Description:
Biosensors are the biological systems that are known to their property of sensing different substances and report their existence in digital (On/Off) or analog (input range) fashion. After their discovery, especially the whole-cell biosensors became a widely used tool for synthetic biology, and manipulated to detect different signals from the environment including nanoparticle toxicity and cancer. By fallowing that fashion, we design and built a whole-cell biosensor that can detect methamphetamine, which is an increasingly popular illegal drug, by expressing a genetically encoded FRET (Forster Resonance Energy Transfer) system.
As one of the classical approaches of designing a biosensor, first you need to find a sensing part to design it. During our literature mining, we found two distinct molecules that are reported to bind methamphetamine with different affinities;
Anti-METH scFv antibody [1]; which composed of two parts, we named them as C1 and C2
https://www.ncbi.nlm.nih.gov/pubmed/24419156
Alpha-synuclein[2]; a truncated model of alpha synuclein that includes amino acids 1-60 used
https://www.ncbi.nlm.nih.gov/pubmed/22771474
Starting with these biological sensor parts, our strategy then based on FRET (Forster Resonance Energy Transfer) technique, which can be simply described as the energy transfer from one excited, fluorescent molecule that results as an emission from the other fluorescent molecule, which is not excited directly by light source but emit energy from the adjacent fluorescent molecule's emission. We choose our FRET couples from the literature as reported to give signals in the most efficient manner. Our constructs formed as shown as the PDB images below;
mRuby2-asn (1-90)-cloverGFP and (Resimgelecek)
CFP-C1-C2-YFP(Resimgelecek)
After designing our biosensors, their initial analyses made by molecular dynamics simulations (shown in Molecular Dynamics section of the web site) and their production made by using commercial pET28-(a) expression plasmid and gene parts provided by IDT sponsorship.
After the production of our parts, tests for methamphetamine detection done by the help of the technical stuff at Turkish Police Narcotic Labs.
