Team:British Columbia/Project

From 2009.igem.org

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<!--- The Mission, Experiments --->
<!--- The Mission, Experiments --->
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== '''The Traffic Light Biosensor: A flexible, modular, and transparent system for multi-level assessment of variable inputs.''' ==
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==Traffic Light Overview==
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Biosensors have a wide variety of uses, from measuring blood glucose levels in diabetes patients to assessing environmental contamination by trace toxins. The majority of these sensors are highly specific for a single input, and their outputs often require specialized equipment such as surface plasmon resonance chips. Our project aims to create a biosensor that recognizes a given target and shifts its output fluorescence from green, to yellow, to red as concentrations approach critical levels (hence, a biological "traffic light").
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Subparts:<br>
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Depending on the concentration of a particular substrate in the medium, E. coli will respond accordingly by producing different coloured fluorescence proteins. A diagram would look like this:
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1. A variable sensitivity biosensor<br>
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2. A lock-and-key logic gate system
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== Biosensor ==
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[[Image:E_coli_Traffic_Light_Subprojects.png|center|thumb||600px|The ''E. coli'' Traffic Light Biosensor is composed of three major subparts: variable arabinose-inducible promoters, RNA lock and key system, and reverse antisense promoters for input detection, color activation and traffic light switching respectively.]]
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Using data derived from random mutagenesis of the pBAD promoter binding sites (5), we intend to construct a library of varying strength pBAD promoters coupled to a reporter gene.
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[[Image:UBC2009-Pbad roadmap.jpg|frame|center|pBAD mutagenesis roadmap]]
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=== The Experiments ===
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Here is what's happening inside our traffic light:
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# Mutagenesis of pBAD promoter sequence.
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# Quantification of mutant promoter-driven RFP fluorescence.
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# BioBrick design and submission.
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=== Results ===
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[[Image:E_coli_Traffic_Light_Step_by_Step.png|thumb|center|850px|Schematic black-box representation of the E. coli Biosensor that detects various concentration inputs and color outputs. The idea is discrete analog outputs based on a user-specified threshold for each range of concentration.]]
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Qualitative examination of RFP development following arabinose addition found that the weak promoter does, as expected, turn red at a significantly slower rate than the wild type promoter. However, the purported strong promoter is both slower to develop colour as well as reaching a lower maximum intensity. We are currently establishing methods for quantification and modeling of RFP induction by arabinose.
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For our ideas to work, we will need:<br>
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1. [https://2009.igem.org/Team:British_Columbia/pBAD A variable sensitivity biosensor]<br>
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2. [https://2009.igem.org/Team:British_Columbia/LockandKey A lock-and-key logic gate system]]<br>
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3. [https://2009.igem.org/Team:British_Columbia/Jammer An antisense "off" switch]
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<!-- !!!!!!!make sure the spacer is enough so that the image doesn't run into the next section -->
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<br><br><br>
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== Logic Gates ==
== Logic Gates ==
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[[Image:UBC2009-Key-lock_roadmap.jpg|700x700px|center]]
[[Image:UBC2009-Key-lock_roadmap.jpg|700x700px|center]]
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==Lock and Key==
==Lock and Key==
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As of the 2009 Jamboree, not all aspects of our project have been completed.  Looking forward, we intend to complete, test, and characterize our lock, key and jammer.  Once this is done we intend to do the same for a second set of lock, key and jammer that have different sequences and will therefore not interfere with the first set.  This second set will be configured to detect a second molecule, distinct from the first.  When this is completed, we will try adding logic gates, allowing us to address nine different combinations: high, medium and low concentrations of arabinose combined with high, medium and low concentrations of whatever we choose as our second molecule, possibly an antibiotic.  When plated on media with two perpendicular gradients, we should be able to independently control each square of a 3x3 grid, possibly displaying 9 different colors of fluorescent proteins, or generating other metabolites, such as indigo.
As of the 2009 Jamboree, not all aspects of our project have been completed.  Looking forward, we intend to complete, test, and characterize our lock, key and jammer.  Once this is done we intend to do the same for a second set of lock, key and jammer that have different sequences and will therefore not interfere with the first set.  This second set will be configured to detect a second molecule, distinct from the first.  When this is completed, we will try adding logic gates, allowing us to address nine different combinations: high, medium and low concentrations of arabinose combined with high, medium and low concentrations of whatever we choose as our second molecule, possibly an antibiotic.  When plated on media with two perpendicular gradients, we should be able to independently control each square of a 3x3 grid, possibly displaying 9 different colors of fluorescent proteins, or generating other metabolites, such as indigo.
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== Miscellaneous Data ==
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:[[Media:Biobricks.zip|Biobricks.zip]] - Fasta file containing every biobrick from [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=List Here]
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== Tools used and produced ==
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To assist our project, we produced a Biobrick digestion engine and Biobrick picture maker to help out the project:
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:http://www.pkts.ca/bb - Biobrick digestion engine - enter the name of a biobrick plasmid and biobrick insert, and this will show you the product of an EcoRI and PstI digestion/ligation as a FASTA file (suitable for viewing in your favorite program).
:http://www.pkts.ca/bb - Biobrick digestion engine - enter the name of a biobrick plasmid and biobrick insert, and this will show you the product of an EcoRI and PstI digestion/ligation as a FASTA file (suitable for viewing in your favorite program).
:http://www.pkts.ca/brickedit/ - Biobrick picture maker - enter a sequence of letters corresponding to the icons, and the program will produce a concatenated file of the Biobrick.
:http://www.pkts.ca/brickedit/ - Biobrick picture maker - enter a sequence of letters corresponding to the icons, and the program will produce a concatenated file of the Biobrick.
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== Links ==
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Also, we generated a handy Fasta file containing every biobrick from [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=List Here]:
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:[[Media:Biobricks.zip|Biobricks.zip]] - Fasta file containing every biobrick
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We also found the following tools very helpful:
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http://rna.tbi.univie.ac.at/ - a package of prediction tools for RNA structures; we used RNAfold to annotate the key and lock structures
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:http://rna.tbi.univie.ac.at/ - a package of prediction tools for RNA structures; we used RNAfold to annotate the key and lock structures
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http://mobyle.pasteur.fr/cgi-bin/portal.py - a set of web-accessible bioinformatics tools including Mfold, which determines 2D RNA structure and draws it
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:http://mobyle.pasteur.fr/cgi-bin/portal.py - a set of web-accessible bioinformatics tools including Mfold, which determines 2D RNA structure and draws it
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http://frodo.wi.mit.edu/ - Primer3, a primer design program
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:http://frodo.wi.mit.edu/ - Primer3, a primer design program

Latest revision as of 03:08, 22 October 2009

Traffic Light Overview

Depending on the concentration of a particular substrate in the medium, E. coli will respond accordingly by producing different coloured fluorescence proteins. A diagram would look like this:

The E. coli Traffic Light Biosensor is composed of three major subparts: variable arabinose-inducible promoters, RNA lock and key system, and reverse antisense promoters for input detection, color activation and traffic light switching respectively.

Here is what's happening inside our traffic light:

Schematic black-box representation of the E. coli Biosensor that detects various concentration inputs and color outputs. The idea is discrete analog outputs based on a user-specified threshold for each range of concentration.

For our ideas to work, we will need:
1. A variable sensitivity biosensor
2. A lock-and-key logic gate system]
3. An antisense "off" switch






Tools used and produced

To assist our project, we produced a Biobrick digestion engine and Biobrick picture maker to help out the project:

http://www.pkts.ca/bb - Biobrick digestion engine - enter the name of a biobrick plasmid and biobrick insert, and this will show you the product of an EcoRI and PstI digestion/ligation as a FASTA file (suitable for viewing in your favorite program).
http://www.pkts.ca/brickedit/ - Biobrick picture maker - enter a sequence of letters corresponding to the icons, and the program will produce a concatenated file of the Biobrick.

Also, we generated a handy Fasta file containing every biobrick from [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=List Here]:

Biobricks.zip - Fasta file containing every biobrick

We also found the following tools very helpful:

http://rna.tbi.univie.ac.at/ - a package of prediction tools for RNA structures; we used RNAfold to annotate the key and lock structures
http://mobyle.pasteur.fr/cgi-bin/portal.py - a set of web-accessible bioinformatics tools including Mfold, which determines 2D RNA structure and draws it
http://frodo.wi.mit.edu/ - Primer3, a primer design program
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