Team:Queens Canada/Results
Results
Experiments: This summer QGEM accomplished a lot of work in the laboratory! We have 1) validated the Engineered NanoLuc luciferase as an choice reporter in our intein-based biosensor construct, 2) We characterized the insolubility of a small-molecule triggered intein splicing system in E. Coli, 3) We validated the nuclear receptor ligand binding as a sensitive domain for analyte detection through the successful assembly of a reagent-less, and continuous FRET-based biosensor with the ability to discern between agonist and antagonists.
1) Validation of NanoLuc Luciferase for application in our Diagnostic Pacifier
To create our desired portable intein-splicing biosensor, we needed to select a signal that could be measured and correlated to the amount of analyte present. Additionally, we had the challenge of selecting a reporter that could be produced and quantified very portably. Our first idea was to use a fluorescent reporter, however with fluorescence, a fluorophore must first be excited by a light, and then a light is given off that needs to filter and its intensity measured. Fluorimeter are bulky devices, and after further investigation, it become apparent that it would be possible the machinery down to fit with a pacifier. However, during our investigations we learned about the elements needed to build a luminometer and found that it would be entirely feasible for us to produce a simplified photon-counter device that could fit into a pacifier! (See Hardware section)To best improve our chances of being able to produce a signal which our portable luminometer could detect we investigated various the relative strength of luciferases. We ultimately decided on testing the novel and very bright NanoLuc Luciferase as the quantitative reporter for our construct. NanoLuc® developed by Promega is a luciferase derived via directed evolution from the luminous shrimp, Oplophorus gracilirostris [4]. The enzyme was obtained from deep-sea shrimp and optimized following the discovery of a novel substrate, furimazine, which allows for the production of visible light with less background activity than other luciferases [4,5]. NanoLuc® is a relatively small, a19.1 kDa monomeric protein that is both soluble and ATP-independent [4]. Compared to firefly (Lampyridae) and sea pansy (Renilla) luciferases, this novel protein offers many advantages reflected by its increased stability, smaller size, and >150-fold increase in luminescence [5]. The unique characteristics of this enzyme construct combined with its high luminescence activity allow for the production of a very sensitive diagnostic assay.
By a generous donation from Promega, we received the promoter-less vector pNL1.1. We then cloned an Anderson Constitutive promoter (Part:BBa_J23100) into the MCS of pNL1.1. To test our construct, we grew an overnight liquid culture of E. Coli expressing the plasmid construct. We pelleted 2-3ml of liquid culture, resuspended in a lytic buffer with lysozyme and protease inhibitor for 30 mins, before sonicating for 10 seconds the placing on ice, twice. We then pelleted the insoluble fraction, collected the supernatant and performed a Luciferase Assay, using Nano-Glo® Luciferase Assay System. Relative light units were measured in a Lumistar Galaxy plate reader. We then took an initial measurement of Relative Light Unit and found a strong result of 38598.2±3385.40 RLU (n=4), compared to 103.5±33.6 RLU (n=5), with our negative control at peak luminescence. Since the ultimate goal of using NanoLuc will be to produce a signal for diagnostic purposes, we sought to further characterize the time-course of NanoLuc Luciferase after the addition of substrate. Through taking measurements every 0.05 seconds, we were able to produce a curve demonstrating the time till plateau of NanoLuc after addition of substrate. Within 10 seconds, NanoLuc had reached its plateau, therefore validating NanoLuc would be an excellent reporter protein for application in our intein-based diagnostic biosensor.
We next sough to characterize the sensitivity of our portable luminometer to the luminescence produced by NanoLuc. The portable luminometer measures differently than our labs luminometer, as the Lumistar Glaxay luminometer in our lab takes an instantaneous measurement of light, in relative light units. Whereas, our built luminometer measures photons/second. Therefore, the two devices cannot be directly compared. For calibration of our luminometer we first confirmed the sample to be luminescing by a measurement with the Lumistar Galaxy Luminometer, we then measured samples diluted 1, 2, and 4-fold and found our luminometer was capable of measuring a linear relationship between the concentrations of our sample. Thus confirming our luciferase construct was functional and rised to plateau quickly, our built portable luminometer was accurate across a range of concentrations, and that Nanoluc Luciferase could serve as a viable signal for our diagnostic assays.
