CH391L/S12/Toggle Switches, Repressilators, and Counters

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Toggle Switches, Repressilators, and Counters


Toggle switches, repressilators, and counters are synthetic biological information processing systems to control gene expression based on environmental cues.[1] Counters use memory and time delay to process the frequency of an event which has applications in recording environmental conditions. Toggle switches use memory by switching into one fixed state following its induction signal which could be applied to detecting pollutant levels in the environment. The toggle switch could keep memory of a certain level of a pollutant and display a reporter gene as a signal.[2] For example, June Medford from Colorado State University has engineered toggle switches in plants that turn off chlorophyll production and turn white when they detect explosive chemicals.

Toggle Switches

Toggle Switch Network[3]
Toggle Switch Network[3]


pTAK Toggle Switch Plasmid

pTAK and pIKE Plasmids[3]
pTAK and pIKE Plasmids[3]

In the pTAK plasmid, the toggle switch consist of the Ptrc-2 promoter which is repressed by lacI and drives the expression of the temperature sensitive λ repressor (R1).[3] R1 represses the second promoter in the switch, PLs1con (P1), which in turn drives the expression of lacI.

Introduction of an IPTG or thermal pulse switches this toggle switch between its two states. The gfpmut3 gene is located downstream of the Ptrc-2 promoter and is used to indicate what state the toggle switch is in as it only expresses fluorescence when the Ptrc-2 promoter is induced. If the P1 promoter is induced, then the Ptrc-2 promoter is repressed and there is no fluorescence; this is called the "low state".

pIKE Toggle Switch Plasmid

Toggle Switch Threshold[3]
Toggle Switch Threshold[3]

The pIKE plasmid toggle switch differs from the pTAK plasmid by the P1 and R1 genes.[3] In pIKE, P1 is the PLtetO-1 promoter and R1 is tetR. This toggle switch is flipped by IPTG or aTc pulses.

Gardner et al designed pIKE and pTAK with different ribosome binding sites to determine bistability under different conditions, and all but one pIKE plasmid conferred bistability which is possibly due to the fact that tetR has less efficiency than the pTAK λ repressor. To test the bistability, the plasmids were induced with IPTG for 6 hours to express fluorescence, called the high state, and then grown 5 hours without IPTG. Plasmids that remained in the high state display bistability and ones that return to low states display monostability. Afterwards, the plasmids were treated with heat or aTc as appropriate for 7 hours to turn off GFP expression then removed for 5.5 hours; plasmids that remained in low state are considered bistable.



Repressilator Network[4]
Repressilator Network[4]

A repressilator is a synthetic gene network that uses the repression of genes in a negative feedback loop to create an oscillating network measured by GFP expression.[4] This network involves three genes, each of which promote the expression of the repressor of the next gene.


GFP Oscillation[4]
GFP Oscillation[4]



The 2010 USTC iGEM team created a model to simulate a repressilator as part of their project.


Synthetic cellular counters count events by expressing a reporter gene, mainly GFP, only after a certain number of pulses of an inducer.[5] Counters are found naturally in systems such as telomere lengthening, and can be applied to tightly control processes like cell growth. Friedland et al constructed two types of synthetic genetic counters that can count up to three.

Riboregulated Transcriptional Cascade

RTC Network[5]
RTC Network[5]

The riboregulated transcriptional cascade (RTC) consists of two promoters each of which is induced by arabinose, and the first promoter expresses a gene that promotes the expression of the second promoter which drives GFP expression.[5] In a two-counter system, the first pulse of arabinose shortly induces the first promoter which encodes for T7 RNAP. The arabinose is then removed and the mRNA metabolized, and whatever small amount of T7 RNAP that was translated transcribes the second promoter to produce little amounts of GFP. Only at the second pulse does GFP expression increase significantly. In the three counters system the same method applies to a set of three promoters: T7 RNAP expression drives T3 RNAP expression which then drives GFP expression.


DNA Invertase Cascade

DIC Network[5]
DIC Network[5]


DIC Multiple Inducer Network[5]
DIC Multiple Inducer Network[5]

A multiple inducer DIC was also designed in which the three arabinose promoters are replaced with three different promoters such as one induced by aTc, one induced by arabinose, and the third induced by IPTG.[5] High GFP expression is only seen when the three inducers are pulsed in that order. This allows a circuit to respond to a chosen sequence of events.



  1. Smolke CD. . pmid:19478174. PubMed HubMed [Smolke2009]
  2. Collins J. . pmid:22378128. PubMed HubMed [Collins2012]
All Medline abstracts: PubMed HubMed
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