Application Of Bioluminescence Research Articles

Biosynthesis-inspired deracemizative production of d-luciferin by combining luciferase and thioesterase

Due to the strict enantioselectivity of firefly luciferase, only d-luciferin can be used as a substrate for bioluminescence reactions. Unfortunately, luciferin racemizes easily and accumulation of nonluminous l-luciferin has negative influences on the light emitting reaction. Thus, maintaining the enantiopurity of luciferin in the reaction mixture is one of the most important demands in bioluminescence applications using firefly luciferase. In fireflies, however, l-luciferin is the biosynthetic precursor of d-luciferin, which is produced from the L-form undergoing deracemization. This deracemization consists of three successive reactions: l-enantioselective thioesterification by luciferase, in situ epimerization, and hydrolysis by thioesterase. In this work, we introduce a deracemizative luminescence system inspired by the biosynthetic pathway of d-luciferin using a combination of firefly luciferase from Luciola cruciata (LUC-G) and fatty acyl-CoA thioesterase II from Escherichia coli (TESB). The enzymatic reaction property analysis indicated the importance of the concentration balance between LUC-G and TESB for efficient d-luciferin production and light emission. Using this deracemizative luminescence system, a highly sensitive quantitative analysis method for l-cysteine was constructed. This LUC-G-TESB combination system can improve bioanalysis applications using the firefly bioluminescence reaction by efficient deracemization of D-luciferin.

Smartphone-based low light detection for bioluminescence application

We report a smartphone-based device and associated imaging-processing algorithm to maximize the sensitivity of standard smartphone cameras, that can detect the presence of single-digit pW of radiant flux intensity. The proposed hardware and software, called bioluminescent-based analyte quantitation by smartphone (BAQS), provides an opportunity for onsite analysis and quantitation of luminescent signals from biological and non-biological sensing elements which emit photons in response to an analyte. A simple cradle that houses the smartphone, sample tube, and collection lens supports the measuring platform, while noise reduction by ensemble averaging simultaneously lowers the background and enhances the signal from emitted photons. Five different types of smartphones, both Android and iOS devices, were tested, and the top two candidates were used to evaluate luminescence from the bioluminescent reporter Pseudomonas fluorescens M3A. The best results were achieved by OnePlus One (android), which was able to detect luminescence from ~106 CFU/mL of the bio-reporter, which corresponds to ~107 photons/s with 180 seconds of integration time.

Reported Bioluminescent Organisms on Land and in the Sea

Some living organisms produce light, thus production of cold light by living organisms is called bioluminescence. Bioluminescence is emission of light as a result of a chemical reaction inside the living organism during the process chemical energy gets converted into light energy. It is a form of chemiluminescence. Bioluminescent organisms are found in marine and terrestrial environments. But bioluminescence is most prevalent in marine organisms. Marine bioluminescence is produced by incredible range of organisms. Bioluminescence producing living organisms produce light and utilize it for vital functions ranging from camouflage, illumination, mimicry, attracting prey, attracting mates, for protection and it may be most common form of communication on the planet. bioluminescence responsible genes are not related in bacteria, algae, fishes, beetles and others because it has evolved independently many times. Bioluminescent organisms can be harnessed to produce natural light in the lamps that could light up our planet one day. In addition to that bioluminescent applications can be used to test extra-terrestrial life on other planets so bioluminescence has scope for many futuristic applications. While, bioluminescence research has offered many potential societal benefits in fields ranging from genetic engineering to medicine. By helping to reveal the evolutionary origins of bioluminescence, we can better know how other complex features arise in nature. So there is greater need study bioluminescence and the organisms producing bioluminescence. In this process scientists continue to discover more bioluminescent species.

New bioluminescent coelenterazine derivatives with various C-6 substitutions.

A series of new coelenterazine analogs with varying substituents at the C-6 position of the imidazopyrazinone core have been designed and synthesized for the extension of bioluminescence substrates. Some of them display excellent bioluminescence properties compared to DeepBlueC™ or native coelenterazine with both in vitro and in vivo biological evaluations, thus placing these derivatives among the most ideal substrates for Renilla bioluminescence applications.

Transcriptome analysis reveals candidate genes involved in luciferin metabolism in Luciola aquatilis (Coleoptera: Lampyridae).

Bioluminescence, which living organisms such as fireflies emit light, has been studied extensively for over half a century. This intriguing reaction, having its origins in nature where glowing insects can signal things such as attraction or defense, is now widely used in biotechnology with applications of bioluminescence and chemiluminescence. Luciferase, a key enzyme in this reaction, has been well characterized; however, the enzymes involved in the biosynthetic pathway of its substrate, luciferin, remains unsolved at present. To elucidate the luciferin metabolism, we performed a de novo transcriptome analysis using larvae of the firefly species, Luciola aquatilis. Here, a comparative analysis is performed with the model coleopteran insect Tribolium casteneum to elucidate the metabolic pathways in L. aquatilis. Based on a template luciferin biosynthetic pathway, combined with a range of protein and pathway databases, and various prediction tools for functional annotation, the candidate genes, enzymes, and biochemical reactions involved in luciferin metabolism are proposed for L. aquatilis. The candidate gene expression is validated in the adult L. aquatilis using reverse transcription PCR (RT-PCR). This study provides useful information on the bio-production of luciferin in the firefly and will benefit to future applications of the valuable firefly bioluminescence system.

RAPD-PCR as a Molecular Discriminative Technique for Human Pathogenic Bacteria - A Review

The application of random amplified polymorphic DNA- polymerase chain reaction (RAPD-PCR) was found to be a simple, cheap and rapid tool to discriminate human pathogenic bacterial isolates especially at intraspecific level. This molecular biological technique relies on the use of random oligonucleotide primers that arbitrarily amplifies specific regions of the genome which gives rise to a unique genomic fingerprint of the strains under investigations. With continued development of novel molecular-based technologies for rapid, high-throughput detection of food borne pathogenic bacteria, the future of conventional microbiological methods such as viable cell enumeration, selective isolation of bacteria on commercial media, and immunoassays seems tenuous. Approaches that enhance recovery of sub lethally injured bacteria, differentiation among species, differentiation among bacteria of interest using biochemical profiling, enumeration using impedance technology, techniques to confirm the presence of target pathogens using immunological methods, and bioluminescence applications for hygiene monitoring are of utmost need in identifying and combating the human pathogenic isolates. The aim of this study is to esti­mate the efficiency of RAPD-PCR technique in assessing the genetic diversity of diseases causing bacterial isolates. The use of RAPD-PCR in evaluating the genomic variability among the pathogenic strains belonging to different genus are also been discussed in the present report.

Plasmid Variance and Nutrient Regulation of Bioluminescence Genes

Bioluminescence, the natural production of light by living organisms, may one day compete with electrical lighting as a form of light production. The limited gene expression of bioluminescent proteins is one hindrance preventing the widespread application of bioluminescence. The purpose of this experiment is to increase expression of Aliivibrio fischeri bioluminescence genes, study the effects of increased expression, and examine the dependence of bioluminescence on nutrient concentration. Discovering the connection between nutrient dependency and light production with A. fischeri could open new possibilities to receive maximal bioluminescence. Expression of A. fischeri genes from a high copy plasmid is expected to increase the gene expression and bioluminescence of Escherichia coli. We hypothesize that because of an increase in the gene expression of the 
 
 A. fischeri lux operon, the production of bioluminescence should be more dependent on nutrients, which provide cofactors and substrates for the bioluminescence reaction. After transfer of the A. fischeri genomic fragment to the high copy plasmid, the bioluminescence of the host E. coli increased significantly. Furthermore, bacterial growth seen in E. coli containing the high copy plasmid decreased. A complex pattern of regulation was seen after exposing the bioluminescent E. coli to various nutrients. Collectively, these findings demonstrate that the higher expression of bioluminescence genes, achieved with a plasmid with a higher copy number, did not render bioluminescent E. coli more dependent on nutrients for bioluminescence.

BLProt: prediction of bioluminescent proteins based on support vector machine and relieff feature selection.

BackgroundBioluminescence is a process in which light is emitted by a living organism. Most creatures that emit light are sea creatures, but some insects, plants, fungi etc, also emit light. The biotechnological application of bioluminescence has become routine and is considered essential for many medical and general technological advances. Identification of bioluminescent proteins is more challenging due to their poor similarity in sequence. So far, no specific method has been reported to identify bioluminescent proteins from primary sequence.ResultsIn this paper, we propose a novel predictive method that uses a Support Vector Machine (SVM) and physicochemical properties to predict bioluminescent proteins. BLProt was trained using a dataset consisting of 300 bioluminescent proteins and 300 non-bioluminescent proteins, and evaluated by an independent set of 141 bioluminescent proteins and 18202 non-bioluminescent proteins. To identify the most prominent features, we carried out feature selection with three different filter approaches, ReliefF, infogain, and mRMR. We selected five different feature subsets by decreasing the number of features, and the performance of each feature subset was evaluated.ConclusionBLProt achieves 80% accuracy from training (5 fold cross-validations) and 80.06% accuracy from testing. The performance of BLProt was compared with BLAST and HMM. High prediction accuracy and successful prediction of hypothetical proteins suggests that BLProt can be a useful approach to identify bioluminescent proteins from sequence information, irrespective of their sequence similarity. The BLProt software is available at http://www.inb.uni-luebeck.de/tools-demos/bioluminescent%20protein/BLProt

High-Throughput Bioluminescence-Based Mutant Screening Strategy for Identification of Bacterial Virulence Genes

A high-throughput bioluminescence screening procedure for identification of virulence genes in bacteria was developed and applied to the fish pathogen Edwardsiella ictaluri. A random transposon mutant library expressing bioluminescence was constructed and robotically arrayed on 384-well plates. Mutants were cultivated and mixed with catfish serum and neutrophils in 96-well plates, and bioluminescence was used to detect mutants that are more susceptible to killing by these host factors. The virulence and vaccine efficacy of selected mutants were determined in channel catfish. Transposon insertion sites in 13 mutants attenuated in the natural host were mapped to the E. ictaluri genome. Ten unique genes were mutated, including genes encoding a negative regulator of sigmaE activity, a glycine cleavage system protein, tricarboxylic acid cycle enzymes, an O polysaccharide biosynthesis enzyme, proteins encoded on the native plasmid pEI1, and a fimbrial chaperon protein. Three of these mutants were found to have potential as live attenuated vaccines. This study demonstrates a novel application of bioluminescence to identify bacterial genes required for host resistance; as a result, efficacious and genetically defined live attenuated vaccine candidates were developed.

Molecular Imaging Provides Novel Insights on Estrogen Receptor Activity in Mouse Brain

Estrogen receptors have long been known to be expressed in several brain areas in addition to those directly involved in the control of reproductive functions. Investigations in humans and in animal models suggest a strong influence of estrogens on limbic and motor functions, yet the complexity and heterogeneity of neural tissue have limited our approaches to the full understanding of estrogen activity in the central nervous system. The aim of this study was to examine the transcriptional activity of estrogen receptors in the brain of male and female mice. Exploiting the ERE-Luc reporter mouse, we set up a novel, bioluminescence-based technique to study brain estrogen receptor transcriptional activity. Here we show, for the first time, that estrogen receptors are similarly active in male and female brains and that the estrous cycle affects estrogen receptor activity in regions of the central nervous system not known to be associated with reproductive functions. Because of its reproducibility and sensitivity, this novel bioluminescence application stands as a candidate as an innovative methodology for the study and development of drugs targeting brain estrogen receptors.

Noninvasive Bioluminescence Imaging in Small Animals

There has been a rapid growth of bioluminescence imaging applications in small animal models in recent years, propelled by the availability of instruments, analysis software, reagents, and creative approaches to apply the technology in molecular imaging. Advantages include the sensitivity of the technique as well as its efficiency, relatively low cost, and versatility. Bioluminescence imaging is accomplished by sensitive detection of light emitted following chemical reaction of the luciferase enzyme with its substrate. Most imaging systems provide 2-dimensional (2D) information in rodents, showing the locations and intensity of light emitted from the animal in pseudo-color scaling. A 3-dimensional (3D) capability for bioluminescence imaging is now available, but is more expensive and less efficient; other disadvantages include the requirement for genetically encoded luciferase, the injection of the substrate to enable light emission, and the dependence of light signal on tissue depth. All of these problems make it unlikely that the method will be extended to human studies. However, in small animal models, bioluminescence imaging is now routinely applied to serially detect the location and burden of xenografted tumors, or identify and measure the number of immune or stem cells after an adoptive transfer. Bioluminescence imaging also makes it possible to track the relative amounts and locations of bacteria, viruses, and other pathogens over time. Specialized applications of bioluminescence also follow tissue-specific luciferase expression in transgenic mice, and monitor biological processes such as signaling or protein interactions in real time. In summary, bioluminescence imaging has become an important component of biomedical research that will continue in the future.

Luciferase from the Italian firefly Luciola italica: Molecular cloning and expression

The cDNA encoding the luciferase from the Italian firefly Luciola italica was cloned using reverse transcriptase-PCR and a gene-specific primer set based on the DNA sequence of Luciola mingrelica. The cDNA sequence of L. italica luciferase was determined to be 1647 base pairs in length with an open reading frame of 548 amino acids. Phylogenetic analysis of the protein sequence demonstrated that this luciferase is closely related to that of other fireflies of the Lampyridae family, particularly within the Luciolinae subfamily, showing 96% homology to luciferases from the fireflies Hotaria unmunsana and Hotaria parvula. The specific activity of the L. italica luciferase was 78% of the North American enzyme, after correction for emission color differences. The bioluminescence emission of the Italian firefly is pH sensitive with maxima at 566 nm and 614 nm at pH 7.8 and 6.0, respectively. Interestingly, the total bioluminescence output was ∼ 2-fold greater than that of P. pyralis luciferase due to differences in turnover characteristics evidenced by extended light emission decay kinetics. We expect that this newly discovered luciferase will be suitable for a wide range of bioluminescence applications including in vivo imaging and multiplex assays.

Bioluminescence regenerative cycle (BRC) system: Theoretical considerations for nucleic acid quantification assays

A novel application of bioluminescence for nucleic acid quantification, the bioluminescence regenerative cycle (BRC), is described in theoretical terms and supported by preliminary experimental data. In the BRC system, pyrophosphate (PPi) molecules are released during biopolymerization and are counted and correlated to DNA copy number. The enzymes ATP-sulfurylase and firefly luciferase are employed to generate photons quantitatively from PPi. Enzymatic unity-gain positive feedback is implemented to amplify photon generation and to compensate for decay in light intensity by self-regulation. The cumulative total of photons can be orders of magnitude higher than in typical chemiluminescent processes. A system level theoretical model is developed, taking into account the kinetics of the regenerative cycle, contamination, and detector noise. Data and simulations show that the photon generation process achieves steady state for the time range of experimental measurements. Based on chain reaction theory, computations show that BRC is very sensitive to variations in the efficiencies of the chemical reactions involved and less sensitive to variations in the quantum yield of the process. We show that BRC can detect attomolar quantities of DNA (10 −18 mol), and that the useful dynamic range is five orders of magnitude. Sensitivity is not constrained by detector performance but rather by background bioluminescence caused by contamination by either PPi or ATP (adenosine triphosphate).

BIOLUMBASE?the database of natural and transgenic bioluminescent organisms

The Institute of Biophysics SB RAS hosts and maintains a specialized collection of luminous bacteria (CCIBSO 836) containing over 700 strains isolated in various regions of the world's oceans. The culture collection is a source of lux genes and biologically active substances. The wide application of bioluminescence in medicine and ecology has given importance to analysing information on the structure and functioning of bioluminescence systems in natural and transgenic microorganisms, as well as on their features that are closely interrelated with bioluminescence. The aims of our BIOLUMBASE database are: gathering information on microorganisms with lux genes, their analysis and free access, and distribution of this data throughout the global network. The database includes two sections, natural and transgenic luminous microorganisms, and is updated by our own experimental results, the published literature and internet resources. For the future, a publicly available internet site for BIOLUMBASE is planned. This will list the strains and provide comprehensive information on the properties and functions of luminous bacteria, the mechanisms of regulation of bioluminescence systems, constructs with lux genes, and applications of bioluminescence in microbiology, ecology, medicine and biotechnology. It is noteworthy that this database will also be useful for evaluation of biological hazards of transgenic strains. Users will be able to carry out bibliographic and strain searches starting from any feature of interest.

A review of conventional detection and enumeration methods for pathogenic bacteria in food

With continued development of novel molecular-based technologies for rapid, high-throughput detection of foodborne pathogenic bacteria, the future of conventional microbiological methods such as viable cell enumeration, selective isolation of bacteria on commercial media, and immunoassays seems tenuous. In fact, a number of unique approaches and variations on existing techniques are currently on the market or are being implemented that offer ease of use, reliability, and low cost compared with molecular tools. Approaches that enhance recovery of sublethally injured bacteria, differentiation among species using fluorogenics or chromogenics, dry plate culturing, differentiation among bacteria of interest using biochemical profiling, enumeration using impedence technology, techniques to confirm the presence of target pathogens using immunological methods, and bioluminescence applications for hygiene monitoring are summarized here and discussed in relation to their specific advantages or disadvantages when implemented in a food microbiology setting.

Applications of bioluminescence- and fluorescence resonance energy transfer to drug discovery at G protein-coupled receptors.

Bioluminescence (BRET)- and fluorescence resonance energy transfer (FRET) techniques have become integral approaches in studies of protein-protein interactions in living cells. They rely on non-radiative transfer of energy between donor and acceptor species that can be appended to the proteins of interest. These techniques display exquisite dependence on distance and orientation between the energy transfer partners. This means they are well suited to measure both small conformational changes in response to ligand binding between partner proteins that remain within a complex or more extensive translocations of proteins between cellular compartments that occur in response to cellular challenge. Introduction of both energy donor and acceptor into a single polypeptide can also allow the detection of ligand-induced conformational switches in monomeric proteins in the millisecond time scale. Many of these approaches are amenable to high throughput screening and the drug discovery process. G protein-coupled receptors (GPCRs) represent a key drug target class. Specific applications of resonance energy transfer techniques to the identification of ligands for this class of protein are highlighted to illustrate general principles.

Basic and Applications of Bioluminescence and Fluorescence Imaging in Bioscience and Physiological Research

Basic and Applications of Bioluminescence and Fluorescence Imaging in Bioscience and Physiological Research

Bioluminescence and chemiluminescence in drug screening

Drug screening, that is, the evaluation of the biological activity of candidate drug molecules, is a key step in the drug discovery and development process. In recent years, high-throughput screening assays have become indispensable for early stage drug discovery because of the developments in synthesis technologies, such as combinatorial chemistry and automated synthesis, and the discovery of an increasing number of new pharmacological targets. Bioluminescence and chemiluminescence represent suitable detection techniques for high-throughput screening because they allow rapid and sensitive detection of the analytes and can be applied to small-volume samples. In this paper we report on recent applications of bioluminescence and chemiluminescence in drug screening, both for in vitro and in vivo assays. Particular attention is devoted to the latest and most innovative bioluminescence and chemiluminescence-based technologies for drug screening, such as assays based on genetically modified cells, bioluminescence resonance energy transfer (BRET)-based assays, and in vivo imaging assays using transgenic animals or bioluminescent markers. The possible relevance of bioluminescence and chemiluminescence techniques in the future developments of high-throughput screening technologies is also discussed.

Bioluminescent bacteria: lux genes as environmental biosensors

Bioluminescent bacteria are widespread in natural environments. Over the years, many researchers have been studying the physiology, biochemistry and genetic control of bacterial bioluminescence. These discoveries have revolutionized the area of Environmental Microbiology through the use of luminescent genes as biosensors for environmental studies. This paper will review the chronology of scientific discoveries on bacterial bioluminescence and the current applications of bioluminescence in environmental studies, with special emphasis on the Microtox toxicity bioassay. Also, the general ecological significance of bioluminescence will be addressed.

Application of bioluminescence and chemiluminescence in biomedical sciences

Application of bioluminescence and chemiluminescence in biomedical sciences