What Is Yeast?

Yeast are a group of unicellular fungi a few species of which are commonly used to leaven bread and ferment alcoholic beverages. Most yeasts belong to the division Ascomycota. A few yeasts, such as Candida albicans can cause infection in humans. More than one-thousand species of yeasts have been described. The most commonly used yeast is Saccharomyces cerevisiae, which was domesticated for wine, bread and beer production thousands of years ago. See Yeast (baking). Yeast physiology can be either obligately aerobic or facultatively fermentative. There is no known obligately anaerobic yeast. In the absence of oxygen, fermentative yeasts produce their energy by converting sugars into carbon dioxide and ethanol (alcohol). In brewing, the ethanol is used, while in baking the carbon dioxide raises the bread and the ethanol evaporates. An example with glucose as the substrate is C6H12O6 (glucose) 2C2H5OH + 2CO2 Yeasts can reproduce asexually through budding or sexually through the formation of ascospores. During asexual reproduction a new bud grows out of the parent yeast when the condition is right, then after the bud reaches an adult size, it separates from the parent yeast. Under low nutrient conditions, yeasts that are capapable of sexual reproduction will form ascospores. Yeasts that are not capable of going through the full sexual cycle are classified in the genus Candida. Yeasts for leavening bread may be produced commercially or caught from the environment. Many yeasts can be isolated from sugar-rich environmental samples. Some good examples include fruits and berries (such as grapes, apples or peaches), exudates from plants (such as plant saps or cacti). Some yeasts are found in association with insects. The use of potatoes, water from potato boiling, eggs, or

sugar in a bread dough accelerates the growth of yeasts. Salt and fats such as butter slow yeast growth down. A common medium used for the cultivation of yeasts is called potato dextrose agar (PDA) or potato dextrose broth. Potato extract is made by autoclaving cut-up potatoes with water for 5 to 10 minutes and then decanting off the broth. Dextrose (glucose) is then added (10 g/L), and the medium is sterilized by autoclaving. Yeast fermentations comprise the oldest and largest application of microbial technology. They are used for beer and wine fermentations and bread production. Beer brewers classify yeasts as top-fermenting and bottomfermenting. This distinction was introduced by the Dane Emil Christian Hansen.

Yeasts are single-celled fungi. As fungi, they are related to the other fungi that people are more familiar with. These include edible mushrooms available at the supermarket, common bakers yeast used to leaven bread, molds that ripen blue cheese and the molds that produce antibiotics for medical and veterinary use. Many consider edible yeast and fungi to be as natural as fruits and vegetables.

Yeast Cells Over 600 different species of yeast are known and they are widely distributed in nature. They are found in association with other microorganisms as part of the normal inhabitants of soil, vegetation, marine and other aqueous environments. Some yeast species are also natural inhabitants of man and animals. While some species are highly specialized and found only in certain habitats at certain times of the year, other species are generalists and can be isolated from many different sources. Bakers yeast is used to leaven bread throughout the world and it is the type of yeast that people are most familiar with. Bakers yeast is produced from the genus and species of yeast called Saccharomyces cerevisiae. The scientific name of the genus of bakers yeast, Saccharomyces, refers to saccharo meaning sugar and myces meaning

fungus. The species name, cerevisiae, is derived from the name Ceres, the Roman goddess of agriculture. Bakers yeast products are made from strains of this yeast selected for their special qualities relating to the needs of the baking industry.

The typical yeast cell is approximately equal in size to a human red blood cell and is spherical to ellipsoidal in shape. Because of its small size, it takes about 30 billion yeast cells to make up to one gram of compressed bakers yeast. Yeast reproduce vegetatively by budding, a process during which a new bud grows from the side of the existing cell wall. This bud eventually breaks away from the mother cell to form a separate daughter cell. Each yeast cell, on average, undergoes this budding process 12 to 15 times before it is no longer capable of reproducing. During commercial production, yeast is grown under carefully controlled conditions on a sugar containing media typically composed of beet and cane molasses. Under ideal growth conditions a yeast cell reproduces every two to three hours. Yeast is the essential ingredient in many bakery products. It is responsible for leavening the dough and imparting a delicious yeast fermentation flavor to the product. It is used in rather small amounts in most bakery products, but having good yeast and using the yeast properly often makes the difference between success and something less than success in a bakery operation.

Yeast
From Wikipedia, the free encyclopedia

Yeast

Yeast of the species Saccharomyces cerevisiae

Scientific classification Domain: Kingdom: Eukaryota Fungi

Phyla and Subphyla Ascomycota Saccharomycotina (true yeasts) Taphrinomycotina Schizosaccharomycet

es(fission yeasts) Basidiomycota Agaricomycotina Tremellomycetes

Pucciniomycotina Microbotryomycetes

Yeasts are eukaryotic micro-organisms classified in the kingdom Fungi, with 1,500 species currently described[1] estimated to be only 1% of all fungal species.[2]Most reproduce asexually by mitosis, and many do so via an asymmetric division process called budding. Yeasts are unicellular, although some species with yeast forms may become multicellular through the formation of a string of connected budding cells known as pseudohyphae, or false hyphae, as seen in mostmolds.[3] Yeast size can vary greatly depending on the species, typically measuring 34 m in diameter, although some yeasts can reach over 40 m.[4] By fermentation the yeast species Saccharomyces cerevisiae converts carbohydrates to carbon dioxide and alcohols - for thousands of years the carbon dioxide has been used in baking and the alcohol in alcoholic beverages.[5] It is also extremely important as a model organism in modern cell biology research, and is one of the most thoroughly researched eukaryotic microorganisms. Researchers have used it to gather information about the biology of the eukaryotic cell and ultimately human biology.[6] Other species of yeast, such as Candida albicans, are opportunistic pathogens and can cause infections in humans. Yeasts have recently been used to generate electricity in microbial fuel cells,[7] and produce ethanol for the biofuel industry. Yeasts do not form a single taxonomic or phylogenetic grouping. The term "yeast" is often taken as a synonym for Saccharomyces cerevisiae,[8] but the phylogenetic diversity of yeasts is shown by their placement in two separate phyla, the Ascomycota and the Basidiomycota. The budding yeasts ("true yeasts") are classified in the order Saccharomycetales.[9]

Contents
[hide]

1 History 2 Nutrition and growth 3 Ecology 4 Reproduction 5 Uses 5.1 Alcoholic

beverages

5.1.1 Beer 5.1.2 Win

o o
n

5.2 Baking 5.3 Bioremediatio

5.4 Industrial

ethanol production

o
beverages

5.5 Nonalcoholic

5.6 Nutritional

supplements

o o
hobby

5.7 Probiotics 5.8 Aquarium

o o o

5.9 Science 5.10 Yeast extract 6 Pathogenic yeasts 7 Food spoilage 8 See also 9 Footnotes 9.1 Cited texts 10 External links

History

See also: History of wine and History of beer The word "yeast" comes to us from Old English gist, gyst, and from the Indo-European root yes-, meaning boil, foam, or bubble.
[10]

Yeast microbes are probably one of the earliest domesticated organisms. People have used yeast for fermentation and baking

throughout history. Archaeologists digging in Egyptian ruins found early grinding stones and baking chambers for yeasted bread, as well as drawings of 4,000-year-old bakeries and breweries.[11] In 1680, the Dutch naturalist Anton van Leeuwenhoek first microscopically observed yeast, but at the time did not consider them to be living organisms, but rather globular structures.[12] In 1857, French microbiologist Louis Pasteur proved in the paper "Mmoire sur la fermentation alcoolique" that alcoholic fermentation was conducted by living yeasts and not by a chemical catalyst.[11][13] Pasteur showed that by bubbling oxygen into the yeast broth, cell growth could be increased, but fermentation was inhibited an observation later called the "Pasteur effect". By the late 18th century, two yeast strains used in brewing had been identified: Saccharomyces cerevisiae, so called top fermenting yeast, and S. carlsbergensis, bottom fermenting yeast. S. cerevisiae has been sold commercially by the Dutch for bread making since 1780; while around 1800, the Germans started producing S. cerevisiae in the form of cream. In 1825 a method was developed to remove the liquid so the yeast could be prepared as solid blocks.[14] The industrial production of yeast blocks was enhanced by the introduction of the filter press in 1867. In 1872, Baron Max de Springer developed a manufacturing process to create granulated yeast, a technique that was used until the first World War.[15] In the United States, naturally occurring airborne yeasts were used almost exclusively until commercial yeast was marketed at the Centennial Exposition in 1876 in Philadelphia, where Charles L. Fleischmann exhibited the product and a process to use it, as well as serving the resultant baked bread.

Nutrition and growth

Yeasts are chemoorganotrophs, as they use organic compounds as a source of energy and do not require sunlight to grow. Carbon is obtained mostly from hexose sugars, such as glucose andfructose, or disaccharides such as sucrose and maltose. Some species can metabolize pentose sugars like ribose,[16] alcohols, and organic acids. Yeast species either require oxygen for aerobiccellular respiration (obligate aerobes), or are anaerobic, but also have aerobic methods of energy production (facultative anaerobes). Unlike bacteria, there are no known yeast species that grow only anaerobically (obligate anaerobes). Yeasts grow best in a neutral or slightly acidic pH environment. Yeasts vary in what temperature range they grow best. For example, Leucosporidium frigidum grows at -2 to 20 C (28 to 68 F), Saccharomyces telluris at 5 to 35 C (41 to 95 F) and Candida slooffiat 28 to 45 C (82 to 113 F).[17] The cells can survive freezing under certain conditions, with viability decreasing over time. Yeasts are generally grown in the laboratory on solid growth media or in liquid broths. Common media used for the cultivation of yeasts include potato dextrose agar (PDA) or potato dextrose broth, Wallerstein Laboratories nutrient (WLN) agar, yeast peptone dextrose agar (YPD), and yeast mould agar or broth (YM). Home brewers who cultivate yeast frequently use dried malt extract (DME) andagar as a solid growth medium. The antibiotic cycloheximide is sometimes added to yeast growth media to inhibit the growth of Saccharomyces yeasts and select for wild/indigenous yeast species. This will change the yeast process.

The appearance of a white, thready yeast, commonly known as kahm yeast, is often a byproduct of the lactofermentation (or pickling) of certain vegetables, usually the result of exposure to air. Although harmless, it can give pickled vegetables a bad flavour and so must be removed regularly during fermentation.[18]

Ecology
Yeasts are very common in the environment, and are often isolated from sugar-rich material. Examples include naturally occurring yeasts on the skins of fruits and berries (such as grapes, apples orpeaches), and exudates from plants (such as plant saps or cacti). Some yeasts are found in association with soil and insects.[19][20] The ecological function and biodiversity of yeasts are relatively unknown compared to those of other microorganisms.[21] Yeasts, including Candida albicans, Rhodotorula rubra, Torulopsis and Trichosporon cutaneum, have been found living in between people's toes as part of their skin flora.[22] Yeasts are also present in the gut flora of mammals and some insects[23] and even deep-sea environments host an array of yeasts.[24][25] An Indian study of seven bee species and 9 plant species found 45 species from 16 genera colonise the nectaries of flowers and honey stomachs of bees. Most were members of the Candida genus; the most common species in honey stomachs was Dekkera intermedia and in flower nectaries, Candida blankii.[26] Yeast colonising nectaries of the stinking hellebore have been found to raise the temperature of the flower, which may aid in attracting pollinators by increasing the evaporation of volatile organic compounds.[21][27] A black yeast has been recorded as a partner in a complex relationship between ants, their mutualistic fungus, a fungal parasite of the fungus and a bacterium that kills the parasite. The yeast have a negative effect on the bacteria that normally produceantibiotics to kill the parasite and so may affect the ants' health by allowing the parasite to spread.[28]

Reproduction

The yeast cell's life cycle: 1. Budding 2. Conjugation 3. Spore

See also: Mating of yeast

Yeasts, like all fungi, may have asexual and sexual reproductive cycles. The most common mode of vegetative growth in yeast is asexual reproduction by budding.[29] Here a small bud (also known as a bleb), or daughter cell, is formed on the parent cell. The nucleus of the parent cell splits into a daughter nucleus and migrates into the daughter cell. The bud continues to grow until it separates from the parent cell, forming a new cell.[30] Some yeasts, including Schizosaccharomyces pombe, reproduce by fission instead of budding.[29] Under high stress conditions, haploid cells will generally die; under the same conditions, however, diploid cells can undergo sporulation, entering sexual reproduction (meiosis) and producing a variety of haploid spores, which can go on to mate (conjugate), reforming the diploid.[31] Some pucciniomycete yeasts, particularly species of Sporidiobolus and Sporobolomyces produce aerially dispersed, asexual ballistoconidia.[32]

Uses
The useful physiological properties of yeast have led to their use in the field of biotechnology. Fermentation of sugars by yeast is the oldest and largest application of this technology. Many types of yeasts are used for making many foods: baker's yeast in bread production; brewer's yeast in beerfermentation; yeast in wine fermentation and for xylitol production.[33] So-called red rice yeast is actually a mold, Monascus purpureus. Yeasts include some of the most widely used model organisms for genetics and cell biology.

Alcoholic beverages
Alcoholic beverages are defined as beverages that contain ethanol (C2H5OH). This ethanol is almost always produced by fermentation the metabolism of carbohydrates by certain species of yeast under anaerobic or low-oxygen conditions. Beverages such as wine, beer, or distilled spirits all use yeast at some stage of their production. A distilled beverage is a beverage containing ethanol that has been purified by distillation. Carbohydrate-containing plant material is fermented by yeast, producing a dilute solution of ethanol in the process. Spirits such as whiskey and rum are prepared by distilling these dilute solutions of ethanol. Components other than ethanol are collected in the condensate, including water, esters, and other alcohols, which account for the flavour of the beverage.

Beer

Beer being fermented by brewers yeast

Brewing yeasts may be classed as "top cropping" (or "top fermenting") and "bottom cropping" (or "bottom-fermenting").[34] Top cropping yeasts are so called because they form a foam at the top of the wort during fermentation. An example of a top cropping yeast is Saccharomyces cerevisiae, sometimes called an "ale yeast".[35] Bottom cropping yeasts are typically used to produce lager-type beers, though they can also produce ale-type beers. These yeasts ferment well at low temperatures. An example of bottom cropping yeast is Saccharomyces pastorianus, formerly known as S. carlsbergensis. Decades ago, taxonomists reclassified S. carlsbergensis (uvarum) as a member of S. cerevisae, noting that the only distinct difference between the two is metabolic. Lager strains of S. cerevisae secrete an enzyme called melibiase, allowing it to hydrolyse the disaccharide melibiose into more fermentable monosaccharides. Top cropping and bottom cropping, cold fermenting and warm fermenting distinctions are largely generalizations used to by the laymen to communicate to the general public. For more on the taxonomical differences, see Terrance M. Dowhanick, B.SC., PH. D "Yeast - Strains and Handling Techiques" in The Practical Brewer, a publication of the Master Brewers Association of the Americas. The most common top cropping brewer's yeast, S. cerevisiae, is the same species as the common baking yeast.[36] Brewer's yeast is also very rich inessential minerals and the B vitamins (except B12).[37] However, baking and brewing yeasts typically belong to different strains, cultivated to favour different characteristics: baking yeast strains are more aggressive, to carbonate dough in the shortest amount of time possible; brewing yeast strains act slower, but tend to produce fewer off-flavours and tolerate higher alcohol concentrations (with some strains, up to 22%). Dekkera/Brettanomyces is a genus of yeast known for their important role in the production of Lambic and specialty sour ales, along with the secondary conditioning of a particular Belgian Trappist beer [38]. The taxonomy of the genus Brettanomyces has been debated since its early discovery and has seen many re-classifications over the years. Early classification was based on a few species that reproduced asexually (anamorph form) through multipolar budding [39]. Shortly after, the formation of ascospores was observed and the genus Dekkera, which reproduces sexually (teleomorph form), was introduced as part of the taxonomy [40]. The current taxonomy includes five species within the genera of Dekkera/Brettanomyces. Those are the anamorphs Brettanomyces bruxellensis,Brettanomyces anomalus, Brettanomyces custersianus, Brettanomyces naardenensis, and Brettanomyces nanus, with teleomorphs existing for the first two species, Dekkera bruxellensis andDekkera anomala [41]. The distinction between Dekkera and Brettanomyces is arguable with Oelofse et al. (2008) citing Loureiro and Malfeito-Ferreira from 2006 when they affirmed that current molecular DNA detection techniques have uncovered no variance between the anamorph and teleomorph states. Over the past decade, Brettanomyces spp. have seen an increasing use in the craft-brewing sector of the industry with a handful of breweries having produced beers that were primary fermented with pure cultures of Brettanomyces spp. This has occurred out of experimentation as very little information exists regarding pure culture fermentative capabilities and the aromatic compounds produced by various strains. Dekkera/Brettanomyces spp. have been the subjects of numerous studies conducted over the past century although a majority of the recent research has focused on enhancing the knowledge of the wine industry. Recent research on 8 Brettanomyces strains available in the brewing industry focused on strain specific fermentations and identified the major compounds produced during pure culture anaerobic fermentation in wort.[42]

Wine

Fresh grapes with visible bloom.

Main article: Fermentation (wine) Yeast is used in winemaking, where it converts the sugars present in grape juice (must) into ethanol. Yeast is normally already present on grape skins (the white powder called "the bloom"). Fermentation can be done with this endogenous "wild yeast,"[43] but this procedure gives unpredictable results, which depend upon the exact types of yeast species present. For this reason, a pure yeast culture is usually added to the must; this yeast quickly dominates the fermentation. The wild yeasts are repressed, which ensures a reliable and predictable fermentation.[44] Most added wine yeasts are strains of S. cerevisiae, though not all strains of the species are suitable.[44] Different S. cerevisiae yeast strains have differing physiological and fermentative properties, therefore the actual strain of yeast selected can have a direct impact on the finished wine.[45] Significant research has been undertaken into the development of novel wine yeast strains that produce atypical flavour profiles or increased complexity in wines.[46][47] The growth of some yeasts, such as Zygosaccharomyces and Brettanomyces, in wine can result in wine faults and subsequent spoilage.[48] Brettanomycesproduces an array of metabolites when growing in wine, some of which are volatile phenolic compounds. Together, these compounds are often referred to as "Brettanomyces character", and are often described as "antiseptic" or "barnyard" type aromas. Brettanomyces is a significant contributor to wine faults within the wine industry.[49] Researchers from University of British Columbia, Canada, have found a new strain of yeast that has reduced amines. The amines in red wine and Chardonnay produce off-flavors and cause headaches and hypertension in some people. About 30 percent of people are sensitive to biogenic amines, such as histamines.[50]

Baking
Main article: Baker's yeast Yeast, most commonly S. cerevisiae, is used in baking as a leavening agent, where it converts the fermentable sugars present in dough into the gas carbon dioxide. This causes the dough to expand or rise as gas forms pockets or bubbles. When the dough is

baked, the yeast dies and the air pockets "set", giving the baked product a soft and spongy texture. The use of potatoes, water from potato boiling, eggs, or sugar in a bread dough accelerates the growth of yeasts. Most yeasts used in baking are of the same species common in alcoholic fermentation. Additionally, Saccharomyces exiguus(also known as S. minor), a wild yeast found on plants, fruits, and grains, is occasionally used for baking. Sugar and vinegar provide the best conditions for yeast to ferment. In bread making, the yeast initially respires aerobically, producing carbon dioxide and water. When the oxygen is depleted, anaerobic respiration begins, producing ethanol as a waste product; however, this evaporates during baking.[51]

A block of fresh yeast

It is not known when yeast was first used to bake bread. The first records that show this use came from Ancient Egypt.[52] Researchers speculate a mixture of flour meal and water was left longer than usual on a warm day and the yeasts that occur in natural contaminants of the flour caused it to ferment before baking. The resulting bread would have been lighter and tastier than the normal flat, hard cake.

Active dried yeast, a granulated form in which yeast is commercially sold

Today, there are several retailers of baker's yeast; one of the best-known in North America is Fleischmanns Yeast, which was developed in 1868. DuringWorld War II, Fleischmann's developed a granulated active dry yeast, which did not require refrigeration and had a longer shelf life than fresh yeast. The company created yeast that would rise twice as fast, reducing baking time. Baker's yeast is also sold as a fresh yeast compressed into a square "cake". This form perishes quickly, and must therefore be used soon after production. A weak solution of water and sugar can be used to determine if yeast is expired. In the solution, active yeast will foam and bubble as it ferments the sugar into ethanol and carbon dioxide. Some recipes refer to this as proofing the yeast as it "proves" (tests)

the viability of the yeast before the other ingredients are added. When using a sourdough starter, flour and water are added instead of sugar; this is referred to as proofing the sponge. When yeast is used for making bread, it is mixed with flour, salt, and warm water or milk. The dough is kneaded until it is smooth, and then left to rise, sometimes until it has doubled in size. Some bread doughs are knocked back after one rising and left to rise again. A longer rising time gives a better flavour, but the yeast can fail to raise the bread in the final stages if it is left for too long initially. The dough is then shaped into loaves, left to rise until it is the correct size, and then baked. Dried yeast is usually specified for use in a bread machine; however, a (wet) sourdough starter can also work.

Bioremediation
Some yeasts can find potential application in the field of bioremediation. One such yeast, Yarrowia lipolytica, is known to degrade palm oil mill effluent,[53]TNT (an explosive material),[54] and other hydrocarbons, such as alkanes, fatty acids, fats and oils.[55] It can also tolerate high concentrations of salt andheavy metals,[56] and is being investigated for its potential as a heavy metal biosorbent.[57]

Industrial ethanol production

See also: Bioethanol The ability of yeast to convert sugar into ethanol has been harnessed by the biotechnology industry to produce ethanol fuel. The process starts by milling a feedstock, such as sugar cane, field corn, or other cereal grains, and then adding dilute sulfuric acid, or fungal alpha amylase enzymes, to break down the starches into complex sugars. A glucoamylase is then added to break the complex sugars down into simple sugars. After this, yeasts are added to convert the simple sugars to ethanol, which is then distilled off to obtain ethanol up to 96% in concentration.[58] Saccharomyces yeasts have been genetically engineered to ferment xylose, one of the major fermentable sugars present in cellulosic biomasses, such as agriculture residues, paper wastes, and wood chips.[59][60] Such a development means ethanol can be efficiently produced from more inexpensive feedstocks, making cellulosic ethanol fuel a more competitively priced alternative to gasolinefuels.[61]

Nonalcoholic beverages

A Kombucha culture fermenting in a jar

Root beer and other sweet carbonated beverages can be produced using the same methods as beer, except the fermentation is stopped sooner, producing carbon dioxide, but only trace amounts of alcohol, and a significant amount of sugar is left in the drink. Kvass, a fermented drink made from rye, is popular inEastern Europe; it has a recognizable, but low alcoholic content. Yeast in symbiosis with acetic acid bacteria is used in the preparation of kombucha, a fermented sweetened tea. Species of yeast found in the tea can vary, and may include: Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii and Zygosaccharomyces bailii.[62] Kombucha is a popular beverage in Eastern Europe and some former Soviet republicsunder the name chajnyj grib ( ), which means "tea mushroom". Kefir and kumis are made by fermenting milk with yeast and bacteria.[63]

Nutritional supplements
See also: Tibicos Yeast is used in nutritional supplements popular with vegans and the health conscious, where it is often referred to as "nutritional yeast". It is a deactivated yeast, usually S. cerevisiae. It is an excellent source of protein and vitamins, especially the Bcomplex vitamins, whose functions are related to metabolism, as well as other minerals and cofactors required for growth. It is also naturally low in fat and sodium. Some brands of nutritional yeast, though not all, are fortified with vitamin B12, which is produced separately by bacteria. Nutritional yeast, though it has a similar appearance to brewer's yeast, is very different and has a very different taste. Brewer's yeast is a good source of B-complex vitamins but, contrary to some claims, it contains little or no vitamin B12.[64] Nutritional yeast has a nutty, cheesy flavor which makes it popular as an ingredient in cheese substitutes. It is often used by vegans in place of Parmesan cheese. Another popular use is as a topping for popcorn. It can also be used in mashed and fried potatoes, as well as in scrambled eggs. It comes in the form of flakes, or as a yellow powder similar in texture to cornmeal, and can be found in the bulk aisle of most natural food stores. In Australia, it is sometimes sold as "savory yeast flakes". Though "nutritional yeast" usually refers to commercial products, inadequately fed prisoners have used "home-grown" yeast to prevent vitamin deficiency.[65]

Probiotics
Some probiotic supplements use the yeast S. boulardii to maintain and restore the natural flora in the gastrointestinal tract. S. boulardii has been shown to reduce the symptoms of acute diarrhea in children,[66][67] prevent reinfection of Clostridium difficile,
[68]

reduce bowel movements in diarrhea-predominant IBS patients,[69] and reduce the incidence of antibiotic,[70] traveler's, and HIV/AIDS[72]associated diarrheas.

[71]

Aquarium hobby
Yeast is often used by aquarium hobbyists to generate carbon dioxide (CO2) to nourish plants in planted aquariums.[73] A homemade setup is widely used as a cheap and simple alternative to pressurized CO2 systems. While not as effective as these, the homemade setup is considerably cheaper for less demanding hobbyists.

There are several recipes for homemade CO2, but they are variations of the basic recipe: Baker's yeast, with sugar, baking soda and water, are added to a plastic bottle. A few drops of vegetable oil at the start reduces surface tension and speeds the release of CO2. This will produce CO2 for about 2 or 3 weeks; the use of a bubble counter determines production. The CO2 is injected in the aquarium via a narrow hose and released through a diffuser that helps dissolve the gas in the water. The CO2 is used by plants in the photosynthesis process.

Science

Diagram showing a yeast cell

Several yeasts, particularly S. cerevisiae, have been widely used in genetics and cell biology. This is largely because S. cerevisiae is a simple eukaryoticcell, serving as a model for all eukaryotes, including humans for the study of fundamental cellular processes such as the cell cycle, DNA replication,recombination, cell division and metabolism. Also, yeasts are easily manipulated and cultured in the laboratory, which has allowed for the development of powerful standard techniques, such as yeast two-hybrid, synthetic genetic array analysis and tetrad analysis. Many proteins important in human biology were first discovered by studying their homologues in yeast; these proteins include cell cycle proteins, signaling proteins, and protein-processing enzymes. On 24 April 1996 S. cerevisiae was announced to be the first eukaryote to have its genome, consisting of 12 million base pairs, fully sequenced as part of theGenome project.[74] At the time, it was the most complex organism to have its full genome sequenced, and took seven years and the involvement of more than 100 laboratories to accomplish.[75] The second yeast species to have its genome sequenced was Schizosaccharomyces pombe, which was completed in 2002.[76][77] It was the sixth eukaryotic genome sequenced and consists of 13.8 million base pairs.

Yeast extract
Main article: Yeast extract

Marmite and Vegemite, products made from yeast extract

Marmite and Vegemite have a distinctive dark colour

Yeast extract is the common name for various forms of processed yeast products that are used as food additives or flavours. They are often used in the same way that monosodium glutamate (MSG) is used, and like MSG, often contain free glutamic acid. The general method for making yeast extract for food products such as Vegemite and Marmite on a commercial scale is to add salt to a suspension of yeast making the solution hypertonic, which leads to the cells shrivelling up. This triggers autolysis, where the yeast's digestive enzymes break their ownproteins down into simpler compounds, a process of self-destruction. The dying yeast cells are then heated to complete their breakdown, after which the husks (yeast with thick cell walls which would give poor texture) are separated. Yeast autolysates are used in Vegemite and Promite(Australia); Marmite, Bovril and Oxo (the United Kingdom, Republic of Ireland and South Africa); and Cenovis (Switzerland).

Pathogenic yeasts

A photomicrograph of Candida albicansshowing hyphal outgrowth and other morphological characteristics.

Some species of yeast are opportunistic pathogens where they can cause infection in people with compromised immune systems. Cryptococcus neoformans is a significant pathogen of immunocompromised people causing the disease termed cryptococcosis. This disease occurs in about 79% of AIDS patients in the USA, and a slightly smaller percentage (36%) in western Europe.[78] The cells of the yeast are surrounded by a rigid polysaccharide capsule, which helps to prevent them from being recognised and engulfed by white blood cells in the human body.

Yeasts of the Candida genus are another group of opportunistic pathogens which causes oral and vaginal infections in humans, known as candidiasis.Candida is commonly found as a commensal yeast in the mucus membranes of humans and other warm-blooded animals. However, sometimes these same strains can become pathogenic. Here the yeast cells sprout a hyphal outgrowth, which locally penetrates the mucosal membrane, causing irritation and shedding of the tissues.[78] The pathogenic yeasts of candidiasis in probable descending order of virulence for humans are: C. albicans, C. tropicalis, C. stellatoidea, C. glabrata, C. krusei, C. parapsilosis, C. guilliermondii, C. viswanathii, C. lusitaniae and Rhodotorula mucilaginosa.[79]Candida glabrata is the second most common Candida pathogen after C. albicans, causing infections of the urogenital tract, and of the bloodstream(candidemia).[80]

Food spoilage
Yeasts are able to grow in foods with a low pH, (5.0 or lower) and in the presence of sugars, organic acids and other easily metabolized carbon sources.[81] During their growth, yeasts metabolize some food components and produce metabolic end products. This causes the physical, chemical, and sensible properties of a food to change, and the food is spoiled.[82] The growth of yeast within food products is often seen on their surface, as in cheeses or meats, or by the fermentation of sugars in beverages, such as juices, and semi-liquid products, such as syrups and jams.[81] The yeast of the Zygosaccharomyces genus have had a long history as a spoilage yeast within the food industry. This is mainly due to the fact that these species can grow in the presence of high sucrose, ethanol, acetic acid, sorbic acid, benzoic acid, and sulphur dioxide concentrations,[83] representing some of the commonly used food preservationmethods. Methylene blue is used to test for the presence of live yeast cells.

What is yeast? Yeast is made up of a single-celled organism, Saccharomyces cerevisiae, which multiplies rapidly when fed sugar in a moist environment. One pound of yeast contains 3,200 billion yeast cells! Yeast also thrives on starch, which it converts to glucose, a simple sugar. This process ferments the sugar, which converts to alcohol and carbon dioxide. The carbon dioxide expands the baked good to produce the light, fluffy texture. Of course, yeast is also a vital part of the making of alchoholic beverages, such asbeer and wine. But that's another story... The minimal alcohol product burns off during the baking process, and the yeast dies also with the heat. The ideal temperature for yeast growth is 100 to 115 degrees F., but for leavening purposes, the ideal temperature is 80 to 95 degrees F. If the yeast grows too quickly, it will produce large bubble pockets in the end product. Yeast begins to die at 120 degrees F. So, it is important to let your yeast dough rise in a spot where the temperature is regulated. One-half an ounce of yeast will raise 4 cups of flour in about 1-1/2 to 2 hours, under ideal conditions. You should also proof your yeast to be sure it is viable before using in a recipe. To check it, mix a bit into 1/4 cup of lukewarm water with 1/4 teaspoon sugar. It should begin to bubble and ferment within about 5 to 10 minutes. If not, the yeast is dead and should be discarded. Salt inhibits the growth of yeast. Never mix yeast into salted water. Since most tap water goes through a filtering process which utilizes salt as a refining/cleaning agent, many cooks use only distilled water for baking. However, if you are baking during the hot summer season and find your dough rising too much, the addition of a little extra salt can control that runaway yeast growth.

Most yeast is sold in single-use packets or bulk bags known as dry active yeast. Compressed yeast is not as widely available, but can be used to the ratio of one standard cake of compressed yeast to one scant tablespoon of dry yeast. If the dry yeast is stored in airtight packaging, in a cool dry place, it is not necessary to refrigerate it. Yeast should always be at room temperature to begin a recipe. Standard single-use packets contain about 2-1/2 teaspoons (1/4 ounce) of yeast granules. Now available on the market is fast-rising active dry yeast, which is smaller-grained than conventional active dry yeast and speeds rising times by as much as fifty percent, often eliminating the need for a second rising period. It may be used interchangeably, measure for measure, with active dry yeast. The best method for using this yeast is to mix it directly with the dry ingredients before adding liquid, instead of adding it to warmed liquid and then adding to dry ingredients. Be aware that compressed fresh yeast consists of 70 percent moisture and must be stored in the refrigerator. Compressed fresh yeast is highly perishable, as opposed to dry active yeast, and loses its vitality within 2 weeks, even when properly stored refrigerated in an airtight container. Compressed yeast can be stored in the freezer, but should be defrosted at room temperature and then used immediately. Compressed fresh yeast is difficult to find in the United States. Dried yeast has become the norm for its staying power in the pantry. Yet, even dry yeast must be stored in an airtight container, with no threat of moisture, and it will lose its life over time. Use 2 teaspoons of dried yeast to a 2/3 ounce compressed yeast cake as a substitution. Yeast measures will also have to be adjusted at higher altitudes. Again, this will take experimentation on your part for your altitude. Why is kneading of yeast breads required? It helps distribute the yeast cells uniformly throughout the dough, so it does not rise unevenly. Kneading also develops a firm gluten structure, providing the framework for the carbon dioxide bubbles. Brewer's yeast has no leavening properties but is added to products for nutritive benefits, as it is rich in the B vitamins. It is also, of course, used in the brewing of beer. Salt and Sugar in Yeast Baked Goods Although salt does inhibit the growth of yeast, it does give a firmer crust, a finer crumb, and adds flavor. Sugars are not essential to leavened baked goods, but they make the product more tender due to postponement of protein coagulation, allowing the dough/batter to grow to a greater volume before being frozen into stasis by the baking process, as well as adding to flavor. If too much sugar is used, it can slow down the growth of the yeast, with a low-rise result. The relationship of sugar to salt to leavening is crucial to a pleasing final product. More About Leaveners: What is yeast? Yeast is made up of a single-celled organism, Saccharomyces cerevisiae, which multiplies rapidly when fed sugar in a moist environment. One pound of yeast contains 3,200 billion yeast cells! Yeast also thrives on starch, which it converts to glucose, a simple sugar. This process ferments the sugar, which converts to alcohol and carbon dioxide. The carbon dioxide expands the baked good to produce the light, fluffy texture. Of course, yeast is also a vital part of the making of alchoholic beverages, such asbeer and wine. But that's another story... The minimal alcohol product burns off during the baking process, and the yeast dies also with the heat.

The ideal temperature for yeast growth is 100 to 115 degrees F., but for leavening purposes, the ideal temperature is 80 to 95 degrees F. If the yeast grows too quickly, it will produce large bubble pockets in the end product. Yeast begins to die at 120 degrees F. So, it is important to let your yeast dough rise in a spot where the temperature is regulated. One-half an ounce of yeast will raise 4 cups of flour in about 1-1/2 to 2 hours, under ideal conditions. You should also proof your yeast to be sure it is viable before using in a recipe. To check it, mix a bit into 1/4 cup of lukewarm water with 1/4 teaspoon sugar. It should begin to bubble and ferment within about 5 to 10 minutes. If not, the yeast is dead and should be discarded. Salt inhibits the growth of yeast. Never mix yeast into salted water. Since most tap water goes through a filtering process which utilizes salt as a refining/cleaning agent, many cooks use only distilled water for baking. However, if you are baking during the hot summer season and find your dough rising too much, the addition of a little extra salt can control that runaway yeast growth. Most yeast is sold in single-use packets or bulk bags known as dry active yeast. Compressed yeast is not as widely available, but can be used to the ratio of one standard cake of compressed yeast to one scant tablespoon of dry yeast. If the dry yeast is stored in airtight packaging, in a cool dry place, it is not necessary to refrigerate it. Yeast should always be at room temperature to begin a recipe. Standard single-use packets contain about 2-1/2 teaspoons (1/4 ounce) of yeast granules. Now available on the market is fast-rising active dry yeast, which is smaller-grained than conventional active dry yeast and speeds rising times by as much as fifty percent, often eliminating the need for a second rising period. It may be used interchangeably, measure for measure, with active dry yeast. The best method for using this yeast is to mix it directly with the dry ingredients before adding liquid, instead of adding it to warmed liquid and then adding to dry ingredients. Be aware that compressed fresh yeast consists of 70 percent moisture and must be stored in the refrigerator. Compressed fresh yeast is highly perishable, as opposed to dry active yeast, and loses its vitality within 2 weeks, even when properly stored refrigerated in an airtight container. Compressed yeast can be stored in the freezer, but should be defrosted at room temperature and then used immediately. Compressed fresh yeast is difficult to find in the United States. Dried yeast has become the norm for its staying power in the pantry. Yet, even dry yeast must be stored in an airtight container, with no threat of moisture, and it will lose its life over time. Use 2 teaspoons of dried yeast to a 2/3 ounce compressed yeast cake as a substitution. Yeast measures will also have to be adjusted at higher altitudes. Again, this will take experimentation on your part for your altitude. Why is kneading of yeast breads required? It helps distribute the yeast cells uniformly throughout the dough, so it does not rise unevenly. Kneading also develops a firm gluten structure, providing the framework for the carbon dioxide bubbles. Brewer's yeast has no leavening properties but is added to products for nutritive benefits, as it is rich in the B vitamins. It is also, of course, used in the brewing of beer. Salt and Sugar in Yeast Baked Goods Although salt does inhibit the growth of yeast, it does give a firmer crust, a finer crumb, and adds flavor. Sugars are not essential to leavened baked goods, but they make the product more tender due to postponement of protein coagulation, allowing the dough/batter to grow to a greater volume before being frozen into stasis by the baking process, as well as adding to flavor. If too much sugar is used, it can slow down the growth of the yeast, with a low-rise result. The relationship of sugar to salt to leavening is crucial to a pleasing final product.

What is yeast?
Yeast is a fungus scientifically referred to as Candida. The specific type of fungus most commonly responsible for vaginitis is Candida albicans. Yeast is commonly present on normal human skin and in areas of moisture, such as the mouth and vagina. In fact, it is estimated that between 20%-50% of healthy women normally carry yeast in the vaginal area.

Artificial blood is a product made to act as a substitute for red blood cells. While true blood serves many different functions, artificial blood is designed for the sole purpose of transporting oxygen and carbon dioxide throughout the body. Depending on the type of artificial blood, it can be produced in different ways using synthetic production, chemical isolation, or recombinant biochemical technology. Development of the first blood substitutes dates back to the early 1600s, and the search for the ideal blood substitute continues. Various manufacturers have products in clinical trials; however, no truly safe and effective artificial blood product is currently marketed. It is anticipated that when an artificial blood product is available, it will have annual sales of over $7.6 billion in the United States alone. Keywords: Blood, artificial blood, Perfluorocarbons

Other Sections

Background
Blood is a special type of connective tissue that is composed of white cells, red cells, platelets, and plasma. It has a variety of functions in the body. Plasma is the extracellular material made up of water, salts, and various proteins that, along with platelets, encourages blood to clot. Proteins in the plasma react with air and harden to prevent further bleeding. The white blood cells are responsible for the immune defense. They seek out invading organisms or materials and minimize their effect in the body. The red cells in blood create the bright red color. As little as two drops of blood contains about one billion red blood cells. These cells are responsible for the transportation of oxygen and carbon dioxide throughout the body. They are also responsible for the typing phenomena. On the membranes of these cells are proteins that the body recognizes as its own. For this reason, a person can use only blood that is compatible with her type. Currently, artificial blood products are only designed to replace the function of red blood cells. It might even be better to call the products being developed now, oxygen carriers instead of artificial blood.

Other Sections

History

There has been a need for blood replacements for as long as patients have been bleeding to death because of a serious injury. According to medical folklore, the ancient Incas were responsible for the first recorded blood transfusions. No real progress was made in the development of a blood substitute until 1616, when William Harvey described how blood is circulated throughout the body. In the years to follow, medical practitioners tried numerous substances such as beer, urine, milk, plant resins, and sheep blood as a substitute for blood. They had hoped that changing a person's blood could have different beneficial effects such as curing diseases or even changing a personality. The first successful human blood transfusions were done in 1667. Unfortunately, the practice was halted because patients who received subsequent transfusions died. Of the different materials that were tried as blood substitutes over the years, only a few met with minimal success. Milk was one of the first of these materials. In 1854, patients were injected with milk to treat Asiatic cholera. Physicians believed that the milk helped regenerate white blood cells. In fact, enough of the patients given milk as a blood substitute seemed to improve that it was concluded to be a safe and legitimate blood replacement procedure. However, many practitioners remained skeptical so milk injections never found widespread appeal. It was soon discarded and forgotten as a blood replacement. Another potential substitute was salt or saline solutions. In experiments done on frogs, scientists found that they could keep frogs alive for some time if they removed all their blood and replaced it with a saline solution. These results were a little misleading, however, because it was later determined that frogs could survive for a short time without any blood circulation at all. After much research, saline was developed as a plasma volume expander.

Artificial Blood

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Artificial blood is a product made to act as a substitute for red blood cells. While true blood serves many different functions, artificial blood is designed for the sole purpose of transporting oxygen and carbon dioxide throughout the body. Depending on the type of artificial blood, it can be produced in different ways using synthetic production, chemical isolation, or recombinant biochemical technology. Development of the first blood substitutes dates back to the early 1600s, and the search for the ideal

blood substitute continues. Various manufacturers have products in clinical trials; however, no truly safe and effective artificial blood product is currently marketed. It is anticipated that when an artificial blood product is available, it will have annual sales of over $7.6 billion in the United States alone.

Background
Blood is a special type of connective tissue that is composed of white cells, red cells, platelets, and plasma. It has a variety of functions in the body. Plasma is the extracellular material made up of water, salts, and various proteins that, along with platelets, encourages blood to clot. Proteins in the plasma react with air and harden to prevent further bleeding. The white blood cells are responsible for the immune defense. They seek out invading organisms or materials and minimize their effect in the body. The red cells in blood create the bright red color. As little as two drops of blood contains about one billion red blood cells. These cells are responsible for the transportation of oxygen and carbon dioxide throughout the body. They are also responsible for the "typing" phenomena. On the membranes of these cells are proteins that the body recognizes as its own. For this reason, a person can use only blood that is compatible with her type. Currently, artificial blood products are only designed to replace the function of red blood cells. It might even be better to call the products being developed now, oxygen carriers instead of artificial blood.

History
There has been a need for blood replacements for as long as patients have been bleeding to death because of a serious injury. According to medical folklore, the ancient Incas were responsible for the first recorded blood transfusions. No real progress was made in the development of a blood substitute until 1616, when William Harvey described how blood is circulated throughout the body. In the years to follow, medical practitioners tried numerous substances such as beer, urine, milk, plant resins, and sheep blood as a substitute for blood. They had hoped that changing a person's blood could have different beneficial effects such as curing diseases or even changing a personality. The first successful human blood transfusions were done in 1667. Unfortunately, the practice was halted because patients who received subsequent transfusions died. Of the different materials that were tried as blood substitutes over the years, only a few met with minimal success. Milk was one of the first of these materials. In 1854, patients were injected with milk to treat Asiatic cholera. Physicians believed that the milk helped regenerate white blood cells. In fact, enough of the patients given milk as a blood substitute seemed to improve that it was concluded to be a safe and legitimate blood replacement procedure. However, many practitioners remained skeptical so milk injections never found widespread appeal. It was soon discarded and forgotten as a blood replacement. Another potential substitute was salt or saline solutions. In experiments done on frogs, scientists found that they could keep frogs alive for some time if they removed all their blood and replaced it with a saline solution. These results were a little misleading, however, because it was later determined that frogs could survive for a short time without any blood circulation at all. After much research, saline was developed as a plasma volume expander. Other materials that were tried during the 1800s include hemoglobin and animal plasma. In 1868, researchers found that solutions containing hemoglobin isolated from red blood cells could be used as blood replacements. In 1871, they also examined the use of animal plasma and blood as a substitute

for human blood. Both of these approaches were hampered by significant technological problems. First, scientists found it difficult to isolate a large volume of hemoglobin. Second, animal products contained many materials that were toxic to humans. Removing these toxins was a challenge during the nineteenth century. A significant breakthrough in the development of artificial blood came in 1883 with the creation of Ringer's solutiona solution composed of sodium, potassium, and calcium salts. In research using part of a frog's heart, scientists found that the heart could be kept beating by applying the solution. This eventually led to findings that the reduction in blood pressure caused by a loss of blood volume could be restored by using Ringer's solution. This product evolved into a human product when lactate was added. While it is still used today as a blood-volume expander, Ringer's solution does not replace the action of red blood cells so it is not a true blood substitute. Karl Landsteiner, who has been called the father of immunology, was the only child of Leopold Landsteiner, a prominent Austrian journalist and editor, and Fanny Hess Landsteiner. Landsteiner was educated at the University of Vienna, where he received his medical degree in 1891. While in medical school, Landsteiner began experimental work in chemistsy, as he was greatly inspired by Ernst Ludwig, one of his professors. After receiving his medical degree, Landsteiner spent the next five years doing advanced research in orgnic chemistry for Emil Fischer, although medicine remained his chief interest. During 1886-1897, he combined these interests at the Institute of Hygiene at the University of Vienna where he researched immunology and serology. Immunology and serology then became Landsteiner's lifelong focus. Landsteiner was primarily interested in the lack of safety and effective-ness of blood transfusions. Prior to his work, blood transfusions were dangerous and underutilized because the donor's blood frequently clotted in the patient. Landsteiner was intrigued by the fact that when blood from different subjects was mixed, the blood did not always clot. He believed there were intrinsic biochemical similarities and dissimilarities in blood. Using blood samples from his colleagues,!he separated the blood's cells frnm its serum, and suspeneed the red blood cells in a saline solution. He then mixed each individual's serum with a sample grom every cell suspension. Clotting occurred in some cares; in others there was no clotting. Landsteiner determined that human beings could be separated into blood groups according to the capacity of their red cells to clot in the presence of different serums. He named his blood classification groups A, B, and O. A fourth group AB, was discovered the following year. The result of this work was that patient and donor could be blood-typed beforehand, making blood transfusion a safe and routine medical practice. This discovery ultimately earned Landsteiner the 1930 Nobel Prize in physiology or medicine. Blood transfusion research did not move forward until scientists developed a better understanding of the role of blood and the issues surrounding its function in the body. During World War I, a gum-saline solution containing galactoso-gluconic acid was used to extend plasma. If the concentration, pH, and temperature were adjusted, this material could be designed to match the viscosity of whole blood, allowing physicians to use less plasma. In the 1920s, studies suggested that this gum solution had some negative health effects. By the 1930s, the use of this material had significantly diminished. World War II reignited an interest in the research of blood and blood substitutes. Plasma donated from humans was commonly used to replace blood and to save soldiers from hemorrhagic shock. Eventually, this led to the establishment of blood banks by the American Red Cross in 1947.

In 1966, experiments with mice suggested a new type of blood substitute, perfluorochemicals (PFC). These are long chain polymers similar to Teflon. It was found that mice could survive even after being immersed in PFC. This gave scientists the idea to use PFC as a blood thinner. In 1968, the idea was tested on rats. The rat's blood was completely removed and replaced with a PFC emulsion. The animals lived for a few hours and recovered fully after their blood was replaced. However, the established blood bank system worked so well research on blood substitutes waned. It received renewed interest when the shortcomings of the blood bank system were discovered during the Vietnam conflict. This prompted some researchers to begin looking for hemoglobin solutions and other synthetic oxygen carriers. Research in this area was further fueled in 1986 when it was discovered that HIV and hepatitis could be transmitted via blood transfusions.

Design
The ideal artificial blood product has the following characteristics. First, it must be safe to use and compatible within the human body. This means that different blood types should not matter when an artificial blood is used. It also means that artificial blood can be processed to remove all diseasecausing agents such as viruses and microorganisms. Second, it must be able to transport oxygen throughout the body and release it where it is needed. Third, it must be shelf stable. Unlike donated blood, artificial blood can be stored for over a year or more. This is in contrast to natural blood which can only be stored for one month before it breaks down. There are two significantly different products that are under development as blood substitutes. They differ primarily in the way that they carry oxygen. One is based on PFC, while the other is a hemoglobin-based product.

Perfluorocarbons (PFC)
As suggested, PFC are biologically inert materials that can dissolve about 50 times more oxygen than blood plasma. They are relatively inexpensive to produce and can be made devoid of any biological materials. This eliminates the real possibility of spreading an infectious disease via a blood transfusion. From a technological standpoint, they have two significant hurdles to overcome before they can be utilized as artificial blood. First, they are not soluble in water, which means to get them to work they must be combined with emulsifiersfatty compounds called lipids that are able to suspend tiny particles of perfluorochemicals in the blood. Second, they have the ability to carry much less oxygen than hemoglobin-based products. This means that significantly more PFC must be used. One product of this type has been approved for use by the Federal Drug Administration (FDA), but it has not been commercially successful because the amount needed to provide a benefit is too high. Improved PFC emulsions are being developed but have yet to reach the market.

Hemoglobin-based products
Hemoglobin carries oxygen from the lungs to the other tissues in the body. Artificial blood based on hemoglobin takes advantage of this natural function. Unlike PFC products where dissolving is the key mechanism, oxygen covalently bonds to hemoglobin. These hemoglobin products are different than whole blood in that they are not contained in a membrane so the problem of blood typing is eliminated. However, raw hemoglobin cannot be used because it would break down into smaller, toxic compounds within the body. There are also problems with the stability of hemoglobin in a solution. The challenge in creating a hemoglobin-based artificial blood is to modify the hemoglobin molecule so these problems are resolved. Various strategies are employed to stabilize hemoglobin. This involves either chemically cross-linking molecules or using recombinant DNA technology to produce modified

proteins. These modified hemoglobins are stable and soluble in solutions. Theoretically, these modifications should result in products that have a greater ability to carry oxygen than our own red blood cells. It is anticipated that the first of these products will be available within one to two years.

Raw Materials
Depending on the type of artificial blood that is made, various raw materials are used. Hemoglobinbased products can use either isolated hemoglobin or synthetically produced hemoglobin. To produce hemoglobin synthetically, manufacturers use compounds known as amino acids. These are chemicals that plants and animals use to create the proteins that are essential for life. There are 20 naturally occurring amino acids that may be used to produce hemoglobin. All of the amino acid molecules share certain chemical characteristics. They are made up of an amino group, a carboxyl group, and a side chain. The nature of the side chain differentiates the various amino acids. Hemoglobin synthesis also requires a specific type of bacteria and all of the materials needed to incubate it. This includes warm water, molasses, glucose, acetic acid, alcohols, urea, and liquid ammonia. For other types of hemoglobin-based artificial blood products, the hemoglobin is isolated from human blood. It is typically obtained from donated blood that has expired before it is used. Other sources of hemoglobin come from spent animal blood. This hemoglobin is slightly different from human hemoglobin and must be modified before being used.

The Manufacturing Process

The production of artificial blood can be done in a variety of ways. For hemoglobin-based products, this involves isolation or synthesization of hemoglobin, molecular modification then reconstitution in an artificial blood formula. PFC products involve a polymerization reaction. A method for the production of a synthetic hemoglobin-based product is outlined below.

Hemoglobin synthesis
1 To obtain hemoglobin, a strain of E. coli bacteria that has the ability to produce human

hemoglobin is used. Over the course of about three days, the protein is harvested and the bacteria are destroyed. To start the fermentation process, a sample of the pure bacteria culture is transferred to a test tube that contains all the nutrients necessary for growth. This initial inoculation causes the bacteria to multiply. When the population is great enough, they are transferred to a seed tank. 2 A seed tank is a large stainless steel kettle that provides an ideal environment for growing bacteria. It is filled with warm water, food, and an ammonia source which are all required for the production of hemoglobin. Other growth factors such as vitamins, amino acids, and minor nutrients are also added. The bacterial solution inside the seed tank is constantly bathed with compressed air and mixed to keep it moving. When enough time has passed, the contents of the seed tank is pumped to the fermentation tank. 3 The fermentation tank is a larger version of the seed tank. It is also filled with a growth media needed for the bacteria to grow and produce hemoglobin. Since pH control is vital for optimal growth, ammonia water is added to the tank as necessary. When enough hemoglobin has been produced, the tank is emptied so isolation can begin.

 4 Isolation begins with a centrifugal separator that isolates much of the hemoglobin. It can be

further segregated and purified using fractional distillation. This standard column separation method is based on the principle of boiling a liquid to separate one or more components and utilizes vertical structures called fractionating columns. From this column, the hemoglobin is transferred to a final processing tank.

Final processing
5 Here, it is mixed with water and other electrolytes to produce the artificial blood. The artificial

blood can then be pasteurized and put into an appropriate packaging. The quality of compounds is checked regularly during the entire process. Particularly important are frequent checks made on the bacterial culture. Also, various physical and chemical properties of the finished product are checked such as pH, melting point, moisture content, etc. This method of production has been shown to be able to produce batches as large as 2,640 gal (10,000 L).

Recombinant DNA
From Wikipedia, the free encyclopedia

Recombinant DNA (rDNA) molecules are DNA sequences that result from the use of laboratory methods (molecular cloning) to bring together genetic material from multiple sources, creatingsequences that would not otherwise be found in biological organisms. Recombinant DNA is possible because DNA molecules from all organisms share the same chemical structure; they differ only in the sequence of nucleotides within that identical overall structure. Consequently, when DNA from a foreign source is linked to host sequences that can drive DNA replication and then introduced into a host organism, the foreign DNA is replicated along with the host DNA. Recombinant DNA molecules are sometimes called chimeric DNA, because they are usually made of material from two different species, like the mythological chimera. The DNA sequences used in the construction of recombinant DNA molecules can originate from any species. For example, plant DNA may be joined to bacterial DNA, or human DNA may be joined with fungal DNA. In addition, DNA sequences that do not occur anywhere in nature may be created by the chemical synthesis of DNA, and incorporated into recombinant molecules. Using recombinant DNA technology and synthetic DNA, literally any DNA sequence may be created and introduced into any of a very wide range of living organisms.

Proteins that result from the expression of recombinant DNA within living cells are termed recombinant proteins. When recombinant DNA encoding a protein is introduced into a host organism, the recombinant protein will not necessarily be produced. Expression of foreign proteins requires the use of specialized expression vectors and often necessitates significant restructuring of the foreign coding sequence. It is important to note that recombinant DNA differs from genetic recombination in that the former results from artificial methods in the test tube, while the latter is a normal biological process that results in the remixing of existing DNA sequences in essentially all organisms.
Contents
[hide]

1 Creating recombinant DNA 2 Expression of recombinant DNA 3 Properties of organisms containing recombinant DNA 4 Applications of recombinant DNA technology 5 History of recombinant DNA 6 Controversy 7 See also 8 References 9 Further reading 10 External links

[edit]Creating

recombinant DNA

Main article: Molecular cloning

Construction of recombinant DNA, in which a foreign DNA fragment is inserted into a plasmid vector. In this example, the gene indicated by the white color is inactivated upon insertion of the foreign DNA fragment.

Molecular cloning is the laboratory process used to create recombinant DNA.[1][2][3][4] It is one of two widely-used methods (along with polymerase chain reaction, abbr. PCR) used to direct the replication of any specific DNA sequence chosen by the experimentalist. The fundamental difference between the two methods is that molecular cloning involves replication of the DNA within a living cell, while PCR replicates DNA in the test tube, free of living cells. Formation of recombinant DNA requires a cloning vector, a DNA molecule that will replicate within a living cell. Vectors are generally derived fromplasmids or viruses, and represent relatively small segments of DNA that contain necessary genetic signals for replication, as well as additional elements for convenience in inserting foreign DNA, identifying cells that contain recombinant DNA, and, where appropriate, expressing the foreign DNA. The choice of vector for molecular cloning depends on the choice of host organism, the size of the DNA to be cloned, and whether and how the foreign DNA is to be expressed.[5] In standard cloning protocols, the cloning of any DNA fragment essentially involves seven steps: (1) Choice of host organism and cloning vector, (2) Preparation of vector DNA, (3) Preparation of DNA to be cloned, (4) Creation of recombinant DNA, (5) Introduction of recombinant DNA into the host organism, (6) Selection of organisms containing recombinant DNA, (7) Screening for clones with desired DNA inserts and biological properties.[4]These steps are described in some detail in a related article (molecular cloning).

[edit]Expression

of recombinant DNA

Main article: Gene expression Following transplantation into the host organism, the foreign DNA contained within the recombinant DNA construct may or may not be expressed. That is, the DNA may simply be replicated without expression, or it may be transcribed and translated so that a recombinant protein is produced. Generally speaking, expression of a foreign gene requires restructuring the gene to include sequences that are required for producing a mRNA molecule that can be used by the host's translational apparatus (e.g. promoter, translational initiation signal, and transcriptional terminator).[6] Specific changes to the host organism may be made to improve expression of the ectopic gene. In addition, changes may be needed to the coding sequences as well, to optimize translation, make the protein soluble, direct the recombinant protein to the proper cellular or extracellular location, and stabilize the protein from degradation.[7]
[edit]Properties

of organisms containing recombinant DNA

In most cases, organisms containing recombinant DNA have apparently normal phenotypes. That is, their appearance, behavior and metabolism are usually unchanged, and the only way to demonstrate the presence of recombinant sequences is to examine the DNA itself, typically using a polymerase chain reaction (PCR) test.[8] Significant exceptions exist, and are discussed below. If the rDNA sequences encode a gene that is expressed, then the presence of RNA and/or protein products of the recombinant gene can be detected, typically using RTPCR or western hybridizationmethods.[8] Gross phenotypic changes are not the norm, unless the recombinant gene has been chosen and modified so as to generate biological activity in the host organism.[9] Additional phenotypes that are encountered include toxicity to the host organism induced by the recombinant gene product, especially if it is overexpressed or expressed within inappropriate cells or tissues. In some cases, recombinant DNA can have deleterious effects even if it is not expressed. One mechanism by which this happens is insertional inactivation, in which the rDNA becomes inserted into a host cells gene. In some cases, researchers use this phenomenon to knock out genes in order to determine their biological function and importance.
[10]

Another mechanism by which rDNA insertion into chromosomal DNA can affect gene

expression is by inappropriate activation of previously unexpressed host cell genes. This can happen, for example, when a recombinant DNA fragment containing an active promoter

becomes located next to a previously silent host cell gene, or when a host cell gene that functions to restrain gene expression undergoes insertional inactivation by recombinant DNA.
[edit]Applications

of recombinant DNA technology

Recombinant DNA is widely used in biotechnology, medicine and research. Today, recombinant proteins and other products that result from the use of rDNA technology are found in essentially every western pharmacy, doctor's or veterinarian's office, medical testing laboratory, and biological research laboratory. In addition, organisms that have been manipulated using recombinant DNA technology, and products derived from those organisms have found their way into many farms, supermarkets, home medicine cabinets and even pet shops. The most common application of recombinant DNA is in basic research, where it is important to most current work in the biological and biomedical sciences.[8] Recombinant DNA is used to identify, map and sequence genes, and to determine their function. rDNA probes are employed in analyzing gene expression within individual cells, and throughout the tissues of whole organisms. Recombinant proteins are widely used as reagents in laboratory experiments and to generate antibody probes for examining protein synthesis within cells and organisms.[2] Many additional practical applications of recombinant DNA are found in human and veterinary medicine, in agriculture, and in bioengineering.[2] Some specific examples are identified below.

Recombinant human insulin. Recombinant insulin has almost completely replaced

insulin obtained from animal sources (e.g. pigs and cattle) for the treatment of insulindependent diabetes. A variety of different recombinant insulin preparations are in widespread use.[11] DrugBank entry.

Recombinant human growth hormone (HGH, somatotropin). Growth hormone is

administered to patients whose pituitary glands generate insufficient quantities to support normal growth and development. Before recombinant HGH became available, HGH for therapeutic use was obtained from pituitary glands of cadavers. This unsafe practice led to some patients developing Creutzfeldt-Jacob disease. Recombinant HGH eliminated this problem, and is now used therapeutically.[12] It has also been misused as a performance enhancing drug by athletes and others.[13] DrugBank entry

Recombinant blood clotting factor VIII. Recombinant factor VIII is a blood-clotting

protein that is administered to patients with forms of the bleeding disorder hemophilia, who are unable to produce factor VIII in quantities sufficient to support normal blood coagulation.[14] Before the development of recombinant factor VIII, the protein was obtained by processing large quantities of human blood from multiple donors, which carried a very high risk of transmission of blood borne infectious diseases, for example HIV and hepatitis B. DrugBank entry

Recombinant hepatitis B vaccine. Prevention of hepatitis B infection is controlled

through the use of a recombinant hepatitis B vaccine, which contains a form of the hepatitis B virus surface antigen that is produced in yeast cells. The development of the recombinant subunit vaccine was an important and necessary development because hepatitis B virus, unlike other common viruses such as polio virus, cannot be grown in vitro. Vaccine information from Hepatitis B Foundation

Diagnosis of infection with HIV. Each of the three widely-used methods

for diagnosing HIV infection has been developed using recombinant DNA. The antibody test (ELISA or western blot) uses a recombinant HIV protein to test for the presence of antibodies that the body has produced in response to an HIV infection. The DNA test looks for the presence of HIV genetic material using reverse transcriptase polymerase chain reaction (RT-PCR). Development of the RT-PCR test was made possible by the molecular cloning and sequence analysis of HIV genomes. HIV testing page from US Centers for Disease Control (CDC)

Golden rice is a recombinant variety of rice that has been engineered to express the

enzymes responsible for -carotene biosynthesis.[9] This variety of rice holds substantial promise for reducing the incidence of vitamin A deficiency in the world's population.
[15]

Golden rice is not currently in use, pending the resolution of intellectual property, Herbicide-resistant crops Commercial varieties of important agricultural crops

environmental and nutritional issues.

(including soy, maize/corn, sorghum, canola, alfalfa and cotton) have been developed which incorporate a recombinant gene that results in resistance to the herbicide glyphosate (trade name Roundup), and simplifies weed control by glyphosate application.[16] These crops are in common commercial use in several countries.

Insect-resistant crops. Bacillus thuringeiensis is a bacterium that naturally

produces a protein (Bt toxin) with insecticidal properties.[15] The bacterium has been applied to crops as an insect-control strategy for many years, and this practice has been widely adopted in agriculture and gardening. Recently, plants have been developed

which express a recombinant form of the bacterial protein, which may effectively control some insect predators. Environmental issues associated with the use of these transgenic crops have not been fully resolved.[17]
[edit]History

of recombinant DNA

Main article: History of biotechnology The idea for recombinant DNA was first proposed by Peter Lobban, a graduate student of Prof. Dale Kaiser in the Biochemistry Department at Stanford University Medical School.
[18]

The first publications describing the successful production and intracellular replication of

recombinant DNA appeared in 1972 and 1973.[19][20][21] Stanford University applied for a US patent on recombinant DNA in 1974, listing the inventors as Stanley N. Cohen and Herbert W. Boyer; this patent was awarded in 1980.[22] The first licensed drug generated using recombinant DNA technology was human insulin, developed by Genentech and Licensed by Eli Lilly and Company.[23]
[edit]Controversy

Scientists associated with the initial development of recombinant DNA methods recognized that the potential existed for organisms containing recombinant DNA to have undesirable or dangerous properties. At the 1975 Asilomar Conference on Recombinant DNA, these concerns were discussed and a voluntary moratorium on recombinant DNA research was initiated for experiments that were thought to be particularly risky. This moratorium was widely observed until the National Institutes of Health (USA) developed and issued formal guidelines for rDNA work. Today, recombinant DNA molecules and recombinant proteins are usually not regarded as dangerous. However, concerns remain about some organisms that express recombinant DNA, particularly when they leave the laboratory and are introduced into the environment or food chain. These concerns are discussed in the articles on genetically-modified organisms and genetically-modified food controversies.

Ribosomal DNA
From Wikipedia, the free encyclopedia

The gene segment of eukaryotic rDNA contains 18S, 5.8S, and 28S tracts and forms a tandem repetitive cluster; the 5S rDNA is coded separately. NTS, nontranscribed spacer, ETS, external transcribed spacer, ITS, internal transcribed spacers 1 and 2, numbered from 5' end.

Ribosomal DNA (rDNA) codes for ribosomal RNA. The ribosome is an intracellular macromolecule that produces proteins or polypeptide chains. The ribosome itself consists of a composite of proteins and rRNA. As shown in the figure, rDNA consists of a tandem repeat of a unit segment, an operon, composed of NTS, ETS, 18S, ITS1, 5.8S, ITS2, and 28S tracts. rDNA has another gene, coding for 5S rRNA, located elsewhere in the genome in most eukaryotes.[1] 5S rDNA is also present in tandem repeats as in Drosophila.[1] In the nucleus, the rDNA region of the chromosome is visualized as a nucleolus which forms expanded chromosomal loops with rDNA. These rDNA regions are also called nucleolus organizer regions, as they give rise to the nucleolus. In the human genome there are 5 chromosomes with nucleolus organizer regions: chromosomes 13,14,15,21 and 22.
[edit]Sequence

homogeneity of the repeat unit

In the large rDNA array, polymorphisms between rDNA repeat units are very low, indicating that rDNA tandem arrays are evolving through concerted evolution.[1] 5S tandem repeat sequences in several Drosophila were compared with each other; the result revealed that insertions and deletions occurred frequently between species and often flanked by conserved sequences.[2] They could occur by slippage of the newly synthesized strand during DNA replication or by gene conversion.[2]
[edit]Sequence

divergence to clarify phylogeny

The rDNA transcription tracts have low rate of polymorphism among species, which allows interspecific comparison to elucidate phylogenetic relationship using only a few specimens. Coding regions of rDNA are highly conserved among species but ITS regions are variable due to insertions, deletions, and point mutations. Between remote species as human and frog comparison of sequences at ITS tracts is not appropriate.[3] Conserved sequences at coding regions of rDNA allow comparisons of remote species, even between yeast and

human. Human 5.8S rRNA has 75% identity with yeast 5.8S rRNA.[4] In cases for sibling species, comparison of the rDNA segment including ITS tracts among species and phylogenetic analysis are made satisfactorily.[5][6] The different coding regions of the rDNA repeats usually show distinct evolutionary rates. As a result, this DNA can provide phylogenetic information of species belonging to wide systematic levels.[7]

The Basics of Recombinant DNA

So What Is rDNA?
That's a very good question! rDNA stands for recombinant DNA. Before we get to the "r" part, we need to understand DNA. Those of you with a background in biology probably know about DNA, but a lot of ChemE's haven't seen DNA since high school biology. DNA is the keeper of the all the information needed to recreate an organism. All DNA is made up of a base consisting of sugar, phosphate and one nitrogen base. There are four nitrogen bases, adenine (A), thymine (T), guanine (G), and cytosine (C). The nitrogen bases are found in pairs, with A & T and G & C paired together. The sequence of the nitrogen bases can be arranged in an infinite ways, and their structure is known as the famous "double helix" which is shown in the image below. The sugar used in DNA is deoxyribose. The four nitrogen bases are the same for all organisms. The sequence and number of bases is what creates diversity. DNA does not actually make the organism, it only makes proteins. The DNA is transcribed into mRNA and mRNA is translated into protein, and the protein then forms the organism. By changing the DNA sequence, the way in which the protein is formed changes. This leads to either a different protein, or an inactive protein.

Now that we know what DNA is, this is where the recombinant comes in. Recombinant DNA is the general name for taking a piece of one DNA, and and combining it with another strand of DNA. Thus, the name recombinant! Recombinant DNA is also sometimes referred to as "chimera." By combining two or more different strands of DNA, scientists are able to create a new strand of DNA. The most common recombinant process involves combining the DNA of two different organisms.

How is Recombinant DNA made?

There are three different methods by which Recombinant DNA is made. They are Transformation, Phage Introduction, and Non-Bacterial Transformation. Each are described separately below. Transformation The first step in transformation is to select a piece of DNA to be inserted into a vector. The second step is to cut that piece of DNA with a restriction

enzyme and then ligate the DNA insert into the vector with DNA Ligase. The insert contains a selectable marker which allows for identification of recombinant molecules. An antibiotic marker is often used so a host cell without a vector dies when exposed to a certain antibiotic, and the host with the vector will live because it is resistant. The vector is inserted into a host cell, in a process called transformation. One example of a possible host cell is E. Coli. The host cells must be specially prepared to take up the foreign DNA. Selectable markers can be for antibiotic resistance, color changes, or any other characteristic which can distinguish transformed hosts from untransformed hosts. Different vectors have different properties to make them suitable to different applications. Some properties can include symmetrical cloning sites, size, and high copy number. Non-Bacterial Transformation This is a process very similar to Transformation, which was described above. The only difference between the two is non-bacterial does not use bacteria such as E. Coli for the host. In microinjection, the DNA is injected directly into the nucleus of the cell being transformed. In biolistics, the host cells are bombarded with high velocity microprojectiles, such as particles of gold or tungsten that have been coated with DNA. Phage Introduction Phage introduction is the process of transfection, which is equivalent to transformation, except a phage is used instead of bacteria. In vitro packagings of a vector is used. This uses lambda or MI3 phages to produce phage plaques which contain recombinants. The recombinants that are created can be identified by differences in the recombinants and non-recombinants using various selection methods.

How does rDNA work?

Recombinant DNA works when the host cell expresses protein from the recombinant genes. A significant amount of recombinant protein will not be produced by the host unless expression factors are added. Protein expression depends upon the gene being surrounded by a collection of signals which provide instructions for the transcription and translation of the gene by the cell. These signals include the promoter, the ribosome binding site, and the terminator. Expression vectors, in which the foreign DNA is inserted, contain these signals. Signals are species specific. In the case of E. Coli, these signals must be E. Coli signals as E. Coli is unlikely to understand the signals of human promoters and terminators. Problems are encountered if the gene contains introns or contains signals which act as terminators to a bacterial host. This results in premature termination, and the recombinant protein may not be processed correctly, be folded correctly, or may even be degraded. Production of recombinant proteins in eukaryotic systems generally takes place in yeast and filamentous fungi. The use of animal cells is difficult due to the fact that many need a solid support surface, unlike bacteria, and have complex growth needs. However, some proteins are too complex to be produced in bacterium, so eukaryotic cells must be used.

Why is rDNA important?

Recombinant DNA has been gaining in importance over the last few years, and recombinant DNA will only become more important in the 21st century as genetic diseases become more prevelant and agricultural area is reduced. Below are some of the areas where Recombinant DNA will have an impact.

Better Crops (drought & heat resistance) Recombinant Vaccines (ie. Hepatitis B) Prevention and cure of sickle cell anemia Prevention and cure of cystic fibrosis

Production of clotting factors Production of insulin Production of recombinant pharmaceuticals Plants that produce their own insecticides Germ line and somatic gene therapy

What is recombinant DNA?

Deoxyribonucleic acid, or DNA, is the blueprint for life. Inside every cell in your body, DNA contains the code that determines who you are and what traits you have. Recombinant DNA is DNA from two different sources that has been combined in vitro (outside living organisms). There are three main reasons for creating recombinant DNA: (i) to create a protein product, (ii) to create multiple copies of genes, and (iii) to insert foreign genes into other organisms to give those organisms a new trait (Campbell & Reece, 2002). Recombinant DNA is used widely today to create large amounts of protein for treating certain illnesses. In 1982, insulin became the first recombinant DNA drug to hit the market (NHGRI, 2003). A person with diabetes does not produce adequate insulin. Insulin, a protein, can now be produced in large quantities by bacteria that have been given the human insulin gene (Hormones, n.d.; Stanford University, 2002; G. Stein & J. Stein, 2002). Another example of a protein that is made by bacteria for medical use is human growth hormone (Hanna, 2004; G. Stein & J. Stein, 2002). The creation of multiple copies of a gene is valuable for genetic research. The availability of multiple copies of a gene has many advantages including the determination of the nucleotide sequence of a gene (Campbell & Reece, 2002). Inserting genes that originated in one organism into another organism is proving indispensable in agriculture and other fields. In agriculture, adding genes to plants to make them draught or insect resistant is already common practice (Anunson et al, 1999; Campbell & Reece, 2002). Another use is the creation of bacteria that will help clean up toxic waste. A bacteria has been created using recombinant technology that can digest oil from an oil spill (Campbell & Reece, 2002). ^Back to top

How does recombinant DNA work?

Here is an overview of how a gene from an organism can be inserted into a bacterium. First, the gene of interest must be identified. For example, the insulin gene would have to be localized in the human genome. Then a plasmid has to be isolated from bacteria cells. A plasmid is a circular, double-stranded DNA sequence that replicates in bacteria and is

separate from the bacterial chromosome. The gene is inserted into the plasmid, and the plasmid is taken up by a bacterium. The bacteria reproduce, and start creating the desired protein (Campbell & Reece, 2002).

A illustration showing showing how human insulin can be produced by bacteria using recombinant DNA (MIT, 1989).

Bacterial Plasmids (Kimball, 2004).

Restriction enzymes, discovered in 1968, are important parts of this process (NHGRI, 2003). In nature, restriction enzymes are a bacteriums self-defense. A restriction enzyme cuts in between a certain sequence of nucleotides, called the restriction sight, which is 4-8 nucleotides long. Every time that sequence occurs in the bacteriums own DNA, methyl groups (-CH3) are added to adenines or cytosines which prevent the restriction enzyme from working. Any time foreign DNA, such as a phage (a bacterial virus), enters the bacterium, the bacteriums restriction enzyme would cut the phages DNA into pieces. Although not all bacteria have restriction enzymes, there are wide varieties of restriction enzymes that have been discovered and continue to be discovered (Campbell & Reece, 2002). Restriction enzymes are used to cut open a plasmid and the same enzyme is used to cut the desired gene out of the chromosome. This makes two matching cuts, and when the gene and plasmid are combined, they form a temporary bond. Another enzyme, DNA ligase, is used to create a permanent seal (Campbell & Reece, 2002).

An example of how a restriction enzyme might work. The restriction site is g-g-a-t-c-c (Kimball, 2004).

This is an example of using restriction enzymes to insert DNA into a plasmid. The restriction site is g-a-a-t-t-c (MIT, 1989).
^Back to top

A Closer Look
To simplify things, the foregoing has described recombinant DNA in terms of one cell, one restriction enzyme, etc. When scientists are attempting to make recombinant DNA, it is done on a much larger scale. Millions of plasmids are mixed with millions of genes. Millions of restriction enzymes are dumped in to make millions of cuts. Plasmids bind to plasmids, plasmids bind to genes, genes bind to genes, plasmids bind to multiple genes, and the whole thing is a mess (Campbell & Reece, 2002). To solve this problem, a cool trick is used. The starting plasmids (the ones that are going to be modified) are called cloning vectors, which is a molecule of DNA that can carry foreign DNA into a cell and replicate there. These plasmids include a gene that confers resistance to ampicillin. They also contain a second gene (for example lacZ). The restriction site, where the restriction enzyme will make a cut, is in the lacZ gene. If foreign DNA is inserted into the lacZ gene

where the restriction enzyme made its cut, the lacZ gene will no longer work. The restriction enzymes are mixed with the plasmids, and then the desired genes, the genes we want to combine with the plasmids, are added. Then the bacteria are induced to take up the plasmids. Changes in heat, or chemicals that are added, are ways to make bacteria do this. After the bacteria are induced to take up the plasmids, the bacteria are grown on a medium of ampicillin and a substance that reacts to the protein product of lacZ. A bacteria colony that took up a plasmid with an intact lacZ gene, meaning that it is not a recombinant plasmid, will turn blue. Bacteria that did not take up any plasmids will not be able to grow on the ampicillin. Therefore, only bacteria that took up a plasmid will be growing, and only bacteria that had something inserted into the lacZ gene will be white (Campbell & Reece, 2002).

The next step is determining which of the remaining non-blue bacteria took up the right gene. We know with certainty that they took up some DNA but we do not know if it was the correct DNA. Scientists can look for the desired protein product (insulin for example) or they can look for the gene. To look for a gene, a sequence of nucleotides that occur in the gene must be known. A complementary sequence of DNA or RNA is created to the known sequence in the gene. This is called a nucleic acid probe. The probe is labeled with a radioactive tag or with a fluorescent tag so that it can be found. The bacterias DNA is denaturized, meaning the DNA is unraveled. This can be done with heat or chemicals. When the tag is added to the bacteria it will bond with complementary DNA (the DNA that is part of the gene we are looking for) inside a bacterium. We then know which bacterium has the desired gene and it can be grown in large tanks to produce the desired protein. This process of using a nucleic acid probe is known as nucleic acid hyperbridization (Campbell & Reece, 2002).