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Revision as of 13:16, 21 July 2017
Project Description
Our project idea is to develop a biosensor to detect the presence of the bacteria Campylobacter. This sensor will utilise the rare sugar xylulose, which is found in the polysaccharide capsule of campylobacter and is released when the bacteria is run through an acidic solution. By exploiting the mannitol operon that is present in the bacteria Pseudomonas fluorescens and expressing this in our chassis organism, Escherichia Coli, we will produce a biosensor that will express the reporter molecule Green Fluorescent Protein (GFP) when xylulose interacts with the repressor molecule of the mannitol operon. Additional sub-projects will include; investigating the quorum sensing mechanisms in campylobacter to increase the specificity of our sensor, developing hardware to produce a functioning biosensor and investigating the legal and ethical issues associated with our project.
The mannitol operon contains a promoter, Pe, which is regulated by xylulose, sorbitol and mannitol. The operon typically regulates the expression of genes required for mannitol consumption. By hijacking this xylulose regulated promoter we hope to develop a construct capable of activating GFP in the presence of xylulose. A schematic of the construct is shown in the diagram opposite. To provide increased specificity to our biosensor, we are aiming to detect autoinducer-2, a quorum sensing molecule released by campylobacter. We aim to insert the LsrA promotor followed by YFP into a plasmid which turns on when autoinducer-2 is present. For our proof of concept, we need to work with xylulose. However, xylulose is rare and, as such, is expensive. Therefore, we will synthesise xylulose by utilising a metabolic step in the bacterial pentose pathway. The enzyme Xylose Isomerase can be purified from E. coli, and used to convert the inexpensive sugar xylose in to xylulose.
For the engineering part of the project, we are aiming to build a functional biosensor that will be able to prove our construct. We will use technologies such as microfluidics and ultrafiltration to produce a sensor that will use our modified E.coli to give a visual indication of the presence of campylobacter. In terms of human practices, we will be adopting a double approach; one that focuses on the legal and political issues concerning EU legislation, which will affect our ability to manufacture a biosensor outside a properly licensed laboratory; and one on ethical issues concerning the impact that food safety has on human health. This second approach will mainly aim to raise awareness among the public on preventative measures in order to avoid campylobacter poisoning.
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