PHERAstar FSX
Powerful and most sensitive HTS plate reader
Our knowledge of the deep-sea, the largest biome on Earth, is in its infancy. Water covers more than 70% of the planet and the deep-sea accounts for more than 300 million km2 of the Earth’s surface. 1,2 Scientists have only scratched the surface of life in the ocean depths but there are intriguing benefits to studying its organisms and ecosystems. In this blog, we look at some of the bioluminescent tools that have been developed from proteins isolated from luminescent organisms flourishing in these environments.
Life under extreme conditions
At first glance, the deep-sea shrimp (Oplophorus gracilirostris) is one of the more typical looking of the 2000 or so species that make up the shrimp family. But this shrimp is no ordinary crustacean. In the dark depths where it typically lives, O. gracilirostris has the canny ability to secrete a luciferase enzyme in “brilliant luminous clouds”, a signal which it uses as a “stay-away-from-me” warning to potential predators.3
Luminescence is thought to have evolved multiple times in different branches of the tree of life. The North American firefly (Photinus pyralis), the click beatle (Pyrophorus plagiophthalamus), the sea pansy (Renilla reniformis), and the deep-sea shrimp are some of the more well studied sources of bioluminescence.
Luminescence is thought to have evolved multiple times in different branches of the tree of life. The North American firefly (Photinus pyralis), the click beatle (Pyrophorus plagiophthalamus), the sea pansy (Renilla reniformis), and the deep-sea shrimp are some of the more well studied sources of bioluminescence.
The deep-sea shrimp is not the only ocean-dwelling organism that is luminescent. The black prince copepod, Gaussia princeps, typically lives in the darkness beginning at depths 200 m below the surface. At nighttime, it feeds predominantly on phytoplankton in more shallow water after making a significant commute from the comforts of its darker haven. G. princeps is the source of GLuc, the shorthand name for its 20-kDa light-emitting luciferase. The Common Piddock mollusc (Pholas dactylus) produces Pholasin®, a photoprotein that can be used to detect the presence of free radicals and reactive species.
The discovery of bioluminescence encouraged scientists to investigate the molecular source of the bioluminescent phenomenon and started a quest to isolate the responsible proteins. Researchers eventually cloned the genes encoding different luciferase enzymes to overexpress these light-emitting proteins for further study. One of the first luciferase genes to be cloned was for the Renilla RLuc.4 The crystal structure of the luciferase from Renilla reniformis was determined in 2007. 5
The usefulness of bioluminescence as a tool to study biology at the molecular level jump started a search for alternative luciferases, in many cases from other marine organisms that were bioluminescent. But what makes the enzymes from marine organisms particularly attractive as molecular probes?
The usefulness of bioluminescence as a tool to study biology at the molecular level jump started a search for alternative luciferases, in many cases from other marine organisms that were bioluminescent. But what makes the enzymes from marine organisms particularly attractive as molecular probes?A source of technological tools
Bioluminescence occurs when luciferase enzymes oxidize the photon-emitting substrate luciferin. Luciferins typically react with molecular oxygen. The net result of the enzyme-catalyzed reaction is an intermediate of the substrate luciferin in an excited state that emits light upon decaying to its ground state.
If you were to select the ideal luciferase as a starting point for engineering a new molecular probe, it might include one that has high sensitivity, bright signal, and small size. These qualities make luciferase suitable for a wide range of applications, nimble enough for example to study protein-protein interactions, protein-small molecule interactions, gene responses or for performing compound screening.
The GLuc and OLuc luciferases from G. princeps and O. gracilirostris are significantly smaller in size (20- and 19-kDa, respectively) compared with Renilla and firefly luciferase. This property alone has prompted researchers to try and engineer them for optimal reporter qualities.
The brightest from the depths: NanoLuc
Inspired by the small size of OLuc-19, a team of scientists set out on the task to engineer a better luciferase. 3,6 The ideal enzyme should be small, monomeric and structurally stable to environmental conditions. Recombinant OLuc-19 while small in size did not retain many of the desirable qualities of its larger native enzyme.
The researchers initiated a three-step program to put this straight: a random mutagenesis step to screen for brighter luminescence; identification of a better substrate to further increase brightness; and screening using the new substrate of another random library of mutants to find even brighter bioluminescence. The endpoint for this work was the engineered luciferase NanoLuc, an enzyme with a novel substrate furimazine and improved physical and chemical features up to 100 times brighter than its Renilla and firefly luciferase counterparts.
Microplate readers: an indispensable tool for luminescence
What types of applications are possible with NanoLuc and other luciferases and how can microplate readers be used for this type of research?
In the application note NanoBRET assay for monitoring of ligand binding to GPCRs in live cells, using the CLARIOstar and the PHERAstar FS, NanoLuc was used together with an acceptor fluorophore in a NanoBRET™ or Nano Bioluminescence Resonance Energy Transfer assay to monitor for ligand binding to G-protein coupled receptors (GPCRs). GPCRs play a crucial role in the human body and many other organisms. They serve as gateways into the cell for a range of external signals, which include hormones, neurotransmitters, and other environmental stimuli. These signals lead to a cascade of intracellular signalling events that trigger downstream processes essential for ensuring the vitality of the cell. You can read more in the blog G protein-coupled receptors.
In the specific assay described in this application note, a GPCR was expressed in live HEK293 cells with NanoLuc at the N-terminus. The receptor ligand was labelled with a different fluorophore suitable for BRET in proximity-based interactions. Only blue emission of NanoLuc is detected if the ligand is not bound to the receptor. In contrast, red fluorescence is additionally detected if the ligand binds to the receptor due to excitation of the fluorescence label by resonance energy transfer from NanoLuc. The tests were performed on the CLARIOstar® Plus or PHERAstar® FSX.
The luciferase NanoBRET system was used in competitive binding experiments involving modified receptors and different fluorescently labelled ligands to determine IC50 and KD values. NanoBRET was shown to be a viable alternative to existing methods for the determination of ligand binding to GPCRs. It has the advantage of being a high-throughput method that does not require the use of radioactivity.
Luciferases also find applications in determining the genotoxic properties of different chemicals. This type of screening is essential to ensure the safety of pharmaceuticals, industrial chemicals and products like those used for personal care.
The application note BlueScreen HC - a luminescence based, high-throughput, in vitro genotoxicity assay describes the use of the GLuc luciferase from G. princeps to measure genotoxicity. In this luciferase assay, exposure to a genotoxic chemical causes increased expression of the GADD45a gene which is coupled to the luciferase reporter system. GADD45a stands for growth arrest and DNA damage inducible alpha. In organisms harboring this gene, its transcript levels increase following stressful growth arrest conditions or treatment with DNA-damaging agents. These properties have been used to construct an in vitro test for mutagenicity which is at the heart of BlueScreen HC™.
BlueScreen HC™ from Gentronix is a high-throughput genotoxicity test that uses a genetically engineered TK6 cell line and is suitable for miniaturization. The cells are engineered to have the GADD45a gene coupled to the G. princeps luciferase. The amount of luciferase produced by the BlueScreen HC cell lines is assessed by injection of the substrate coelenterazine, which results in a short-lived flash luminescence signal.
![Fig.5: BlueScreen HC S9 assay positive cytotoxicity (a) and genotoxicity (b) results for 20 µg/ml benzo[a]pyrene. Error bars show ±1 standard deviation based on 4 replicate analyses on separate microplates.](https://www.bmglabtech.com/hubfs/1_Webseite/5_Resources/Blogs/deep-sea-blog-Fig.5.webp)
Exposure to a genotoxic chemical gives a dose-dependent increase in the production of luciferase from the GADD45a reporter gene.
In addition, reduced cell proliferation, a measure of cytotoxicity, was assessed either by changes in absorbance in BlueScreen HC or in fluorescence from a DNA stain in BlueScreen HC S9. Genotoxicity and cytotoxicity were measured simultaneously using flash luminescence, absorbance, and fluorescence measurements. BlueScreen HC and BlueScreen HC S9 are fully compatible with BMG Labtech microplate readers using the three detection modes and reagent injector systems. The combination of microplate reader and assay kits provided fast, accurate and reproducible results for the detection and quantification of genotoxic liability for chemicals with different potencies and modes of action.
A crucial part of making accurate and reliable luciferase measurements is the need to maintain cell health. The Atmospheric Control Unit (ACU) on the CLARIOstar is ideal for this purpose and helps maintain cells under precisely defined conditions so that long-term cell-based assays can be carried out under different experimental conditions.
In the application note The new Atmospheric Control Unit (ACU) for the CLARIOstar provides versatility in long-term cell-based assays. The CLARIOstar microplate reader and ACU was able to fully sustain the normal proliferation and health of untreated K562 cells for the entire duration of experiments. This was essential to permit the detection of time-dependent and dose-dependent effects on cell proliferation and cytotoxicity due to the tested compounds.
In this case, NanoLuc luciferase was used to measure cell viability (RealTime-Glo® MT Cell Viability Assay). The RealTime-Glo® MT Cell Viability Assay is a bioluminescent test that relies on the metabolic (MT) reducing potential of cells. NanoLuc luciferase and cell-permeant pro-NanoLuc® substrate were added to cells in culture. Viable cells reduce the pro-NanoLuc® substrate which diffuses into the cell medium. In the medium, the substrate is rapidly converted by NanoLuc luciferase to emit luminescence proportional to the number of viable cells. 
In the study, the effect of the tyrosine kinase inhibitor bosutinib was measured over time for its impact on cytotoxicity and cell viability. The ACU was shown to sustain cells for 72 hours which was the duration of the subsequent experiments with the inhibitor.
GLuc and NanoLuc like other luciferases can be used in dual luciferase reporter assays which are widely used to investigate gene transcription and regulation. Today researchers have several options to take advantage of dual-reporter formats and you can read more about the different options in the BMG LABTECH blog Wavelength based dual glow reporter genes.
BMG LABTECH microplate readers allow simultaneous dual detection and can therefore measure emission at two wavelengths at the same time. This reduces the read time in half which is a significant advantage over microplate readers that require sequential measurements. Simultaneous dual detection also helps to reduce variability due to bubbles, temperature effects and fluid movement. This makes BMG Labtech microplate readers the ideal choice for dual luciferase reporter assays.
BMG LABTECH solutions
What is the preferred BMG LABTECH microplate reader for specific needs and applications related to luminescence?
Luminescence detection can be performed on BMG LABTECH´s dedicated luminescence plate reader LUMIstar® Omega, and multi-mode microplate readers including the PHERAstar FSX, CLARIOstar Plus, VANTAstar® and FLUOstar® Omega.
All of our luminescence plate readers that are certified for Dual-Luciferase Reporter assays utilize luminescence-optimized low-noise PMT (photomultiplier tube) and can be equipped with high-precision reagent injectors.
All BMG LABTECH microplate readers have exceptionally fast reading capabilities. Collectively, these multi-mode readers combine high performance with miniaturized assays, short measurement times, and offer considerable savings on materials and other resources.
Configure your microplate reader and get an initial recommendation!
References
- National Geographic, The five major types of biomes, https://education.nationalgeographic.org/resource/five-major-types-biomes/
- Miller KA, Thompson KF, Johnston P, Santillo D (2018) An overview of seabed mining including the current state of development, environmental impacts, and knowledge gaps. Frontiers in Marine Science https://doi.org/10.3389/fmars.2017.00418
- Hall MP, Unch J, Binkowski BF, Valley MP, Butler BL, Wood MG, Otto P, Zimmerman K, Vidugiris G, Machleidt T, Robers MB, Benink HA, Eggers CT, Slater MR, Meisenheimer PL, Klaubert DH, Fan F, Encell LP, Wood KV. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol. 2012 Nov 16;7(11):1848-57. doi: 10.1021/cb3002478. Epub 2012 Aug 30. PMID: 22894855; PMCID: PMC3501149.
- Lorenz WW, McCann RO, Longiaru M, Cormier MJ. Isolation and expression of a cDNA encoding Renilla reniformis luciferase. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4438-42. doi: 10.1073/pnas.88.10.4438. PMID: 1674607; PMCID: PMC51675.
- Loening AM, Fenn TD, Gambhir SS. Crystal structures of the luciferase and green fluorescent protein from Renilla reniformis. J Mol Biol. 2007 Dec 7;374(4):1017-28. doi: 10.1016/j.jmb.2007.09.078. Epub 2007 Oct 3. PMID: 17980388; PMCID: PMC2700051.
- England CG, Ehlerding EB, Cai W. NanoLuc: A Small Luciferase Is Brightening Up the Field of Bioluminescence. Bioconjug Chem. 2016 May 18;27(5):1175-1187. doi: 10.1021/acs.bioconjchem.6b00112. Epub 2016 Apr 19. PMID: 27045664; PMCID: PMC4871753.
- Stoddart LA, Kilpatrick LE, Briddon SJ, Hill SJ. Probing the pharmacology of G protein-coupled receptors with fluorescent ligands. Neuropharmacology. 2015 Nov;98:48-57. doi: 10.1016/j.neuropharm.2015.04.033.
