Tristable/Intro to Tristable

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Tri-stable Toggle Switch

The Tri-Stable switch three distinct and stable outputs in response to three distinct inputs. These three inputs are three separate chemicals which will each induce one state of the switch.
The Tri-stable Toggle Switch Architecture
In order to achieve this goal, we are constructing three constructs, each of which consists of a repressible, constitutively-on promoter attached to two repressors. Specifically, our three constructs are:

pBAD->LacI->TetR,

pLacI->AraC->TetR and

pTet->AraC->LacI,

where [http://en.wikipedia.org/wiki/L-arabinose_operon AraC] represses pAraC/BAD, [http://en.wikipedia.org/wiki/Lac_repressor LacI] represses pLac and [http://en.wikipedia.org/wiki/Tetracycline_controlled_transcriptional_activation TetR] represses pTet.


Each of the three repressors are inactivated by one of three chemicals, the three inducer chemicals mentioned earlier. These three([http://en.wikipedia.org/wiki/Arabinose L-arabinose], [http://en.wikipedia.org/wiki/IPTG IPTG] (Isopropyl β-D-1-thiogalactopyranoside) and [http://en.wikipedia.org/wiki/Tetracycline Tetracycline], respectively), cause conformational changes in their respective repressor proteins which keeps them from binding to DNA in an inhibitory manner which leads to gene expression. For example, in the presence of arabinose, AraC cannot repress pAraC/BAD so LacI and TetR are produced which in turn repress pTet and pLac and the pAraC/BAD construct is turned on.


AraC/BAD

The gene AraC, one of several genes (AraA, AraB, AraD, etc) originally for the metabolism of arabinose.[http://www.mun.ca/biochem/courses/3107/Topics/Ara_operon.html]

Dimer structure with arabinose on the left (yellow)
The left image shows the araC dimer repressing transcription, while the right conformation enables transcription
The protein forms a dimer with and without arabinose but the structural change activates or represses the pAraC/BAD.






LacI

In nature, LacI represses pLac which promotes the LacYZA genes that metabolize lactose. Thus LacI represses pLac except in the presence of lactose (or lactose mimics, eg IPTG).
Image[http://www.mun.ca/biochem/courses/3107/Topics/Lac_genetics.html]. LacI forms a tetramer and represses pLac. IPTG causes a conformation change in LacI.
Lactose causes a conformational change which inhibits LacI from binding to the operator site of pLac. Four LacI proteins form a tetramer to inhibit pLac and four inducer molecules are required to cause the full conformational change in the repressor.[http://www.mun.ca/biochem/courses/3107/Topics/Lac_genetics.html]









TetR

TetR represses the constitutive promoter pTet. In the presence of tetracycline, an antibiotic, a conformational change in TetR inhibits the protein from binding to the operator region. In nature, pTet promotes TetR and TetA. The latter of which acts to pump tetracycline out of the cell, thus the pump is only activated in the presence of Tetracycline.
A tetracycline molecule binds to each of the two TetR monomers to form a dimer
[http://en.wikipedia.org/wiki/Tetracycline_controlled_transcriptional_activation]

The TetR, as it turns out is a very tight repressor and a range of 0 to 1 ug/ml has been shown to cause a 5 order of magnitude change in luciferase production.[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=1319065&query_hl=1&itool=pubmed_docsum]


We will be using anhydrotetracycline (aTc) as the inducer molecule as it has been shown to exhibit stronger binding to TetR and is not an antibiotic that will compromise our system.


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