Team:British Columbia/Project

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

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.

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").

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

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.

Lock and Key

The key is a short RNA segment that is the reverse-complement of the lock, which binds to the lock, unfolding the hairpin that conceals the ribosome binding site. Ribosomes should then start translating the RNA that was locked.

Jammer

Our analog biosensor requires a component to turn off designed lights (i.e. reporter genes) when the target passes a certain threshold. To do so, we have designed a modular, endogenous RNA knockdown system using antisense RNA hybridization. We have independently conceived of using a reverse promoter, that was first described by O’Connor and Timmis (1987), to generate an antisense transcript that hybridizes to the sense transcript, to reduce gene expression.

This is accomplished at the RNA-level through both transcription and translation interfaces. First, when the reverse promoter recruits RNA polymerases, they transcribe in the antisense direction. This causes the sense and anti-sense oriented polymerases to collide, thus reducing transcription (Ward and Murray, 1979; Crampton et al. 2006).

Roadmap

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.

Miscellaneous Data

We also produced a couple tools to help out the project:

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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-== Biosensor ==
-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.
-[[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.]]
-<!-- there used to be an old roadmap file: UBC2009-Pbad roadmap.jpg -->
-=== The Experiments ===
-# Mutagenesis of pBAD promoter sequence to create a stronger promoter (Strong pBAD) and a weaker promoter (Weak pBAD).
-# Quantification of mutant promoter-driven RFP fluorescence.
-# BioBrick design and submission.
-=== Results ===
-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.
 == Logic Gates ==