Let’s take a look at the original Wu xing system again.




        The “水”inhibit the“火”，“木”inhibit “土”，“火”inhibit“金”，“土”inhibit “水”，“金” inhibit “木”.
        If we use device 1 to represent “火”，device 2 to represent “土”，device 3 to represent “金”，device 4 to represent “水”，device 5 to represent “木”, we will get a restraining cycle as below.




        Let’s take a look at the deviceⅠ, namely the “火”（red）.
        The lasI in promoting circuit in deviceⅠ(火)produces 3-O-C12-HSL, which will act as the inhibiting signal to device Ⅲ (金) , and the deviceⅠwill accept the inhibiting AHL signal C6-HSL, C7-HSL, C8-HSL (produced by rhiI )from device Ⅳ.
        Likely, The cinI in promoting circuit in deviceⅡ(土)produces 3-OH-C14:1-HSL, which will act as the inhibiting signal to device Ⅳ (水) , and the deviceⅡwill accept the inhibiting AHL signal 3-O-C6-HSL (produced by luxI )from device Ⅴ.
The sinI in promoting circuit in deviceⅢ(金) produces C12-HSL、3-Oxo-C14-HSL、C16:1-HSL、3-Oxo-C16:1-HSL、C18-HSL, which will act as the inhibiting signal to device Ⅴ (木) , and the device Ⅲ will accept the inhibiting AHL signal 3-O-C12-HSL (produced by lasI )from deviceⅠ.
        The rhiI in promoting circuit in device Ⅳ(水) produces C6-HSL、C7-HSL、C8-HSL, which will act as the inhibiting signal to device Ⅰ (火) , and the device Ⅳ will accept the inhibiting AHL signal 3-OH-C14:1-HSL (produced by cinI )from deviceⅡ.
        The luxI in promoting circuit in device Ⅴ(木) produces 3-O-C6-HSL, which will act as the inhibiting signal to device Ⅱ (土) , and the device Ⅴ will accept the inhibiting AHL signal C12-HSL、3-Oxo-C14-HSL、C16:1-HSL、3-Oxo-C16:1-HSL、C18-HSL (produced by sinI )from device Ⅲ.