IGEM:Brown/2007/Tri-Stable Switch

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===Tri-stable Toggle Switch===
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==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.  [[Image:Tristable_Toggle_Switch_2007.jpg|thumb|left|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] (Isopropyl β-D-1-thiogalactopyranoside) 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.
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.  [[Image:Tristable_Toggle_Switch_2007.jpg|thumb|left|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] (Isopropyl β-D-1-thiogalactopyranoside) 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.
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====AraC/BAD====
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===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]  [[Image:Two_Dimers_of_AraC.jpg|thumb|left|Dimer structure with arabinose on the left (yellow) [http://www.bactregulators.org/structure/arac-3d.php] ]]
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]  [[Image:Two_Dimers_of_AraC.jpg|thumb|left|Dimer structure with arabinose on the left (yellow) [http://www.bactregulators.org/structure/arac-3d.php] ]]
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[[Image:AraC_Promoters.gif|left|thumb|The left image shows the araC dimer repressing transcription, while the right conformation enables transcription [http://www.bactregulators.org/structure/arac-3d.php]]]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).
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[[Image:AraC_Promoters.gif|thumb|The left image shows the araC dimer repressing transcription, while the right conformation enables transcription [http://www.bactregulators.org/structure/arac-3d.php]]]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).
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====LacI====
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===LacI===
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).  [[Image:LacI_repressor.gif|thumb|left|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.]]    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 inhibitor.[http://www.mun.ca/biochem/courses/3107/Topics/Lac_genetics.html]
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).  [[Image:LacI_repressor.gif|thumb|left|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.]]    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 inhibitor.[http://www.mun.ca/biochem/courses/3107/Topics/Lac_genetics.html]
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====TetR====
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===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 which acts to pump tetracycline out of the cell, thus the pump is only activated in the presence of Tetracycline.[http://en.wikipedia.org/wiki/Tetracycline_controlled_transcriptional_activation]
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 which acts to pump tetracycline out of the cell, thus the pump is only activated in the presence of Tetracycline.[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]
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]
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Tetracycline is highly diffusable through cell membrane (permeation coeficient or 5.6±1.9 * 10^-9 cm/s or half equilibrium time = 35 ± 15 min) and TetR shows a very high affinity for the molecule. The binding constant of TetR to [tc-Mg+] is Ka ~ 10^9 M^-1.  When bound to tc, TetR has a low binding level to DNA of 10^5 M^-1. [http://content.febsjournal.org/cgi/content/abstract/270/15/3109]
Tetracycline is highly diffusable through cell membrane (permeation coeficient or 5.6±1.9 * 10^-9 cm/s or half equilibrium time = 35 ± 15 min) and TetR shows a very high affinity for the molecule. The binding constant of TetR to [tc-Mg+] is Ka ~ 10^9 M^-1.  When bound to tc, TetR has a low binding level to DNA of 10^5 M^-1. [http://content.febsjournal.org/cgi/content/abstract/270/15/3109]
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===Modeling===
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==Modeling==
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===Model 1.0===
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Brown iGEM 2006 Matlab model code [[Media:tristable2006.txt]]
Brown iGEM 2006 Matlab model code [[Media:tristable2006.txt]]

Revision as of 08:58, 22 June 2007

Contents

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
The Tri-stable Toggle Switch Architecture
Our three constructs are pBAD->LacI->TetR, pLacI->AraC->TetR, and pTet->AraC->LacI, where AraC represses pBAD, LacI represses pLac and TetR represses pTet. The three chemicals (arabinose, IPTG (Isopropyl β-D-1-thiogalactopyranoside) and 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.[4]
Dimer structure with arabinose on the left (yellow) [1]
Dimer structure with arabinose on the left (yellow) [1]
The left image shows the araC dimer repressing transcription, while the right conformation enables transcription [2]
The left image shows the araC dimer repressing transcription, while the right conformation enables transcription [2]
The protein forms a dimer in with and without arabinose but the structural change activates or represses the pBAD (Bcl-2-associated death promoter, an apoptotic regulator in humans).







LacI

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).
Image[3]. LacI forms a tetramer and represses pLac.  However, an inducer, such as IPTG, causes a conformation change that removes LacI from the operator site.
Image[3]. LacI forms a tetramer and represses pLac. However, an inducer, such as IPTG, causes a conformation change that removes LacI from the operator site.
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 inhibitor.[5]









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 which acts to pump tetracycline out of the cell, thus the pump is only activated in the presence of Tetracycline.[6] 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.[7]

A tetracycline molecule binds to each of the two TetR monomers to form a dimer
A tetracycline molecule binds to each of the two TetR monomers to form a dimer

Tetracycline is highly diffusable through cell membrane (permeation coeficient or 5.6±1.9 * 10^-9 cm/s or half equilibrium time = 35 ± 15 min) and TetR shows a very high affinity for the molecule. The binding constant of TetR to [tc-Mg+] is Ka ~ 10^9 M^-1. When bound to tc, TetR has a low binding level to DNA of 10^5 M^-1. [8]



Modeling

Model 1.0

Brown iGEM 2006 Matlab model code Media:tristable2006.txt

Initial Table of Constants

Derivation of Model Equations

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