Brown
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===Tri-stable Toggle Switch=== | ===Tri-stable Toggle Switch=== | ||
| - | The Tri-stable Toggle Switch | + | The purpose of the Tri-stable Toggle Switch is to produce three distinct, continuous, and stable outputs for each of three inputs. These three inputs are three separate chemicals which will each induce one state of the switch. [[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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[[Image:Tc_bound_to_TetR.jpg|thumb|left|A tetracycline molecule binds to each of the two TetR monomers to form a dimer]] | [[Image:Tc_bound_to_TetR.jpg|thumb|left|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. [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=== | ===Modeling=== | ||
Revision as of 18:59, 15 August 2007
Contents |
Welcome to our World
Our 2 Projects
Lead Detector
Lead poisoning is a public health concern - there is lead in soil, paint, water, and dust. Lead Poisoning is often caused by ingesting contaminated drinking water, or soil. It can cause neurological and gastrointestinal disorders, especially among children.
The legal limit of lead in drinking water is 15 parts per billion.
Current ways of testing for lead either require expensive chemical lab analysis or involve inaccurate home kits.
A lead detector, based on E. Col, is cheap, sensitive, quick, and specific
Our system involves a Lead detecting promoter, an amplifier, and an output of GFP. We're also working on a system to remove false positives.
Tri-stable Toggle Switch
The purpose of the Tri-stable Toggle Switch is to produce three distinct, continuous, and stable outputs for each of three inputs. These three inputs are three separate chemicals which will each induce one state of the switch.
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]
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).
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] 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]
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]
Modeling
Brown iGEM 2006 Matlab model code Media:tristable2006.txt
[http://parts2.mit.edu/wiki/index.php/Table_of_preliminary_model_constants Initial Table of Constants]
[http://parts2.mit.edu/wiki/index.php/Derivation_of_the_Model_Equations Derivation of Model Equations]
A simpler model based on the bistable paper was developed that takes the relative transcription/translation rates into account Media:tristable2007.txt.
