Yeast Fermentation in the Classroom

Fig. 1 - Requirements for Fermenting Yeast

Fermentation is a process carried out in living cells as a means of obtaining energy from carbohydrates in the absence of oxygen. It is essentially, therefore, a form of anaerobic respiration. In all cases, the molecule pyruvate is formed as an intermediate product, but the final chemicals produced depend largely on the type of organism carrying out fermentation.

In both wine making and bread making the microorganism involved is yeast, a unicellular fungus. In each process the yeast converts glucose to carbon dioxide and ethanol (see figure 1), according to the following equation:

glucose → ethanol + carbon dioxide

When bread is produced the carbon dioxide is used to make the dough rise and the ethanol evaporates in the cooking process. In wine production, however, the carbon dioxide is allowed to escape (except in sparkling wines) while the ethanol is retained.

Producing Ethanol - Materials and Teaching Method

This experiment is best suited to a Science laboratory but could be improvised adequately in a normal classroom. Students may not be familiar with the odour of alcohol when asked to smell each flask, but it could be suggested to them that it is similar to methylated spirits or the smell of felt-tipped pens.

The purpose of the cotton wool wadding is to provide a warm environment for the yeast to multiply in the presence of glucose and water in the glucose solution.The materials required per four students are as follows:

•                2 conical flasks fitted with a cork, glass tubing and about 10cm attached rubber tubing

•                2 normal test tubes

•                limewater

Fig. 2 - Carbon dioxide Prodiced in Fermentation

•                10% glucose solution (can be made by dissolving 5 glucose tablets in 300ml water)

•                1 teaspoon dried yeast

•                Cotton wool wadding

•                Test tube rack

•                cellotape

Students should be instructed to copy down the following directions, which should be followed by a teacher-led explanation.

•                Add 100ml glucose solution to each conical flask.

•                Place 1 teaspoon dried yeast in one of the flasks.

•                Wrap both flasks in cotton wool and fasten with cellotape.

•                Place the cork and tubing on each flask. Submerge the free ends of each tube in two separate test tubes containing 10 ml limewater.

•                Allow to stand for 12 hours.

The following questions could be written on the board after students write up the experiment and their observations.

Fig. 3 - Test for Carbon Dioxide

1.             Describe any differences you noticed in the limewater in each experimental set-up.

2.             In which flask did you observe foaming and bubbles?

3.             Smell the contents of each conical flask. Which one has an alcoholic smell?

4.             Suggest why both flasks were wrapped in cotton wool.

5.             Complete this conclusion: ________ and glucose are needed for fermentation to occur. The products of fermentation are ________ gas and ethanol. Carbon dioxide gas turns limewater ________ (see figure 2).

Ethanol Production Follow-Up Activities

Students could repeat the experiment, this time stretching a balloon over the mouth of each conical flask instead of connecting the flasks to a test tube of limewater. The flask with yeast in it should produce carbon dioxide gas, which blows up the balloon (see figure 3).

Making Bread - Materials and Teaching Method

In this activity a crockpot or bread making machine would both be ideal for use in a classroom, but if neither are available the dough can be prepared in the room and then taken to a school oven. These ingredients make one medium sized loaf.The class could work together to make one loaf or, if multiple cookers are available, could work in smaller groups. The materials required are:

•                Electric crockpot or bread maker

•                290 ml warmwater

•                2 tablespoons oil

•                1 1/2 teaspoons salt

•                2 tablespoons sugar

•                3 cups plain flour

•                2 tablespoons milk

•                2 teaspoons dried yeast

•                mixing bowl

Students should be instructed to copy down the following directions, which should be followed by a teacher-led explanation.

•                Grease the inside of the crockpot.

•                Mix all ingredients together in the mixing bowl.

•                Remove the resulting dough and knead it for 5 minutes.

•                Allow dough to rest for 10 minutes. It should start to rise during this time.

•                Place dough inside the crockpot. Cook on the highest setting for around 2 hours or until golden brown.

The following questions could be discussed or perhaps written on the board to be copied and answered by the students.

1.             What causes the bread dough to rise?

2.             Write the word equation for the fermentation reaction (involving yeast and sugar) that occurs in bread making.

3.             If ethanol is one of the products of yeast fermentation, why isn't bread alcoholic?

Bread Making Follow-Up Activities

Students could use the same recipe to make two batches of bread at home. They could determine the cooking time required for a large loaf compared to the time needed for the same amount of dough made into several small rolls. Investigations could also be made into the effectiveness of using fresh yeast as opposed to dried yeast. Fresh yeast is readily available at supermarkets or Health Food outlets.

A further lesson , where students research the history of alcohol production and bread making, may be helpful in enhancing their understanding of the fermentation process. A minimum goal should be to ensure all students grasp the concept of fermentation, the specific chemical reaction that occurs when yeast ferments and the varied uses for its products.

References

"The Science of Bread," 2008. Accessed 16/4/2010

Penicillium and Strain Isolation Procedures

Fig. 1 - Fleming's Plate

Sir Alexander Fleming discovered the effects of Penicillin in 1928. Purely by chance, he noticed that Staphyloccus bacteria would not grow in the vicinity of cultures of the fungus Penicillium notatum that were growing on experimental petri dishes.

Fleming suggested that that the fungus was producing a chemical that prevented the development of cell walls in bacteria (see figures1 and 2). He named this chemical penicillin (see figure 3), and it soon became apparent, in the prevailing climate of the 1940s, that it had the potential to be used as a drug to combat bacterial infections resulting from war injuries. A method needed to be devised quickly to allow penicillin to be produced on a large scale.

Mass Production of Penicillin

Fig. 2 - Penicillium Culture

It soon became clear, however, that Penicillium notatum was not suited to large scale production. Scientists Howard Florey and Ernst Chain attempted to resolve this by exposing the fungus to X-rays to produce mutant strains that could be cultured more successfully.

This, combined with the use of huge aerated fermentation tanks containing corn-steep liquor, and strain isolation techniques, allowed them to achieve a ten-fold increase in penicillin production. Moreover, the discovery of Penicillium chrysogenum, a new and more productive strain of the fungus, resulted in an increase in the yield of penicillin by a factor of 1000. Chain also developed a method of crystallising and concentrating the drug, with the result that by D-Day ,June 6, 1944, there was enough penicillin to meet demands.

What is Strain Isolation?

Strain isolation is a method commonly used by microbiologists to isolate, purify and culture a desired strain of a particular microbe. It can be as simple as picking and re-streaking the microbe on a nutrient agar plate until a pure culture is obtained (the 'dilution' method).

Other methods include investigating the nutrient requirements of the desired microbe and providing only those nutrients on successive culture plates until eventually only the microbe in question remains (the 'selective medium' technique). Fleming used a combination of both these methods to isolate and purify his cultures of Penicillium notatum. Florey and Chain also employed similar methods to culture their mutated Penicillium strains.

Fig. 3 - Chemical structure of Penicillin G

Isolation of Microbes Using the Dilution Method

The original ‘dilution’ method pioneered by Joseph Lister attempts to isolate a single cell of the desired microbe so that it can be cultured on a nutrient medium. Successive dilutions of the microbe in nutrient broths were the earliest technique used to do this, but it has now largely been replaced by a dilution method using a solid substrate.

Here, a sterile needle draws the sample across a culture plate several times. The needle is re-sterilised and a small amount of the sample is taken from the original streaks and spread at right angles to them. This procedure is repeated until the final streaks represent a diluted quantity (ideally single cells) of the sample microbe, which is then cultured.

Isolation of Microbes Using the Selective Medium Technique

This method utilizes the fact that individual microbe species prefer specific nutrients on which to grow. By choosing a particular nutrient medium some cultures will be encouraged to grow while others are inhibited.

Examples include the isolation of the bacterium Pseudomonas fluorescens on a medium containing ammonia and lactate, and the purification of Steptomycetes bacteria using glycerol and arginine substrates. Penicillium chrysogenum has itself been isolated and purified by Rafi and Rahman using either glucose or yeast agar media.

Producing, Isolating and Testing Mutated Strains of Penicillium

New strains of Penicillium mould are still being produced by mutating selected colonies which are then isolated and tested for their effectiveness. Although Florey and Chain used X- rays, mutagens can also include ultraviolet (UV) irradiation or chemicals such as colchicine and ethyl methanesulphonate (EMS).

Penicillin is extracted from colonies of these strains and plated out in the presence of Staphylococcus bacteria. The zones of growth inhibition that result are measured and compared to those of a standard penicillin sample to give an indication of the penicillin’s strength. An alternative method of determining penicillin concentration is to use HPLC (High Performance Liquid Chromatography) assaying techniques.

Fleming's discovery, coupled with Florey and Chain's work in purifying and concentrating penicillin, earned each of them the Nobel Prize in Physiology or Medicine in 1945. Their techniques have been further developed by other scientists in recent decades, with the result that penicillin production is now efficient and economical.

References

Bowden, M.,2002, Howard Florey and Ernst Chain, "Pharmaceutical Achievers," chemheritage.org, accessed 30/3/2010

Mailer, J. and Mason, B.,2001, "Penicillin: Medicine's Wartime Wonder Drug and its Production at Peoria, Illinois," Illinois Periodicals Online, niu.edu

Rafi, M. and Rahman,2002, "Isolation and Identification of Indigenous Penicillium chrysogenum Series," fspublishers.org, accessed 1/5/2010

Proteomics: Making Use of the Human Genome

fug. 1 - Crystal structure of the Anthrax Protein

Proteomics, the study of proteins, is an extension of genomics. It recognises the significant role proteins play in all cellular activities.

Most metabolic activities are regulated by proteins (see fig.1), in the form of either enzymes, neurotransmitters, antibodies or control elements in cellular reproduction and gene expression. Founders of the Human Genome Project realised the importance of proteins and their relationship to the genes that code for them.

The Project therefore set out to map all the genes in the human genome, deduce the DNA sequence of each gene and to identify disease-causing genes, in an attempt to gain knowledge about cellular functions, possible causes of disease and to use this knowledge to identify possible drug targets .The Human Genome Project was effectively completed in 2000.

The Need for Proteomics

Knowledge of the human genome alone, however, cannot tell us everything we need to know about cellular activities. This is because although proteins are expressed by genes through the processes of transcription and translation, there are far more intracellular proteins throughout the life of an organism than there are genes coding for them.

One reason for the fact that there is a greater number of proteins than genes is the ability of proteins to undergo 'post translational modifications' after they have been expressed. Post translational modifications (PTMs) occur when proteins undergo various modifications after or during the translation stage of protein synthesis. It has therefore become necessary to develop 'Proteomics', a new discipline that studies the expression, function, identification, interaction and structure of proteins.

Fig.2 - Proteins in a 1D Gel Electrophoresis

Proteomics and the Study of Disease

Proteomics has become a useful tool in the study of disease. When proteins are isolated on electrophoresis gels (see fig. 2), those involved in a particular disease are often either over or under-expressed. These proteins are known as 'biomarkers'. Once these biomarkers have been identified, they can be targeted with drugs or used as drugs themselves if they are found to be absent in diseased tissue.

In sufferers of prostate cancer, for instance, the protein biomarker PSA is over expressed. This information can therefore prove useful in diagnosing sufferers of this disease. Another example can be seen in the recent discovery of a protein that occurs in people who suffer toxic reactions to chemotherapy drugs. Because it has been identified, the development of a drug targeting this protein is now possible.

A non-invasive screening method for breast cancer is now also feasible because of the discovery, through proteomic techniques, of a protein in human tears (lacryglobin).This protein is identical to one that is over expressed in breast cancer.

Other Applications of Proteomics

Another application of proteomics is in the identification of biomarkers in heat tolerant varieties of wheat. When subjected to heat stress a certain wheat variety called 'Fang' expresses proteins that are thought to be responsible for its heat resistant qualities. The identification of these proteins and the genes responsible for them provides breeders with the information needed to cultivate wheat varieties with this desirable characteristic.

Proteomics also provides scientists with information about protein-protein interactions. Knowledge of these interactions is of great relevance because they are often the basis of complex chemical signaling pathways in cells. In addition,the location of a particular protein within a cell can be determined using proteomic techniques. This is important because it often indicates the type of metabolic processes the protein is involved in.

Proteomics in Industry

Numerous proteomics companies have emerged in the last decade as a response to the need to identify and characterise proteins associated with disease or other metabolic manifestations of interest. Matritech, for instance, uses proteomic techniques to develop diagnostic tests for breast, bladder, cervical and prostate cancers. Its NMP22 bladder cancer test is FDA (Food and Drug Administration) approved and is currently being used by urologists.

Companies such as MDS Proteomics operate in conjunction with pharmaceutical organisations to discover drug targets that can be treated with small molecules or antibodies. Although still in the pre-clinical trial stage for many of their products, these organisations are providing the technology needed to achieve effective therapeutic solutions in the near future. Some of this technology includes electrophoresis and mass spectrometry systems, coupled with advanced computing and drug screening techniques.

Fig. 3 - PTM Showing Phosphorylation

Other companies provide protein databases that can be accessed by the medical and pharmaceutical industries for protein identification purposes. Scimagix, for instance, places gel electrophoresis images of proteins in a database for use by proteomic scientists when searching for 'protein signatures' similar to the ones on their own gels.

The applications of proteomics are extensive: not such a surprising revelation when the significance of proteins in cellular metabolism is considered. Moreover, proteomics offers a more efficient pathway towards drug discovery than genomics, as it studies the proteins responsible for disease rather than the genes that code for them. Continued expansion of this rapidly growing area of biotechnology is therefore to be expected.

References

Graves,P. and Haystead,T., 2002, 'Molecular Biologist's Guide to Proteomics', mmbr.asm.org, accessed 12/5/2010

Fuji-Keizai, 2003, 'Post Genome Project Era Proteomics R&D Competition', fuji-kezai.com, accessed 10/5/2010

Protein Science.com, 2007, 'Companies', proteinscience.com, accessed 13/5/2010

DNA Fingerprinting in Forensic Science

Gel Electrophoresis Fingerprint - Mnolf

Comparing the DNA from a crime scene with that of suspects and victims is a powerful tool in criminal investigations. Forensics relies on several methods.

First developed in 1984 by Sir Alec Jeffreys, DNA fingerprinting (or profiling), involves comparing specific non-coding regions (introns) of the DNA molecule from various samples (saliva, skin, blood or semen) found at crime scenes or taken from suspects. These non coding regions make up about 30% of the DNA molecule, and consist of numerous repeated base sequences (variable number tandem repeats, or VNTRs). These intron sequences can be highly variable from one person to the next.

RFLP Fingerprinting

Restriction Fragment Length Polymorphism (RFLP) fingerprinting, where the variable fragments are relatively long (up to 35000 bases in length) was the original technique used until more efficient methods were developed around a decade ago. This procedure involves using specific restriction enzymes to cut DNA at points representing the targeted tandem repeat sequences.

Because a particular VNTR can vary with respect to the number of repeats from person to person, this results in DNA fragments of different lengths. The DNA strands are separated on electrophoresis gels, which consist of an agarose or polyacrylamide gel subjected to an electric potential difference (see fig. 1). Because DNA is negatively charged, it will migrate towards the positive end of the gel. Longer fragments, however, will not move as fast as shorter ones, resulting in the effective separation of any variable tandem repeats present.

DNA samples from several sources are run along separate lanes on the same gel. After electrophoretic separation has been achieved, the DNA is denatured into single strands by heating or applying chemicals. The resulting fragments are then transferred to a nitrocellulose or nylon membrane using the Southern Blot technique.

Radioactively labelled DNA strands of different lengths that are complementary to the different VNTR fragments are then introduced. These 'probes' will bind to complementary strands on the membrane and thus indicate their presence on X- ray film when the membrane is exposed to autoradiography (see fig. 2).

Typically, DNA fingerprinting compares several fragments from each sample, each containing a different tandem repeat sequence. During Bill Clinton's impeachment trial, for instance, seven different variable repeat sequences were used from Clinton's DNA and the DNA from the semen stains on Monica Lewinsky's dress. The bands on the gel matched exactly, representing a one in eight trillion probability that they were not from the same person.

PCR/STR Fingerprinting

The more traditional RFLP profiling procedure is now being replaced by the polymerase chain reaction(PCR) method, which often involves the use of shorter DNA segments known as short tandem repeats (STRs). This method is faster and requires less DNA. Shorter DNA strands are also less vulnerable to degradation.

Moreover, STR fragments are more suited to being amplified by the polymerase chain reaction, which can produce millions of copies of one initial STR fragment within the space of around two hours. The process involves repeated heating and cooling amid a mix of free nitrogen bases, primers and the enzyme DNA polymerase. As a result, even the smallest DNA samples - as little as one billionth of a gram - can be utilised.

Although STR separation can be carried out on electrophoretic gels, capillary electrophoresis, in which a fused silica capillary is used instead of a gel slab, is now being used more frequently . While applying the same principles of separation as the more traditional gel slab electrophoresis, it is more rapid and has a higher resolution.

Uses of DNA Fingerprinting in Forensic Science

The more STRs that are compared from each DNA sample, the less likely the chance will be that they have arisen from the same source. The likelihood of any two specimens being identical is calculated using the 'product rule'. Here, the probability of the frequency of occurrence of each STR in a population is multipled by that of the other STRs separated from the DNA sample.

When all thirteen STRs used in the U.S. national database, CODIS, are combined, the likelihood that the DNA has arisen from another source can be as little as 1 in 575 trillion for Caucasian Americans and 1 in 900 trillion for African Americans.

During the O.J. Simpson trial in 1995, for instance, O.J.’s guilt was suggested by STR tests carried out on his blood and blood from the crime scene, which revealed the probability of his being innocent was1 in 240 000. Further RFLP tests narrowed this down to 1 in 57 billion. The main reason he wasn’t convicted was due to the seed of doubt the defence sowed in the minds of the jurors about possible interference with the evidence by the FBI.

In addition to its use in providing evidence to incriminate suspects, DNA profiling can also be of use in exonerating suspects accused of crimes. About 25% of violent crime cases in the U.S. since 1989 have resulted in the exoneration of suspects because of DNA profiling procedures. By 1996, over 108 post-conviction exonerations had in fact occurred in the USA using DNA profiling.

Because STRs and VNTRs are inherited from each of our parents, DNA profiling can also be used to establish paternity in cases involving custody and child support. Other uses in forensics include the identification of victims of catastrophes such as the September 11 attacks.

It must be remembered that these tests are not always foolproof and should be used in conjunction with other evidence where possible. DNA fingerprinting has nevetheless affected the outcome of criminal investigations in a revolutionary way.

References

Australian Government, 2001, 'DNA Profiling', Biotechnology Online, biotechnologyonline.gov.au, accessed 24/5/2010

Brinton, K. and Liebermann, K,1994 "Basics of DNA Fingerprinting-Southern Blot', washington.edu, accessed 22/5/2010

Saferstein, R., 2004, 'Criminalistics, an Introduction to Forensic Science', Pearson