"

From 2012.igem.org

Single Population Toggle Switch Design

<img width="750" src="">

The future of our work with this three population signal propagation system is bright. We have designed a way to adapt these three populations in such a way that will create a self regulating oscillating system. In order for this to happen, we will have to add a system of repressors to each population. This repressor system will use the same quorum sensing molecule outputs of our current system and use them to stop the fluorescence expression of particular populations at a time. This Positive and Negative System design for each population is detailed as follows.

Population 1:

Positive Feedback: The sequence begins with the synthesis of LsrK. When LsrK binds with Ai-2 produced by the third population, it creates a phosphorylated dimer, referred to as “Ai-2 +P”, that binds to the PLsrA promoter. This promotes the transcription of both Luxl and the yellow fluorescence protein, YFP. Also, LuxI, when bound with S-adenosylmethionine (SAM), produces AHL.
· Negative Feedback: In population 1, LasR is constitutively promoted. When it binds with Ai-1, it creates a dimer that, in turn, binds with PLasR and promotes the transcription of araC. AraA then binds with PBad to repress the production of LsrK, AHL and YFP.

Population 2:

Positive Feedback: The second population in the sequence is induced by the AHL produced by Population 1. When AHL is in the presence of the bacteria, it creates a dimer with LuxR that binds to PLuxR. PLuxR then promotes LasI which when bound with SAM produces Ai-1. RFP is attached to the end of the LasI gene, so that when LasI is produced, the bacterium also fluoresces red.

Negative Feedback: In population 2, LsrK is constitutively promoted. When it binds with Ai-2, it creates a dimer, binds with PLsrA and promotes LacI. LacI then binds with PLac to repress the production of LuxR, Ai-1 and RFP. This inhibits the red fluorescence with the populations

Population 3:

Positive Feedback: The third population in the sequence is induced by the Ai-1 molecules produced by Population 2. When Ai-1 is in the presence of the bacteria, it creates a dimer with LasR and binds to PLasR. PLasR then promotes LuxS which when bound with SAM produces Ai-2. CFP is attached to the end of the LuxS gene, so that when LuxS is produced, the bacterium fluoresces cyan.

Negative Feedback: In Population 3, LuxR is constitutively promoted. When it binds with AHL, it creates a dimer which binds with PLuxR and promotes TetR. TetR then binds with PTetR to repress the production of LasR, LuxS, and CFP.