Team:British Columbia/Project

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The Traffic Light Biosensor: A flexible, modular, and transparent system for multi-level assessment of variable inputs.

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").

Subparts:
1. A variable sensitivity biosensor
2. A lock-and-key logic gate system

Biosensor

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.

pBAD mutagenesis roadmap

The Experiments

1. Mutagenesis of pBAD promoter sequence.
2. Quantification of mutant promoter-driven RFP fluorescence.
3. BioBrick design and submission.

Results

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.

Logic Gates

In 2004, Isaacs et al. (4) constructed a lock and key system termed the riboregulator. Briefly, the riboregulator consists of a lock sequence immediately 5' of and complementary to the RBS; upon transcription, the lock binds to the RBS and prevents translation of the mRNA. This binding can be "unlocked" by the introduction of a "key" mRNA, which disrupts the binding between the lock and RBS and allows translation to resume.

We intend to convert the lock and key as described by Isaacs et al. into standardized BioBrick parts and integrate them with our library of variable strength promoters in order to create a multi-level logic gate system. We also hope to design an additional component, termed the "jammer", which prevents the key from unlocking the lock.

Roadmap

Lock and Key

The lock is a short DNA segment that is the reverse-complement of the lock.

Miscellaneous Data

Biobricks.zip - Fasta file containing every biobrick from [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=List Here]

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.

Links

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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