Sunday, November 11, 2018

Bioluminescence

About 150 million years ago, some living creatures started evolving ways to make their own light, and this is what bioluminescence is: The ability of an organism to create and emit light. Having your own "flashlight" happens to be very useful. Depending on the creature, it is used for mimicry, for camouflage, to warn away predators, for communication among one's species and to find mate.

Just as bioluminescence has a multitude of functions, it appears to have evolved in a multitude of different ways. In bioluminescent bacteria, the bioluminescent machinery appears to be borrowed from its machinery for cellular respiration. In marine organisms, it seems to have evolved from once-essential cellular detoxification machinery. In at least one dinoflagellate species, the photosynthesis mechanism has been fine-tuned toward light production. In the firefly, molecules that once broke down fatty acids for energy storage now emit that energy as light instead. In this article we will explore the mystery of how bioluminescence evolved and how it offers important life advantages of non-glowing relatives.

What Is Bioluminescence (And What Isn't)?

Although bioluminescence is quite rare if you measure it as a percentage of the total number of all of Earth's species, it is quite common among marine species and it is surprisingly diverse among different distantly related groups of organisms, including land organisms. Most bioluminescent species (about 70%) are marine and these include ocean-living bacteria, dinoflagellates (these are the tiny creatures that make disturbed ocean water sparkle with tiny green-blue lights), marine crustaceans such as some krill and shrimp, some echinoderms such as sea stars, sea lilies and sea cucumbers, as well as sea squirts, and a great many species of fish, and even a few sharks. Bioluminescence has also found its expression on land but it is not as common. Land species include some bacteria and fungi as well as some insects, annelids (worms) and arachnids (spiders, scorpions, ticks and mites etc.). Groups that do not contain any bioluminescent species are the land vertebrate classes such as amphibians, reptiles, birds and mammals to which we belong.

There are, however, several notable fluorescent land animals, such as the polka dot tree frog, a reddish yellow frog that glows bright green under ultraviolet light. Biofluorescence is also seen in many fish and corals, in jellyfish, butterflies, parrots, spiders and even in the flowers of the common four o'clock plant (Mirabilis jalapa). Unlike bioluminescent species, which make their own light, these organisms never glow in complete darkness. They absorb light and then emit it immediately once it is absorbed, usually at a longer (lower energy) wavelength. It is important to distinguish between a fluorescent species and a bioluminescent species. They are easily mixed up.

NOT BIOLUMINESCENT:

Jonbeebe;Wikipedia

All scorpions such as this (actually black) one fluoresce bright aqua blue under an ultraviolet lamp. They glow more faintly in nature, in the dim ultraviolet light reflected by the moon. Their exquisitely sensitive eyes also happen to see this particular colour best.

Biologist Edie Widder offers us a taste of bioluminescence across the marine world with some incredible footage of bioluminescent marine creatures in this 13-minute video, part of her TED talk in 2013:



BIOLUMINESCENT:

art farmer:Wikipedia

Common Eastern Firefly (Photinus pyralis)

This firefly species is common in North America. At twilight, males use flashes of greenish-yellow light to attract females, who will respond with an answering flash of their own.

BIOLUMINESCENT:

Wofl-commonswiki; Wikipedia

Female glowworm (Lampyris noctiluca)

This glowworm is actually a beetle because it has a hard shell or carapace. In this case it is the much larger wingless female rather than the male that uses light to attract males. The brighter her glow, the more fertile she is.

In addition to these well-known land examples, bioluminescence has found widespread use among marine species that utilize it in ocean water. They use it near the water surface on dark moonless nights or very deep in the ocean, where sunlight can't penetrate.

NOT BIOLUMINESCENT (trick example):

Sierra Blakely;Wikipedia

Crystal Jelly (Aequorea victoria)

This jellyfish is commonly found floating and swimming off the west coast of Canada and the northern United States, especially in Puget Sound. This photograph shows you what the colourless animal looks like but you are actually seeing light reflected from the camera, a common misconception in such photographs found online. Its bioluminescence is only visible in its outer ring, as a faint blue-green glow (which is emitted only when it is disturbed), shown below.

Photo taken by Osamu Shimomura

Dr. Shimomura, a famous organic chemist and marine biologist, isolated two luminescent proteins from this species of jellyfish. One of them is a bioluminescent protein called aequorin. It emits blue light. He also found trace amounts of another protein, and this one is fluorescent rather than bioluminescent. It's called green fluorescent protein (GFP). This protein absorbs the blue light emitted from the aequorin complex and re-emits it as green light. The aequorin complex mentioned here is a substrate/enzyme complex - more specifically a luciferin/luciferase complex. We will get better acquainted with these two evocative-sounding words later on. GFP is now widely used in biological and medical research. It is this discovery for which Dr. Shimomura received the Nobel Prize in Chemistry in 2008.

Exactly what does this jellyfish uses bioluminescence for? That remains a mystery. Perhaps it's a warning to potential predators. Researchers know that individuals do not flash at each other and they do not glow continuously. They can be stimulated to glow when they are disturbed but it is rarely observed in undisturbed individuals.

Marine bioluminescence is very prevalent among those species that live in very deep pitch-black ocean water were no sunlight penetrates, a kilometre or more beneath the ocean surface. One could imagine that these animals would have lost their sight over time, like blind cave-dwelling salamanders did. Many of them have, relying on pressure changes and smell instead. However, some species evolved extreme light sensitivity to the faint bioluminescent light shows they encounter. Down here, water is cold, pitch-black and under tremendous pressure. Here in this inhospitable environment, a myriad of organisms communicate and navigate through a wide variety of beautiful and ephemeral displays of coloured lights.

Some interesting examples include the 246 species of lanternfish (members of the Myctophidae family), which live deep in the all of the world's oceans, making up an astonishing 600 million metric tonnes of biomass. This is about 10 times the world's yearly catch of fish! These abundant but small (most are just 6 cm long) deep-sea fish play an essential ecological role as food for larger fish. Myctophum punctatum, pictured below, lives a vertically mobile life. It rises over a kilometre to reach waters near the ocean surface every sundown, following the also vertical daily migration of zooplankton, its food source.

Edd48;Wikipedia
Lanternfish (Myctophum punctatum)

Although not readily visible in the photograph above, most of these species luminesce through photophores (light-producing organs) arranged in rows along the belly (they are not the reflective blue upper dorsal spots you see above). Photos of its bioluminescence are rare but the digital model of this species shown below offers an idea of how these bioluminescent photophores are arranged.

JBD5;Turbosquid

3D Digital  Model of Myctophum punctatum

Myctophum punctatum uses its bioluminescence as a special type of camouflage called counter-illumination. The fish regulates the brightness of the bluish light emitted by its photophores to match the blue wavelengths of the faint sunlight light streaming from above. This masks its silhouette from predators swimming underneath it. Some species also emit green or yellow light, which might be used for communication or courtship.

Just as fascinating are the anglerfish, an order of fish (Lophoformis) comprised of more than 200 species. All anglerfish are carnivores that use bioluminescence as a type of fishing lure. Representatives of these macabre-looking fish are shown below.

Masaki Maya et al., Wikipedia

Representatives of Anglerfish Order Lophoformis

Most, but not all, of these species live deep in the ocean, where the water is pitch black, extremely cold and under intense pressure. In these species, a piece of dorsal spine has evolved into a protruding "fishing pole" tipped with a luminous bulb. Not all individuals of each species have a fishing pole, however. Those that possess one are all females. Males are much smaller and have no need to fish for food because they are completely parasitic upon the females. (Click this link: what you first see is awesome!) Bulb-less, a male latches onto a female with its sharp teeth and eventually fuses with the female's body. It connects to her bloodstream, and eventually loses its eyes and internal organs, everything except its testes. Females are known to carry more than six males on their bodies at one time.

Some rare footage of an anglerfish mating pair reveals that these strange fish not only have bioluminescent lures but a surrounding net of fine bioluminescent filaments as well. They were filmed in 2016 at 792 metres deep off Portugal's coast.



The 3-minute video above was published by Science Magazine in 2018.

A vast number of species (in the thousands) that use bioluminescence, as well as a large number of variations in the chemical reaction that produces the light, suggest that bioluminescence evolved independently many different times throughout history. Experts estimate that it evolved at least 40 different times starting at least 150 million years ago, near the beginning of the Cretaceous Period.

In fish alone, bioluminescence evolved at least 27 different times. All this convergent evolution is a testament to its usefulness. Bioluminescence is a very useful jack-of-all-trades system, having numerous functions such as lures for prey, predator warning systems, mate attraction and communication. Bioluminescence is autogenic in many fish, which means that the animal itself produces the light. Examples are the lanternfish, which emit light from light-production organs called photophores. Many other marine species have instead left the job of light production to internalized luminescent bacteria. To further enhance this symbiotic relationship, they have co-evolved mechanisms within their bodies to turn these bacteria off and on at their will. They evolved a handy light switch for them in other words. Anglerfish belong to this group. Their mutually beneficial relationship with their luminescent bacteria is especially interesting. Recent genetic studies reveal that these bacteria have lost almost half of their genome compared to their free-living close relatives, an example of adaptive gene loss, a use-it-or-lose-it principle in genetic evolution. The bioluminescent bacteria appear to be able to swim in and out of the bulb freely using their flagella, but they have lost all the genes associated with sensing and digesting food sources. The fish, instead, supply all the cellular nutrients the bacteria requires while the bacteria provide light to help the fish lure food.

Perhaps one of the eeriest examples of bioluminescence is foxfire or faerie fire. Produced by some species of fungi in decaying wood, the light might be used to attract insects to spread the fungus? spores or as a warning to any foraging animals nearby. Omphalotus olearius, by day, is an ordinary looking (but poisonous!) European mushroom that looks much like a chanterelle:

Abbatiello;Wikipedia

Fungus Omphalotus olearius During the Daytime

By night, it reveals why it is also called the jack-o-lantern mushroom. Its orange gills glow an eerie green. It's easy to imagine faeries holding their mysterious night-meetings here.

Noah Siegel;Wikipedia

Omphalotus olearius at Night

How Does Bioluminescence Work? Where Did It Originally Come From?

Like a glow stick, bioluminescent animals utilize a chemical reaction that produces light. Because bioluminescence evolved independently in a wide range species across the evolutionary spectrum, different species use different chemical reactions to make light. The general mechanism, however, is the same throughout. It involves three essential ingredients: a light-emitting molecule, an enzyme, and molecular oxygen, O2. Inside the light-emitting cell, a special protein reacts with a charged oxygen ion and undergoes an oxidation reaction. During the reaction, an intermediate molecule in an excited state is produced. What does this mean? A molecule enters an excited state when one or more electrons in its atoms absorb enough energy to jump outward to a higher energy quantum state. Light is produced during the reaction when an excited electron in the intermediate molecule emits a photon of visible light. By dong so, it loses just the right amount of energy to drop to its ground (resting or lowest energy) state. This scenario isn't unusual in biochemistry. These chemical reactions often involve a short-lived intermediate molecule. It is usually highly reactive and it is often in an excited state. What is unique here is that the intermediate complex releases its excitation energy is as a photon rather than sequestering it in the potential energy of one or more new chemical bonds. The special protein in this reaction is generically called a luciferin (which means a light-emitting substance*). The light-emission reaction is catalyzed by a protein enzyme, generically called a luciferase. The trigger for the reaction can be a mechanical, neurological or chemical change.

*I was so curious I had to look up "Lucifer." How is this word also the word for devil? According to Wikipedia, Lucifer  originated as the Latin word for "bringer of light?" and as a metaphor for "morning star," which, in Christianity, also appears to have been the original name for Satan before his fall from grace. It then became a common byword for Satan or Devil.

Luciferin in Many Marine Organisms

The shape and size of the luciferin molecule varies widely among the various bioluminescent phyla. However, some luciferins are more commonly found than others. For example, a luciferin called coelenterazine is found in most bioluminescent marine organisms. It may have evolved into its luciferin role from an earlier role in detoxifying cells. Oxygen is essential for most life on Earth, but inside cells it is a hazardous molecule, especially when it is in the form of an even more reactive oxygen free radical. Coelenterazine is a strong anti-oxidant. It reacts with free oxygen radicals and neutralizes them before they can react with and seriously damage proteins and DNA inside cells. This might have been coelenterazine's original function. When ancient ancestors of marine organisms moved down into deeper sea habitats, where it is cold and oxygen levels are low, the metabolic rate of organisms would have gone down, requiring less intercellular coelenterazine. It was a molecule, already present, ready to evolve into its new luciferin function.

The enzyme luciferase, which catalyzes the oxidation of the luciferin, also varies widely. Each unique luciferin/luciferase system represents a unique evolutionary origin of bioluminescence. How did these molecules arise inside living cells? As suggested above, they appear to have mutated over time from biological molecules already present in the organism. Molecules very similar to the luciferin molecules seem to have already been present in many non-luminescent living organisms, doing some other function.

Luciferin in Some Dinoflagellates

Another intriguing example to illustrate this theory is a bioluminescent marine dinoflagellate. The luciferin used by most of these tiny unicellular organisms closely resembles chlorophyll, an even more ancient and biologically important molecule. Chlorophyll is also present because many dinoflagellates are photosynthetic. In some of these species, luciferin even seems to retain some of the light-absorption function of chlorophyll. Some species, such as P. lunula, bioluminesce only at night, and after sunny days they glow more brightly. The luciferin of this species might be a photo-oxidized chlorophyll. This chlorophyll molecule fluoresces blue when it is exposed to UV light (a part of sunlight) but the fluorescence eventually stops when all the chlorophyll molecules become degraded by photo-oxidation (oxidation in the presence of sunlight). Once it is photo-oxidized, it?s function changes and it becomes a bioluminescent luciferin, which glows blue. Even though this species offers a tantalizing clue about the origin of bioluminescence, researchers know that several other bioluminescent dinoflagellate species don't use this biochemical mechanism.

Luciferase in Fireflies

Luciferase, like luciferin, seems to have evolved from molecules that were already present in the organism. Over time, the molecule's function shifted toward to light-production, offering a new selective advantage to those individuals. This process of evolution toward bioluminescence is still far from understood but some headway has been made from studying coelenterazine luciferin evolution in marine organisms and by studying the chlorophyll-like mechanism for bioluminescence in a dinoflagellate species. Luciferases in beetles such as the firefly have also been the subject of close study, and again, the bioluminescent machinery might be borrowed from the cell's general biochemistry machinery. The firefly luciferase enzyme seems to have evolved from another enzyme called AMP-CoA-ligase. This means that somehow this enzyme evolved from breaking down fatty acids into an oxygenase/light-production function. Just to note here, the original ligase with its original function still operates in the organism. Its ligase role is still essential to its cellular biochemistry, especially in a number of regulative cellular functions. We can assume that the change in function toward producing light gave bioluminescent beetles such as fireflies a significant evolutionary advantage over their non-bioluminescent relatives, by enhancing their reproductive success. The numerous evolutionary paths of luciferin and luciferase are wonderful examples of Mother Nature re-purposing items at hand into new and amazing living tools.

Two things are fairly certain: First, bioluminescence evolved independently at least 40 separate times and second, it first evolved many millennia ago. There is evidence that it evolved in marine fish between 150 and 60 million years ago. In just under half of these marine fish, bioluminescence evolved not inside of them but in their symbiotic bioluminescent bacteria.

Evolution of Bioluminescence in Bacteria

It's possible, but not proven by any means, that bioluminescence first appeared in bacteria living in the early Cretaceous period (about 145 million years ago). Aerobic bacteria evolved once Earth accumulated enough oxygen in the atmosphere to support them. These bacteria had a metabolic advantage over their anaerobic cousins. They could now use the high-energy chemical bonds in oxygen to oxidize glucose (cellular food) during cellular respiration. Those products could then be used to make ATP (adenosine triphosphate), a very important energy molecule used by all cells.

Aerobic bacteria, and most, but not all, living organisms, also use oxygen to obtain energy from other molecules in addition to glucose, such as from fatty acids. Fatty acids, which I very briefly mentioned earlier, are all-around energy storage molecules in aerobic cells. Their energy, too, can be ultimately captured in ATP. When the aerobic pathway isn't used in the cell, anaerobic processes use fatty acids to make a variety of important molecules like phospholipids, messengers and hormones. Even the aerobic eukaryotic cells of multicellular animals carry out anaerobic processes.

Early aerobic bacterial species, living about a billion years ago at the end of the Great Oxygenation Event, likely lived in a changing environment where oxygen levels went up and down. This environmental stress might have been the first trigger for the evolution for bioluminescence. When oxygen levels fell, a mutation in an enzyme called riboflavin oxygenase (think of this molecule as an early luciferase) might have allowed these organisms to oxidize aldehyde molecules, which would accumulate under those conditions. By oxidizing the aldehydes, they could be turned into useful fatty acids. A supply of these molecules under low oxygen conditions would be a great evolutionary advantage. These bacteria would have that extra energy boost to keep reproducing and accumulating in numbers until oxygen became plentiful again. If some of these mutants also used another molecule, the already abundantly present reduced flavin mononucleotide (FMNH2) as a substrate or cofactor (an early luciferin in other words), then they could have been "accidentally" luminous, because this reaction creates an excited intermediate molecule that gives off light when it returns to its ground state.

If these early individuals were also light-sensitive, as many bacteria are, luminance among them might have been very useful in helping individuals recognize the presence of others nearby. It might have offered them a brand new selective advantage by helping them to disperse and/or colonize more easily. In bacteria, this behaviour, called quorum sensing, allows bacteria to detect and respond to their neighbours through gene regulation. The bacteria can switch genes on or off in order to optimize the population for changing conditions. Offspring might start producing a biolfilm, for example, so they can stick to an optimal rock surface. Or the population might switch to spore encapsulation when conditions become harsh. You could think of this new advantage like a military troop receiving radio communication on the battlefield - very useful under quickly changing conditions. All bioluminescent bacteria have a few characteristics in common: they are rod-shaped, gram-negative, they have flagella to move around with, and most importantly to the evolution argument I just laid out, they are all facultative anaerobes, which means they can live and grow when oxygen levels are high and also when they are low or zero.

The Genetics of Bioluminescence

Even though a single general pathway toward bioluminescence might have originated in facultative anaerobic bacteria, the chemistry of bioluminescence in bacteria varies depending on the bacterial strain or species. This suggests that bacterial bioluminescence evolved independently numerous times just as it did in other phyla. While the chemistry each time might be unique, all the luminescent bacterial species share the same gene sequence called the lux gene sequence, or lux operon, again offering an argument for the evolution of a single general pathway toward bioluminescence, after which the chemistry evolved independently numerous times. The lux operon might be the code left over for an ancient DNA repair system. Now this sequence codes for all the proteins involved in the luminescent mechanism. It is a short fragment of DNA just 9 kilobases long, and it contains just 5 genes that code for the proteins required for bioluminescence. A few additional genes regulate the operon (they turn it on or off). Several other enzymes, substrates and co-factors used in the production of light are already present in the cell. This short gene sequence can be isolated and inserted into normally non-luminescent bacterial and eukaryotic cells to make them bioluminescent. A non-luminous bacterium, such as Escherichia coli for example, can be transformed into a bioluminescent one simply by the insertion of the lux gene sequence. As you can imagine, this has a myriad of potential uses in medicine such as imaging and in research.

Bacteria tend to evolve fast because their life cycle is short and there are many of them. If a random mutation in the genetic code introduces a new advantage, survivors pass it on and it quickly increases in the population. The lux operon sequence is strongly conserved (which means it stays much the same with few surviving mutations) among bioluminescent bacteria. This suggests that it must have a significant selective advantage even though it is an expensive option for a cell to choose. There is a very high energy cost to emit light. A green light photon, for example, has about the same energy as the chemical bonds of 8 ATP molecules. That is a significant energy commitment for a microscopically small organism like a bacterium.

Bioluminescence Chemistry

Let's focus now on the light-producing reaction itself. The light-emitting reaction in bacteria has been studied extensively. Reduced riboflavin phosphate (FMNH2) and a long-chain fatty aldehyde (RCHO) are both oxidized, and oxygen diffused from the environment into the cell is the perfect oxidizer to do the job. During this oxidation reaction, blue-green light is emitted. The reaction is catalyzed by various enzymes called luciferases. The luciferase used depends on the species. FMNH2 and RCHO are already present in all aerobic bacteria (and eukaryotic cells) because they are part of the electron transport chain.

I was inspired to research bioluminescence after exploring the electron transport chain in the previous article, Ozone. This chain is part of the process of aerobic cellular respiration. "Aerobic" means requiring oxygen. Through aerobic cellular respiration, cells use food and oxygen and turn it into the energy needed to grow, multiply and move. The electron chain carries out one of the processes of aerobic respiration, called oxidative phosphorylation. Its main purpose is to make an important energy molecule called ATP.

The electron transport chain in bacteria (or in mitochondria in eukaryotic cells like ours) transfers electrons from donor molecules to acceptor molecules in a series of redox reactions. It is coupled with the movement of protons (H+ ions) pumped back across the cell membrane so that the cell and its environment remain neutrally charged. The chain drives the production of energy-rich ATP and it oxidizes a variety of enzymes and other proteins along the way. The final electron acceptor is oxygen, which is the perfect molecule for this because it is a powerful oxidizer (or "electron-grabber" if you want).

In non-luminescent bacteria, FMNH2 (riboflavin phosphate) simply diffuses into the cytoplasm. In bioluminescent bacteria this is where the luciferase enzyme and enzymes that catalyze the creation of the intermediate complex are also present. They channel FMNH2 into forming part of a light-emitting complex. In the process, FMNH2 is reduced to FMN and a long-chain fatty aldehyde (RCHO) is reduced to a carboxyl acid (RCOOH). ("R" is organic chemistry shorthand for any carbon-hydrogen group)

This is what the generic reaction looks like:

FMNH2 + RCHO + O2 → FMN + H2O + RCOOH + hv (490 nm)

The products of the reaction are water, RCOOH, a carboxyl acid, and flavin mononucleotide (FMN), which is an electron carrier in the electron transport chain of every living cell. Energy is released in the reaction in the form of a blue-green photon [hv (490 nm)].

During the reaction, the about-to-be-reduced FMNH2 binds to the luciferase enzyme. An enzyme is a biological catalyst. It increases the rate of the reaction but it isn't consumed during it. The luciferase catalyzes the reaction by reacting with oxygen and then interacting with the aldehyde to form a fairly stable intermediate complex in an excited state. It decays, or returns to ground state, slowly. This means that light can be emitted over a significant period of time by numerous complexes rather than just in a single brief flash (although some species do flash). The particular enzyme utilized by each specific species can have a significant effect on the decay rate (duration of light production) and the turnover rate (how soon it can glow again) of this light-emitting complex.

Although bioluminescence might have evolved during exposure to low-oxygen environments, molecular oxygen (O2) is essential for the light emission reaction pathway. Marine organisms get it from the oxygen in seawater. Land organisms get it from air. The reduction of oxygen ultimately transforms potential chemical bond energy into light energy.

Although this reaction produces blue-green light, bioluminescence can show up in a variety of colours. Blue-green or blue light is most common. In a marine environment where light levels are very low, blue light is the only visible wavelength short enough to remain streaming through the water after longer wavelengths have been scattered by water molecules. This makes light in the blue spectrum very useful for communication in dark deep marine waters.

Single mutations in luciferase can modify the chemical binding sites on the light-emitting complex just enough to distort the emission colour. Some bacteria also carry fluorescent proteins that change the emission colour, for example, to yellow. These proteins absorb blue light and re-emit it as less energetic wavelengths like yellow. A few organisms emit red. In each case, the basic chemical reaction that produces the light remains the same.

Bioluminescence Is Useful To Humans

Now that the mechanics of luminescence in various species are being worked out, an increasing number of new technologies are being developed to take advantage of it. Bioluminescent imaging, for example, allows scientists to non-invasively study biological processes while they are taking place inside live subjects. One especially promising new idea is to use it to track the progress of cancer metastasis in the living body in order to understand the progress of cancer better. Bioluminescent DNA machinery, the lux operon, can be inserted into various types of non-luminous cells.

To start, the lux operon can be spliced into a virus's genome. The virus then infects a cell and inserts that DNA into the cellular DNA. The cell can then translate and transcribe the luciferase/luciferin proteins that emit detectable light. If, for example, the lux operon is inserted into the cancer cells of a primary tumour of a laboratory animal such as a mouse, that tumour will light up and glow. As the cancer cells spread into the blood stream and invade other organs over days and weeks, the process can be observed and studied simply by observing where the mouse glows. Of course, visible light can?t pass through an animal but researchers got around this problem by culturing bioluminescent cells and selecting for lux operon mutations that emit near-infrared light rather than blue or yellow light. Cancer cells emitting this wavelength of light can be imaged and tracked over time using an infrared camera placed outside the mouse's body. This kind of imaging can be so sensitive it can detect the glow from a single cell. It is a way to see exactly where cancers spread in the living body over time.

Bioluminescence can also be used as a very sensitive assay in genetic research. Researchers can take the lux genetic code for firefly luciferin/luciferase, for example. This genetic sequence can be isolated and cloned onto any DNA sequence of interest and then inserted into a virus, which has all the cellular machinery to make proteins from the inserted genetic code. The protein that is transcribed can be measured itself or its enzymatic activity can be measured by the intensity of its bioluminescence, in this case, a yellow glow. Each protein molecule transcribed from the DNA sequence of interest, perhaps it is code for a specific enzyme or a regulatory protein, now has a glowing tag attached to it. It is a very sensitive assay.

The idea of using tiny glowing markers to visualize specific protein molecules in a petri dish or in tissue or in a living organism is not new. I already briefly mentioned green fluorescent protein (GFP), a breakthrough assay protein developed in 2008, and now widely used in medical and scientific research. The genetic code from GFP is also used as a genetic assay, but in its case the marker protein fluoresces green when exposed to blue to ultraviolet light.  Both bioluminescent protein complexes and fluorescent proteins emit visible light as the result of an excited electron in an energized molecule returning to its ground state. The bioluminescent marker protein, however, luminesces in total darkness and does not need to be in a lit environment. This means there is no background to eliminate when measuring the light emitted from an assay result and this makes it up to a thousand times more sensitive than GFP assays. It means as well that extremely small changes in light emission now become measurable by using ultra-sensitive cameras to detect changes too small for our eyes to detect.

A Glimpse at Future Technologies

Bioluminescence has fascinated humans for millennia. Technologies that use bioluminescence are probably still in their infancy and the future looks bright (sorry). Think of trees gently lighting future streets at night  à la Avatar. What a way for a city to go green and save electricity costs! The myriad possibilities of bioluminescent technologies are limited only by the imagination.

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