Brown

From 2007.igem.org

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== Welcome to our World ==
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[[/Outreach]] (Tito, 10/20/07)
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[[Image:brown_website.jpg|frame|left| [http://www.brownigem.com The Website: Our Public Image]]]
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[[Image:brown_oww.jpg|frame|right| [http://openwetware.org/wiki/IGEM:Brown/2007 The Resource: Our Calculations]]]
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==Our 2 Projects==
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===Lead Detector===
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<a href="home"><image src="home_pressed.gif" alt="Home" border=0></a>
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<a href="lead"><image src="lead_reg.gif" alt="Lead Sensor" border=0></a>
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<a href="tristable"><image src="tri_reg.gif" alt="Tristable Switch" border=0></a>
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<span style="font-size:18pt">Celluar Lead Sensor</span>
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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.
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.
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The legal limit of lead in drinking water is 15 parts per billion.
The legal limit of lead in drinking water is 15 parts per billion.
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Current ways of testing for lead either require expensive chemical lab analysis or involve inaccurate home kits.
Current ways of testing for lead either require expensive chemical lab analysis or involve inaccurate home kits.
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A lead detector, based on E. Col, is cheap, sensitive, quick, and specific
A lead detector, based on E. Col, is cheap, sensitive, quick, and specific
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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.
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.
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<span style="font-size:18pt">Tristable Switch</span>
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The purpose of the Tri-stable Toggle Switch is to produce three distinct, continuous, 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.
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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 AraC represses pBAD, LacI represses pLac and TetR represses pTet. Each of the three repressors are inactivated by one of three chemicals, the three inducer chemicals mentioned earlier. These three(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.
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<span style="font-size: 14pt; color: #4b8ada">&nbsp;News Updates
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<b>08.28.07</b><hr size=1 color=#696969>
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Nanodrop donates ND-1000 Spectrophotometer and ND-3300 Fluorospectrophotometer to Brown iGEM
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<p>
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<b>08.22.07</b><hr size=1 color=#696969>
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Brown iGEM Team presents its August Update to faculty and friends
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<p>
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<b>08.20.07</b><hr size=1 color=#696969>
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Nanodrop it like it's hot! (YouTube Link)
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<p>
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<b>08.08.07</b><hr size=1 color=#696969>
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We'll be hosting several iGEM Teams here at Brown University on August 18th for the iGEM 2007 Meetup. Teams will present their progress to each other and discuss the future of Synthetic Biology.
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<b>06.07.07</b><hr size=1 color=#696969>
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$25,000 grant from Pfizer for equipment purchases
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<p>
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<b>06.01.07</b><hr size=1 color=#696969>
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Labnet sponsors Brown iGEM with vortex mixers, a microcentrifuge, pipettes, and autopipettes.
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===Tri-stable Toggle Switch===
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The purpose of the Tri-stable Toggle Switch is to produce three distinct, continuous, 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.  [[Image:Tristable_Toggle_Switch_2007.jpg|thumb|left|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/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.  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 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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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)]]
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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]]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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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 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]
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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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[[Image:Tc_bound_to_TetR.jpg|thumb|left|A tetracycline molecule binds to each of the two TetR monomers to form a dimer]]
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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]
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===Modeling===
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Brown iGEM 2006 Matlab model code [[Media:tristable2006.txt]]
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[http://parts2.mit.edu/wiki/index.php/Table_of_preliminary_model_constants Initial Table of Constants]
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[http://parts2.mit.edu/wiki/index.php/Derivation_of_the_Model_Equations Derivation of Model Equations]
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A simpler model based on the bistable paper was developed that takes the relative transcription/translation rates into account [[Media:tristable2007.txt]].
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Revision as of 20:22, 20 October 2007

Brown University
Home Lead Sensor Tristable Switch Community Supplemental About us
Celluar Lead Sensor

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.

Tristable Switch

The purpose of the Tri-stable Toggle Switch is to produce three distinct, continuous, 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.

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 AraC represses pBAD, LacI represses pLac and TetR represses pTet. Each of the three repressors are inactivated by one of three chemicals, the three inducer chemicals mentioned earlier. These three(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.
 News Updates
08.28.07
Nanodrop donates ND-1000 Spectrophotometer and ND-3300 Fluorospectrophotometer to Brown iGEM

08.22.07

Brown iGEM Team presents its August Update to faculty and friends

08.20.07

Nanodrop it like it's hot! (YouTube Link)

08.08.07

We'll be hosting several iGEM Teams here at Brown University on August 18th for the iGEM 2007 Meetup. Teams will present their progress to each other and discuss the future of Synthetic Biology.

06.07.07

$25,000 grant from Pfizer for equipment purchases

06.01.07

Labnet sponsors Brown iGEM with vortex mixers, a microcentrifuge, pipettes, and autopipettes.

Retrieved from "http://2007.igem.org/wiki/index.php/Brown"