From 2007.igem.org
Welcome to our World
[http://www.brownigem.com The Website: Our Public Image]
[http://openwetware.org/wiki/IGEM:Brown/2007 The Resource: Our Calculations]
Projects
Tri-stable Toggle Switch
The Tri-stable Toggle Switch will be able to produce three distinct, continuous (stable) outputs for each of the three inputs. A chemical will induce the system to "lock into" one state while repressing the other two states.
The Tri-stable Toggle Switch Architecture
Our three constructs are pBAD->LacI->TetR, pLacI->AraC->TetR, and pTet->AraC->LacI, where [http://en.wikipedia.org/wiki/Bcl-2-associated_death_promoter AraC] represses pBAD, [http://en.wikipedia.org/wiki/Lac_repressor LacI] represses pLac and [http://en.wikipedia.org/wiki/Tetracycline_controlled_transcriptional_activation TetR] represses pTet. The three chemicals ([http://en.wikipedia.org/wiki/Arabinose arabinose], [http://en.wikipedia.org/wiki/IPTG IPTG] and [http://en.wikipedia.org/wiki/Tetracycline Tetracycline], respectively), cause conformational changes in their respective repressor proteins which leads to gene expression. For example, in the presence of arabinose, AraC cannot repress pBAD so LacI and TetR are produced which in turn repress pTet and pLac.
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 in with and without arabinose but the structural change activates or represses the pBAD ([http://en.wikipedia.org/wiki/Bcl-2-associated_death_promoter Bcl-2-associated death promoter], an apoptotic regulator in humans).
LacI
Image[http://www.mun.ca/biochem/courses/3107/Topics/Lac_genetics.html]. LacI forms a tetramer and represses pLac. However, an inducer, such as IPTG, causes a conformation change that removes LacI from the operator site.
In nature, LacI represses pLac which promotes LacYZA genes that metabolize lactose, thus LacI represses pLac except in the presence of lactose (or lactose mimics, eg IPTG). Lactose causes a conformational change which inhibits LacI from binding to the operator site of pLac.
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, which acts to pump tetracycline out of the cell, thus the pump is only activated in the presence of Tetracycline.
