Team:British Columbia/Project

From 2009.igem.org

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<!--- The Mission, Experiments --->
<!--- The Mission, Experiments --->
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== 'Overview of 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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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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[[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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[[Image:E_coli_Traffic_Light_Subprojects.png|thumb||400px|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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Here is what's happening inside our traffic light:
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Biosensors have a diverse variety of real-world functions, ranging from measuring blood glucose levels in diabetes patients to assessing environmental contamination of 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 specific target and alters its output fluorescence from green, to yellow, to red as a function of concentration up to critical levels (hence, a biological "traffic light").
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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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Subparts:<br>
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For our ideas to work, we will need:<br>
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1. A variable sensitivity biosensor<br>
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1. [https://2009.igem.org/Team:British_Columbia/pBAD A variable sensitivity biosensor]<br>
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2. A lock-and-key logic gate system
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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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<br><br><br><br><br><br><br><br><br><br><br>
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<br><br><br>
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== Biosensor ==
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Using data derived from the systematic mutagenesis of the pBAD promoter binding sites (5), we constructed a library of varying strength pBAD promoters coupled to a reporter gene.
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[[Image:E_coli_Traffic_Light_Step_by_Step.png|thumb|right|600px|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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<!-- there used to be an old roadmap file: UBC2009-Pbad roadmap.jpg -->
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=== The Experiments ===
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# Mutagenesis of pBAD promoter sequence to create a stronger promoter (Strong pBAD) and a weaker promoter (Weak pBAD).
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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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We have been able to successfully show that at low arabinose concentrations, the activity of the Strong pBAD promoter and Weak pBAD promoter following arabinose induction is, as expected, greater and lesser respectively then the Wild Type pBAD promoter. By examining the development of a RFP reporter, it is observed that the Strong pBAD promoter has both a faster rate of development and reaches a higher maximum value compared to the Wild Type sequence. Similarly, the Weak pBAD promoter develops slower and to a lower maximum intensity. Additionally, we have shown that Strong pBAD and Weak pBAD are more and less responsive respectively to lower concentrations of arabinose then the Wild Type promoter. Therefore, they could be suitable to be used in conjunction as a bio-sensor.
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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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