Bioreporters are intact, living microbial cells

Cell (biology)

The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....

 that have been genetically engineered to produce a measurable signal in response to a specific chemical or physical agent in their environment

Environment (biophysical)

The biophysical environment is the combined modeling of the physical environment and the biological life forms within the environment, and includes all variables, parameters as well as conditions and modes inside the Earth's biosphere. The biophysical environment can be divided into two categories:...

. Bioreporters contain two essential genetic elements, a promoter gene and a reporter gene

Reporter gene

In molecular biology, a reporter gene is a gene that researchers attach to a regulatory sequence of another gene of interest in cell culture, animals or plants. Certain genes are chosen as reporters because the characteristics they confer on organisms expressing them are easily identified and...

. The promoter gene is turned on (transcribed

Transcription (genetics)

Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...

) when the target agent is present in the cell’s environment. The promoter gene in a normal bacterial cell is linked to other genes that are then likewise transcribed and then translated into proteins that help the cell in either combating or adapting to the agent to which it has been exposed. In the case of a bioreporter, these genes, or portions thereof, have been removed and replaced with a reporter gene. Consequently, turning on the promoter gene now causes the reporter gene to be turned on. Activation of the reporter gene leads to production of reporter proteins that ultimately generate some type of a detectable signal. Therefore, the presence of a signal indicates that the bioreporter has sensed a particular target agent in its environment.

Originally developed for fundamental analysis of factors affecting gene expression

Gene expression

Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...

, bioreporters were early on applied for the detection of environmental contaminants and have since evolved into fields as diverse as medical diagnostics

Medical diagnosis

Medical diagnosis refers both to the process of attempting to determine or identify a possible disease or disorder , and to the opinion reached by this process...

, precision agriculture

Precision agriculture

Precision farming or precision agriculture is a farming management concept based on observing and responding to intra-field variations.It relies on new technologies like satellite imagery, information technology, and geospatial tools...

, food safety

Food safety

Food safety is a scientific discipline describing handling, preparation, and storage of food in ways that prevent foodborne illness. This includes a number of routines that should be followed to avoid potentially severe health hazards....

 assurance, process monitoring and control, and bio-microelectronic computing. Their versatility stems from the fact that there exist a large number of reporter gene

Reporter gene

In molecular biology, a reporter gene is a gene that researchers attach to a regulatory sequence of another gene of interest in cell culture, animals or plants. Certain genes are chosen as reporters because the characteristics they confer on organisms expressing them are easily identified and...

 systems that are capable of generating a variety of signals. Additionally, reporter genes can be genetically inserted into bacterial, yeast

Yeast

Yeasts are eukaryotic micro-organisms classified in the kingdom Fungi, with 1,500 species currently described estimated to be only 1% of all fungal species. Most reproduce asexually by mitosis, and many do so by an asymmetric division process called budding...

, plant

Plant

Plants are living organisms belonging to the kingdom Plantae. Precise definitions of the kingdom vary, but as the term is used here, plants include familiar organisms such as trees, flowers, herbs, bushes, grasses, vines, ferns, mosses, and green algae. The group is also called green plants or...

, and mammalian cells, thereby providing considerable functionality over a wide range of host vectors.

Reporter gene systems

Several types of reporter genes are available for use in the construction of bioreporter organisms, and the signals they generate can usually be categorized as either colorimetric, fluorescent, luminescent, chemiluminescent or electrochemical. Although each functions differently, their end product always remains the same – a measurable signal that is proportional to the concentration of the unique chemical or physical agent to which they have been exposed. In some instances, the signal only occurs when a secondary substrate

Substrate (chemistry)

In chemistry, a substrate is the chemical species being observed, which reacts with a reagent. This term is highly context-dependent. In particular, in biochemistry, an enzyme substrate is the material upon which an enzyme acts....

 is added to the bioassay

Bioassay

Bioassay , or biological standardization is a type of scientific experiment. Bioassays are typically conducted to measure the effects of a substance on a living organism and are essential in the development of new drugs and in monitoring environmental pollutants...

 (luxAB, Luc, and aequorin). For other bioreporters, the signal must be activated by an external light source (GFP and UMT), and for a select few bioreporters, the signal is completely self-induced, with no exogenous substrate or external activation being required (luxCDABE). The following sections outline in brief some of the reporter gene systems available and their existing applications.

Bacterial luciferase (Lux)

Luciferase

Luciferase

Luciferase is a generic term for the class of oxidative enzymes used in bioluminescence and is distinct from a photoprotein. One famous example is the firefly luciferase from the firefly Photinus pyralis. "Firefly luciferase" as a laboratory reagent usually refers to P...

 is a generic name for an enzyme

Enzyme

Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...

 that catalyzes a light-emitting reaction. Luciferases can be found in bacteria, algae, fungi, jellyfish, insects, shrimp, and squid, and the resulting light that these organisms produce is termed bioluminescence. In bacteria, the genes responsible for the light-emitting reaction (the lux genes) have been isolated and used extensively in the construction of bioreporters that emit a blue-green light with a maximum intensity at 490 nm. Three variants of lux are available, one that functions at < 30°C, another at < 37°C, and a third at < 45°C. The lux genetic system consists of five genes, luxA, luxB, luxC, luxD, and luxE. Depending on the combination of these genes used, several different types of bioluminescent bioreporters can be constructed.

luxAB Bioreporters

luxAB bioreporters contain only the luxA and luxB genes, which together are responsible for generating the light signal. However, to fully complete the light-emitting reaction, a substrate

Substrate (chemistry)

In chemistry, a substrate is the chemical species being observed, which reacts with a reagent. This term is highly context-dependent. In particular, in biochemistry, an enzyme substrate is the material upon which an enzyme acts....

 must be supplied to the cell. Typically, this occurs through the addition of the chemical decanal

Decanal

Decanal is an organic compound with the chemical formula C9H19CHO. It is the simplest ten-carbon aldehyde. Decanal occurs naturally and is used in fragrances and flavoring. Decanal occurs in nature and is an important component in citrus along with octanal, citral, and sinensal...

 at some point during the bioassay procedure. Numerous luxAB bioreporters have been constructed within bacterial, yeast, insect, nematode, plant, and mammalian cell systems.

luxCDABE Bioreporters

Instead of containing only the luxA and luxB genes, bioreporters can contain all five genes

Gênes

Gênes is the name of a département of the First French Empire in present Italy, named after the city of Genoa. It was formed in 1805, when Napoleon Bonaparte occupied the Republic of Genoa. Its capital was Genoa, and it was divided in the arrondissements of Genoa, Bobbio, Novi Ligure, Tortona and...

 of the lux cassette, thereby allowing for a completely independent light generating system that requires no extraneous additions of substrate nor any excitation

Excited state

Excitation is an elevation in energy level above an arbitrary baseline energy state. In physics there is a specific technical definition for energy level which is often associated with an atom being excited to an excited state....

 by an external light source. So in this bioassay, the bioreporter is simply exposed to a target analyte

Analyte

An analyte, or component , is a substance or chemical constituent that is of interest in an analytical procedure. Grammatically, it is important to note that experiments always seek to measure properties of analytes—and that analytes themselves can never be measured. For instance, one cannot...

 and a quantitative

Quantitative property

A quantitative property is one that exists in a range of magnitudes, and can therefore be measured with a number. Measurements of any particular quantitative property are expressed as a specific quantity, referred to as a unit, multiplied by a number. Examples of physical quantities are distance,...

 increase in bioluminescence results, often within less than one hour. Due to their rapidity and ease of use, along with the ability to perform the bioassay repetitively in real time

Real-time data

Real-time data denotes information that is delivered immediately after collection. There is no delay in the timeliness of the information provided. Real-time data is often used for navigation or tracking....

 and on-line, makes luxCDABE bioreporters extremely attractive. Consequently, they have been incorporated into a diverse array of detection methodologies ranging from the sensing of environmental contaminants to the real-time monitoring of pathogen infections in living mice.

Nonspecific lux Bioreporters

Nonspecific lux bioreporters are typically used for the detection of chemical toxins. They are usually designed to continuously bioluminesce. Upon exposure to a chemical toxin, either the cell dies or its metabolic activity is retarded, leading to a decrease in bioluminescent light levels. Their most familiar application is in the Microtox http://www.sdimicrotox.com/ assay where, following a short exposure to several concentrations of the sample, the decreased bioluminescence

Bioluminescence

Bioluminescence is the production and emission of light by a living organism. Its name is a hybrid word, originating from the Greek bios for "living" and the Latin lumen "light". Bioluminescence is a naturally occurring form of chemiluminescence where energy is released by a chemical reaction in...

 can be correlated to relative levels of toxicity

Toxicity

Toxicity is the degree to which a substance can damage a living or non-living organisms. Toxicity can refer to the effect on a whole organism, such as an animal, bacterium, or plant, as well as the effect on a substructure of the organism, such as a cell or an organ , such as the liver...

.

Firefly luciferase (Luc)

Firefly luciferase catalyzes a reaction that produces visible light in the 550 – 575 nm range. A click-beetle luciferase is also available that produces light at a peak closer to 595 nm. Both luciferases require the addition of an exogenous substrate (luciferin) for the light reaction to occur. Numerous luc-based bioreporters have been constructed for the detection of a wide array of inorganic

Inorganic compound

Inorganic compounds have traditionally been considered to be of inanimate, non-biological origin. In contrast, organic compounds have an explicit biological origin. However, over the past century, the classification of inorganic vs organic compounds has become less important to scientists,...

 and organic compound

Organic compound

An organic compound is any member of a large class of gaseous, liquid, or solid chemical compounds whose molecules contain carbon. For historical reasons discussed below, a few types of carbon-containing compounds such as carbides, carbonates, simple oxides of carbon, and cyanides, as well as the...

s of environmental concern. Their most promising application, however, probably lies within the field of medical diagnostics. Insertion of the luc genes into a human cervical carcinoma cell line (HeLa

HeLa

A HeLa cell is a cell type in an immortal cell line used in scientific research. It is the oldest and most commonly used human cell line. The line was derived from cervical cancer cells taken on February 8, 1951 from Henrietta Lacks, a patient who eventually died of her cancer on October 4, 1951...

) illustrated that tumor-cell clearance could be visualized within a living mouse by simply scanning with a charge-coupled device camera, allowing for chemotherapy

Chemotherapy

Chemotherapy is the treatment of cancer with an antineoplastic drug or with a combination of such drugs into a standardized treatment regimen....

 treatment to rapidly be monitored on-line and in real-time. In another example, the luc genes were inserted into human breast cancer cell lines to develop a bioassay for the detection and measurement of substances with potential estrogenic and antiestrogenic activity.

Aequorin

Aequorin

Aequorin

Aequorin is a photoprotein isolated from luminescent jellyfish and a variety of other marine organisms...

 is a photoprotein isolated from the bioluminescent jellyfish Aequorea victoria

Aequorea victoria

Aequorea victoria, also sometimes called the crystal jelly, is a bioluminescent hydrozoan jellyfish, or hydromedusa, that is found off the west coast of North America. This species is thought to be synonymous with Aequorea aequorea of Osamu Shimomura, the discoverer of green fluorescent protein . ...

. Upon addition of calcium

Calcium

Calcium is the chemical element with the symbol Ca and atomic number 20. It has an atomic mass of 40.078 amu. Calcium is a soft gray alkaline earth metal, and is the fifth-most-abundant element by mass in the Earth's crust...

 ions (Ca2+) and coelenterazine, a reaction occurs whose end result is the generation of blue light in the 460 - 470 nm range. Aequorin has been incorporated into human B cell

B cell

B cells are lymphocytes that play a large role in the humoral immune response . The principal functions of B cells are to make antibodies against antigens, perform the role of antigen-presenting cells and eventually develop into memory B cells after activation by antigen interaction...

 lines for the detection of pathogenic bacteria and viruses in what is referred to as the CANARY assay (Cellular Analysis and Notification of Antigen Risks and Yields). The B cells are genetically engineered to produce aequorin. Upon exposure to antigens of different pathogens, the recombinant B cells emit light as a result of activation of an intracellular signaling cascade that releases calcium ions inside the cell.

Green fluorescent protein (GFP)

Green fluorescent protein

Green fluorescent protein

The green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...

 (GFP) is also a photoprotein isolated and cloned from the jellyfish Aequorea victoria

Aequorea victoria

Aequorea victoria, also sometimes called the crystal jelly, is a bioluminescent hydrozoan jellyfish, or hydromedusa, that is found off the west coast of North America. This species is thought to be synonymous with Aequorea aequorea of Osamu Shimomura, the discoverer of green fluorescent protein . ...

. Variants have also been isolated from the sea pansy Renilla reniformis. GFP, like aequorin, produces a blue fluorescent signal, but without the required addition of an exogenous substrate. All that is required is an ultraviolet light source to activate the fluorescent properties of the photoprotein. This ability to autofluoresce makes GFP highly desirable in biosensing assays since it can be used on-line and in to monitor intact, living cells. Additionally, the ability to alter GFP to produce light emissions besides blue (i.e., cyan, red, and yellow) allows it to be used as a multianalyte detector. Consequently, GFP has been used extensively in bioreporter constructs within bacterial, yeast, nematode, plant, and mammalian hosts.

Uroporphyrinogen (urogen) III methyltransferase (UMT)

Uroporphyrinogen (urogen) III methyltransferase (UMT) catalyzes a reaction that yields two fluorescent products which produce a red-orange fluorescence in the 590 - 770 nm range when illuminated with ultraviolet light. So as with GFP, no addition of exogenous substrates is required. UMT has been used as a bioreporter for the selection of recombinant plasmids, as a marker for gene transcription

Transcription (genetics)

Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...

 in bacterial, yeast, and mammalian cells, and for the detection of toxic salts such as arsenite

Arsenite

In chemistry an arsenite is a chemical compound containing an arsenic oxoanion where arsenic has oxidation state +3.The different forms of the anion are the next ones:* ortho-arsenite: AsO33-* meta-arsenite: AsO2-...

 and antimonite

Antimonite

In chemistry, an antimonite refers to salts of antimony, such as NaSb4 and NaSbO2 which can be prepared by reacting alkali with antimony oxide, Sb2O3...

.