What is a Transgenic Species?

Fig.1 - Crown Gall

A transgenic species is an organism that has had part of another organism's DNA (often known as a 'transgene') transferred into it using recombinant DNA technology.

The resulting organism is often known as a genetically modified organism (GMO). Examples include transgenic crops that have had genes inserted into them to improve features such as resistance to insects or disease, shelf life or nutritional value, and genetically modified bacteria which can produce human proteins on a large scale.

Other uses for GMOs include medical research and the manufacture of pharmaceuticals. In the pharmaceutical industry, GMOs such as goats, rabbits, pigs and bacteria can be used to manufacture insulin, vaccines, growth hormones, human protein C, anticoagulant and haemoglobin.

Inserting Genes Into Bacteria

Bacteria can be transformed when plasmids (loops of bacterial DNA) containing the desired gene are introduced into them. The genes on the plasmid are then expressed, and can be passed on to all subsequent bacterial generations.This process begins by using a specific restriction enzyme to remove the desired gene from human DNA. The same enzyme is then used to cut a bacterial plasmid in one place. The human gene is inserted into the gap in the plasmid, and an enzyme called DNA ligase then re-joins the 'sticky' ends that were formed by the restriction enzyme.

The recombinant plasmid is then introduced into a bacterial cell in the presence of cold calcium chloride, which makes the cell walls of the bacterium more porous. Exposure to an an electric potential difference, also known as ‘electroporation’, may also be used to successfully introduce the plasmid into the host cell. Recombinant bacteria are currently used to produce large amounts of human insulin and human growth hormone for pharmaceutical use.

Inserting Genes Into Animals

DNA microinjection is the most widely used transgenesis method in animals and involves the microinjection of a gene into the nucleus of a fertilised ovum. This is a random process and may not always lead to expression of the desired gene. As a result, transgenic animals are mated to ensure that their offspring acquire the transgene.

Embryonic stem cell-mediated gene transfer is another method used to introduce genes into animal cells. Here, the modified DNA is incorporated into embryonic stem cells of the chosen animal. These stem cells are then introduced into an embryo, resulting in a chimeric organism (that is, one where only some cells contain the desired gene).

Retrovirus mediated gene transfer is also used in some instances. Retroviruses, viruses capable of inserting their genetic material into a host cell, are modified to transfer the desired gene into the animal. Not all cells of the animal will have the gene incorporated into them, so the animal will again be chimeric. The transgene will only be genetically transmitted if the retrovirus invades the animal's sex cells. Alternatively, chimeric animals are inbred for up to 20 generations until the desired gene is present in every cell.

Transgenic animals currently being researched include transgenic cattle, sheep and pigs that can produce milk that contains human insulin, human collagen, human fertility hormones and monoclonal antibodies.

Inserting Genes Into Plants

Agrobacterium tumefaciens (crown gall bacteria) is often used as a vector to carry the desired transgene into plant cells, as its normal infection process involves inserting a circular DNA plasmid into the host cell. The plasmid is initially modified by cutting it in two places with a restriction enzyme and inserting the desired gene. It is then incorporated into the targeted plant cell via the crown gall bacteria (see fig. 1) or is introduced manually.

Examples of GM plants produced using this method include Bt cotton (fig.2), which has had a gene inserted into it to make it toxic to pests such as bollworms, and GM soybeans, which incorporate a gene from the Salmonella bacterium to make them resistant to 'roundup' insecticide.

Fig.2 - BT Cotton

Most crop plants, including the cereals and grasses, are monocotyledonous: that is, their leaves have parallel veins and they don't have a tap root system. Inserting genes into this type of plant can prove to be more difficult than with higher plants.Transgenic monocotyledons can instead be produced using a particle gun. This fires a gold bullet, coated with the desired DNA, into the nucleus of the target plant. Examples of GM monocotyledonous crops include herbicide resistant GM Canola (rapeseed), corn and sugar cane, and insect resistant Bt corn.

Ethical and Environmental Concerns Linked to GMOs

One concern associated with GM plants and animals has always been the risk of horizontal gene transfer; that is, the transgene may become incorporated into related, or even unrelated species. Herbicide resistance could, for example, spread to weeds growing alongside GM Canola with the result that they can no longer be eradicated by farmers.

Transgenic organisms may also escape to the wild and compete with native species for resources - the superior 'Sumo Salmon', a transgenic fish containing the gene for human growth hormone, is an example of this. In addition, social inequity could result from the use of GMOs. For instance, large corporations may end up owning the rights to the most productive crops, so restricting access to those most in need of them.

Thorough testing of GM0s and their products is also needed to avoid any risk to public health - the need for this was recently highlighted by a study linking Monsanto's GM corn to organ damage in rats.

Despite these concerns, the use of transgenic species in agriculture, medicine and the pharmaceutical industry has already revolutionised our lives in countless ways.

References

Goldstein, K, 2010, ‘Monsanto’s GMO Corn Linked to Organ Failure, Study Reveals’, Huffington Post, huffingtonpost.com, accessed 21/3/2010

Jigar Nare, S, 2008, 'Transgenic Animals- A Boon by Biotechnology', pharmainfo.net, accessed 23/3/2010

Pighin, J, 2003, 'Transgenic Crops: How Genetics is Providing New Ways to Envision Agriculture', scq.ubc.ca, accessed 21/3/2010

Recombinant Vaccines

Fig.1 - Recombinant DNA

Recombinant DNA technology, in which a section of DNA from one species is inserted into the DNA of another (see figure 1), is proving to be useful in the manufacture of vaccines that contain only the desired antigen - usually one or several proteins - without the need for attenuated toxins or modified versions of the disease causing virus or bacterium. This results in the production of an immune response without the risk of actual contraction of the original disease, and is potentially more cost effective and commercially viable.

How Are Recombinant Vaccines Delivered?

Recombinant vaccines can be delivered to recipient organisms via a live vector such as the Vaccinia virus (which carries a gene for a rabies glycoprotein), or in the expressed protein form as in Tickgard cattle vaccine or the Gardasil cervical cancer vaccine. In such cases a purified protein preparation is usually injected into the muscles.

To produce such vaccines containing actual antigen proteins, the desired gene is often inserted into bacteria or yeasts, which are cultured on a large scale. The proteins produced from these transgenic species are then incorporated into a vaccine preparation. Some animal vaccines are actually now being trialled in the form of an oral vaccine containing an antigenic protein which can be introduced in the animal’s feed.

How Are Recombinant Vaccines Made?

An example of how the process works can be seen in the production of Tickgard vaccine. Here, genes responsible for the manufacture of the gut protein Bm86 in the cattle tick, Rhipicephalus microplus, are inserted into the DNA of microbes. These microbes are cultured, and express the Bm86 protein on a large scale. The protein can then be purified and injected into cattle, with the result that their immune system will protect them against the Bm86 antigen introduced in a real tick bite.

Current research has found that, although the Bm86 gene can be adequately expressed in recombinant E. coli bacteria and strains of the fungus Aspergillus, the yeast, Pichia pastoris has proved to be the most successful at secreting the protein. One reason for this is its rapid growth rate and its ability to grow on inexpensive media.

Recombinant Vaccines Used by Humans

Fig.2 - Gardasil Vaccine

Besides Gardisal cervical cancer vaccine, one of the only other few recombinant vaccines currently used in humans is the Hepatitis B Virus (HBV) vaccine, which contains a surface protein from the hepatitis virus. This protein is produced by recombinant yeast cells and then purified for injection. The HBV vaccine is much safer to use than a weakened form of the actual virus, which, if it reverts back to its original form, could cause liver cancer or hepatitis.

Another recombinant vaccine that has been successfully trialled for human use is the recombinant influenza vaccine, although it has not yet been produced on a commercial basis. Feder, 2009, in fact argues that the US should have had batches of this recombinant vaccine prepared in advance during the swine flu (H1N1) outbreak in 2009, as it is fast to produce and obviates the reliance on vaccines from overseas. Recombinant influenza vaccines are composed of hemagglutinin, a protein present in various strains of the influenza virus. This protein is expressed by recombinant cell cultures and later purified to produce the vaccine.

Recombinant Vaccines For Animal Use

Several commercially used recombinant vaccines used on animals employ a vector based delivery system. These include the VRG vaccine, which protects animals against rabies, and the Purevax recombinant feline leukaemia vaccine.

As mentioned above,the VRG vaccine consists of a recombinant Vaccinia virus that carries the gene for a rabies glycoprotein.The virus has been modified in several ways, one of which involves the removal of the thymidine kinase gene, making it safer to administer than in its original form. Studies have in fact shown it has not caused any side effects in over 10 avian species and 35 mammalian species.

The Purevax leukaemia vaccine contains a harmless recombinant canarypox virus that incorporates the FeLV gene. This gene produces a protein identical to that produced by the FeLV (feline leukaemia) virus, with the result that the cat's immune response is triggered without the danger of the actual virus being introduced. The canarypox virus is also used as a vector in dog and ferret vaccines.

The Future of Recombinant Vaccines

Further research is likely to increase the availability and effectiveness of recombinant vaccines, resulting in safer preparations that are tailor made to specific diseases in both animals and humans.Vaccines against non-infectious diseases such as diabetes and for protection against parasitic organisms in animals may also become a possibility. Methods of delivery will also undergo improvement, especially in relation to orally introduced treatments.

References

Canales, et.al., 2008, 'Expression of Recombinant Rhipicephalus (Boophilus) microplus, R. annulatus and R. decoloratus Bm86 orthologs as secreted proteins in Pichia pastoris', Biomedcentral.com, accessed 5/4/2010

Feder, N., 2009, 'Recombinant Vaccine for Pandemic Flu: Why is it Not in Large Scale Production?', Pogo.org, accessed 6/4/2010

World Health Organisation, 2010, 'Recombinant Vaccines for Oral Immunisation of Wildlife', Who.int, accessed 3/4/2010

This was helpful 

Yoghurt Making in the Classroom

Home-made Yoghurt

Making Yoghurt in the Classroom

This activity combines a study of the history and science of yoghurt making with a practical activity that can be performed in most classroom settings.

Yoghurt making is an effective method of introducing scientific concepts to students. Aimed towards middle secondary students, the information below can be used to create a worksheet or Powerpoint presentation that can be introduced before the practical activity.

The History of Yoghurt

The exact origins of yoghurt are relatively hazy, but it is thought to have originated accidentally over 4500 years ago when milk was left to become sour. It was definitely used as a food in the Middle East and Turkey from the second century AD. Yoghurt was introduced into the Western world as a health food by a scientist named Mechnikov, who suspected that it was responsible for the longevity of the Bulgarian people.

Yoghurt and Biotechnology

Biotechnology is defined as the use of living things to make products beneficial to mankind. As such, yoghurt making and other fermentation processes can be regarded as among the most important and early examples of this science.

Fermentation involves the use of bacteria and other microbes to manufacture alcohol, lactic acid or other products useful to man. Yoghurt is the result of specialised lactic acid bacteria (Lactobacillus bulgaricus and Streptococcus thermophilus) converting the lactose in milk to lactic acid and energy. Because this process does not require oxygen it is known as anaerobic respiration.

Lactobacillus bulgaricus and Streptococcus thermophilus are both ‘homofermenters’, which means that lactic acid is their only fermentation product. Other species of bacteria, however, are ‘heterofermenters’, producing carbon dioxide and ethanol in addition to lactic acid. These additional products can add flavour to various fermented foods.

The word equation for the production of lactic acid by homofermenters is:

Glucose/lactose → lactic acid

The word equation for the production of lactic acid by heterofermenters is:

Glucose/lactose → lactic acid + ethanol + carbon dioxide

The Characteristics of Yoghurt

The lactic acid produced in the production of yoghurt acts to curdle milk and enhance its flavour. It also performs a natural sterilising role by helping to reduce the growth of bacteria that cause food poisoning. The lactic acid bacteria are also "gut friendly," which means they foster the growth of helpful intestinal microbes and also counteract toxins.

Ironically, many commercial yoghurts are pasteurised, which destroys the live bacteria present in the yoghurt and their beneficial properties. As a consequence, the best yoghurts to buy are the organic and natural ones.

Other Uses for Lactic Acid Fermentation

Lactic acid fermentation is also used to produce foods such as pickles, cheeses, sauerkraut, sourdough breads, kefir, kimchi (a Korean dish of pickled vegetables) , cassava and nham (Thai fermented fresh pork). Again, the lactic acid creates a unique flavour and also serves a preservative function.

Making Natural Yoghurt: Materials and Ingredients

Note that the materials and ingredients listed are based on the requirements of one group of 4-6 students

•                Portable hotplate or portable microwave oven

•                Thermometer

•                1 teaspoon natural yoghurt to use as a starter culture

•                Small saucepan, 500ml beaker (or a plastic bowl if using the microwave oven)

•                250 ml milk

•                1 plastic cup

•                Incubator or Thermos flask

Method for Making Natural Yoghurt

1.             Pour the milk into the saucepan, beaker or plastic bowl.

2.             Heat the milk to 90°C and maintain it roughly at this temperature for 10 minutes.

3.             Now allow the milk to cool. When it has reached 40°C, mix in 1 teaspoon of natural yoghurt.

4.             Pour the mixture into a plastic cup and incubate it for at least 12 hours at around 30-40°C. If a school incubator is not available a Thermos flask or other insulated container could be used.

5.             The mixture should now have a thick consistency and the characteristic smell and taste of yoghurt (see figure below).

Follow Up Work for Students

Students can investigate why the milk needed to be heated to a certain temperature then cooled to 40°C. Note that this is essentially because high temperatures kill off any harmful bacteria and also restructure the milk proteins so that when they set they won’t form lumpy curds. Cooling is required so that the lactic acid bacteria don’t get killed when they are added to the mixture.

References

Eat Yoghurt, "History of Yoghurt," eatyoghurt.com, 2010. Accessed 8/4/2010

Tempeh info, "Lactic Acid Fermentation," tempeh.info, 2010. Accessed 9/4/2010

Wordpress, "Yoghurt Making," wordpress.com, 2008. Accessed 9/4/2010

Fermentation and its Uses

Fig. 1 - Glycolysis

Fermentation, according to Louis Pasteur, is essentially respiration in the absence of oxygen. This, however, is an oversimplification of the process.

More technically, fermentation involves the breakdown of carbohydrate molecules to form an intermediate product, pyruvate and a small amount of energy. This stage of the process is known as glycolysis, and it occurs inside all living cells (see figure 1).

The pyruvate molecule may then be changed into products such as ethanol, lactic acid, carbon dioxide, hydrogen, citric or acetic acid, depending on the type of organism and the conditions. Further metabolism of these products may occur if oxygen becomes available – one example of this is the oxidation of ethanol to acetic acid when wine is spoiled.

The Production of Alcohol

Beers, wines and spirits are all produced by fermenting various carbohydrates. Yeasts do this naturally to sugars; a property that has been utilised by humans for thousands of years. Wine was probably first produced in the Middle East as long ago as 5000 BC, and evidence of a process involving crushed grapes dating from 6500 years ago has been found in Macedonia.

Fig, 2 - Fermentation Vats

Modern wine makers combine additional yeast varieties to those present naturally in the harvested grapes, and the process of fermentation from sugar to ethanol and carbon dioxide occurs in large steel or plastic vats or oak barrels (see figure 2). The carbon dioxide produced is either allowed to escape, or trapped to form sparkling wines.

Yeasts are also instrumental in the production of beer, manufactured since around 6000 BC. Unlike wine making, the source of carbohydrates is partly germinated malt barley rather than grape sugar (see figure 3). The reason malt grain needs to be germinated is that this procedure produces enzymes that begin the conversion of starches into sugars. Hops are added to produce a bitter flavour during the brewing process, and also to act as a preservative.

Spirits such as whiskey, brandy and Cognac are produced using distillation to further concentrate the ethanol to around 37-43% (as opposed to around 12% in wines). Additional sugar may be added to produce liqueurs.

Ethanol is also produced industrially on a large scale for use as a biofuel. This has traditionally involved a two step fermentation procedure using aerated tanks containing the yeast Saccharomyces cerevisciae and substrate carbohydrates. Recent studies have shown that the bacterium Zymomonas mobilis produces a larger quantity of ethanol and, unlike yeast, does not become inhibited by high ethanol concentrations. Interestingly, Z. mobilis can also be genetically modified to convert a larger range of sugars into ethanol.

The Production of Citric Acid

Fig. 3 - Germinated Malt Barley

Citric acid is a useful product in both the food and pharmaceutical industries; it is used in food as a preservative and to produce an acidic, sour taste in soft drinks and other beverages. In the pharmaceutical industry it can be used as a buffering agent and to clean equipment.

Citric acid is formed by the fermentation of a molasses substrate by the fungus Aspergillus niger. The biochemical pathway involved includes the production of pyruvate in glycolysis, followed by its conversion to citric acid via the condensation of acetyly co-enzyme A and oxaloacetate. The presence of specific amounts of trace elements such as iron have proved to be important in this process.

Acetic Acid Production

In the presence of the Acetobacter bacterium and oxygen, fermented carbohydrates, ciders or wines can be converted to vinegar (acetic acid). The result is usually a 5% solution of acetic acid.

Acetic acid is used in diluted form in the food industry as a condiment and pickling agent. It is also employed in industry as a solvent and an important reagent in many organic synthesis reactions.

Lactic Acid Production

Again, this product is formed after pyruvate has been produced in the glycolysis pathway. The presence of lactic acid bacteria (Lactobacillus bulgaricus, Streptococcus thermophilus or similar species) usually results in the conversion of two pyruvate molecules to lactic acid or lactic acid, ethanol and carbon dioxide.

In the food industry, lactic acid fermentation is used in the production of yoghurt, sauerkraut, pickles and cheeses. Other uses for lactic acid include tanning leather, manufacturing lacquers and inks, as a moisturising agent in cosmetics and in the production of polylactic acid. Polylactic acid, which can be used to make biodegradable plastics, is finding many uses in the biomedical industry, as well as in areas such as clothing and food packaging.

A Versatile Reaction

Fermentation certainly produces a diverse range of chemicals and is obviously a key reaction in many industries. The one thing all these processes have in common is an initial culture containing carbohydrates and a particular species of microorganism. Although in all cases pyruvate is produced as an intermediate step, the microorganisms - whether bacteria, yeasts or fungi - will each determine the specific end fermentation products.

References

Gunasekaran, P., and Chandra Raj.,K., 'Ethanol Fermentation Technology', Madurai Kamaraj University, ias.ac.in, accessed 14/4/2010

Mudgeee Grape Grower's Association, 2010, 'How is Wine Made?', mudgeewine.com, accessed 13/4/2010

Temph.info, 2010, 'Acetic Acid Fermentation', tempeh.info, accessed 12/4/2010