From 2007.igem.org

Contents 1 Welcome to our World 2 Our 2 Projects 2.1 Lead Detector 2.2 Tri-stable Toggle Switch 2.2.1 AraC/BAD 2.2.2 LacI 2.2.3 TetR 2.3 Modeling

Welcome to our World

/Outreach (Tito, 10/20/07)

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 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 [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.

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] 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

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. 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]

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.