Team:UCC Ireland/methanol biosensor

Methanol Biosensor Background: Organisms can experience exposure to genotoxic chemicals like formaldehyde and have evolved systems to detect and protect from exposure to formaldehyde. For our biosensor, we decided to exploit this. The FrmR protein is a DNA-binding transcriptional regulator that is specifically inactivated in the presence of formaldehyde and is naturally found in bacteria. FrmR is part of the E. coli frmRAB operon. It is the first gene and is known as EcFrmR, a member of the CsoR/RcnR family of transcriptional repressors. It binda a formaldehyde dehydrogenase FrmA and an S-formylgluthathione hydrolyase FrmB. Normally EcFrmR forms a complex with the frmRAB promoter (Pfrm). The formaldehyde-mediated cross-linking remodels the surface of the tetrameric EcFrmR resulting in the de-repression of the frmRAB expression by promoting dissociation from the complex. The complex normally regulates transcription of downstream DNA in the absence of formaldehyde. Many experiments have been shown to corroborate this mechanism (Katie, J Denby et. al.). Experimental Approach: Our experimental approach for testing the methanol biosensor was planned to take place in three stages in order to answer the following questions following creation of our biosensor (outlined below) in pRSFduet vector. 1. Is the production of sfGFP repressed in the absence of methanol? 2. What concentrations of methanol induce production of sfGFP? 3. Is the biosensor sensitive enough to produce detectable levels of sfGFP when placed in alcoholic beverages? This approach was planned in parallel to our erythromycin biosensor to examine the same aspects of its functionality. We aimed firstly to show that our system worked and production of sfGFP could be specifically induced by methanol. Secondly, we aimed to examine the sensitivity of our biosensor and determine a suitable range of methanol concentration that the system could be induced with. Finally, we wished to examine whether our biosensor worked in working conditions i.e. in an alcoholic beverage. This approach was designed to systematically validate the design of our biosensor and use it as a platform to further refine the detection of methanol within the context of our universal biosensor system. Construct design: Fig. 1. Methanol inducible expression of sfGFP. Both MDH and FrmR are constitutively expressed by the J23101 promoter. MDH breaks down methanol when it enters the system, forming formaldehyde. In the absence of formaldehyde, FrmR is thought to bind to the Fmr(A) inducible promoter, inhibiting expression of sfGFP. When formaldehyde enters the system following breakdown of methanol by MDH, FrmR will be displaced from the promoter, allowing expression of sfGFP. Experiments: Unfortunately, our experiments with the methanol biosensor were stalled at the first hurdle. Like the erythromycin biosensor construct, both the MDH and FrmR repressed system were successfully inserted into the pRSFduet vector which was confirmed by sequencing. However, upon induction with methanol and formaldehyde, no fluorescence was observed. Due to time constraints, we were not successful in achieving a response to methanol or formaldehyde from the system. Future Plans: Once the construct is successfully inserted into the pRSFduet vector and responds to methanol, the range of concentrations that are detectable will be determined using fluorescence assays. If the legal limit of methanol lies outside of this range, site-directed mutagenesis experiments will be carried out to adjust the level of sensitivity of the system. In accordance with our overall biosensor strategy, the plasmid will be introduced to a cell free chassis and optimised for use by home distillers and microbreweries/microdistilleries in a lypholised paper system. References: Denby, K., Iwig, J., Bisson, C., Westwood, J., Rolfe, M., Sedelnikova, S., Higgins, K., Maroney, M., Baker, P., Chivers, P. and Green, J. (2016). The mechanism of a formaldehyde-sensing transcriptional regulator. Scientific Reports, 6(1).