Overview
In the design of our Campylobacter biosensor we decided to employ a genetic logic circuit. This would require the presence of two transcriptional input signals from Campylobacter-associated molecules before an output signal in the form of GFP fluorescence could be produced. We aimed to build and characterise two genetic AND-gates; one based on a split-GFP fluorescence system; and the other based on a splitting the Enterobacteria phage T7 RNA polymerase. We wished to quantify the responsiveness of both AND-gates with well-characterised small-molecule regulated promoters from the iGEM registry, and then proceed to utilising our own Campylobacter sensing transcriptional systems to drive the AND-gate biosensor. Both split-GFP and split-T7 RNAP gates were constructed from BioBrick parts and tested, however DNA sequence errors appear to have prevented the proper function of either system.
Introduction
After brainstorming our Campylobacter biosensor project idea, we began investigating any molecules that are present within - or secreted by – Campylobacter. In an ideal scenario, we would detect a small molecule that is associated with gastroenteritis-causing Campylobacter only - not in any other non-pathogenic subspecies of the Campylobacter genus – nor present in any other bacteria that might be inhabit a poultry-carcass environment. Additionally, in this ideal scenario, a well-characterised E. coli gene regulatory system with a strong output expression level would already exist.
Sensation
We identified two molecules of interest from Campylobacter for detection. One was xylulose, a 5-carbon ketopentose sugar. The second was the quorum sensing molecule autoinducer-2. Xylulose is a rare sugar most commonly associated with the pentosephosphate pathway, where xylulose-5-phosphate is an intermediate step. Interestingly, xylulose was found in the polysaccharide capsule of Campylobacter jejuni strain RM1221 (Gilbert et al., 2007). The presence of xylulose is not common in bacterial polysaccharide capsules, and the glyosidic bonds which incorporate xylulose were found to be extremely acid-labile. One sub-project aimed to exploit a xylulose-associated transcriptional activator system (mtlR-mtlE) from Pseudomonas fluorescens (mtlR). Another subproject aimed to mutagenize the well-characterised arabinose transcriptional regulatory system (araC-pBAD) from E. coli and modify its ligand specificity to the closely related structure of xylulose (araC). Our engineering subproject designed a functional prototype biosensor device that simplified a process of dissolving a swabbed input sample, processing the solution through acid and high temperature to release xylulose, and then delivering the sample to a waiting strain of genetically modified bacterial carrying the biosensor circuit for detection (hardware, applied design). Finally, purified xylulose is extremely expensive to purchase commercially, so for the testing of our biosensor we attempted to biosynthesise and purify the sugar ourselves (biosynthesis). The other molecule we identified as a marker for Campylobacter was autoinducer-2 (AI-2), a secreted quorum sensing molecule (quorum). AI-2 is a significantly less specific biomarker, as many varied gram-positive and gram-negative bacterial species sense their population density and surrounding bacterial environment using this molecule (Miller and Bassler, 2001). On the other hand, this ubiquity meant that AI-2 gene regulation was well characterised, with prior iGEM teams having worked on the natural E. coli AI-2 quorum sensing regulatory system.
The AND-gate
We hypothesised that building a combination biosensor requiring the input of two Campylobacter-associated molecules would reduce the risk of false-positives. To do this we envisaged a biosensor genetic circuit in the form of an AND-gate. In electronic circuitry, an AND-gate is a form of switch that only activates an output in the presence of two positive input signals (Figure 1).
References
