Genetic transformation is a process which entails taking a gene from one organism, and inserting it into another so as to alter or add a trait. Uses of genetic transformation include modifying plants to resist frost or pests, strengthening agriculture; altering bacteria to help in environmental disasters by giving them the capability to digest oil; and gene therapy, in which sick cells are transformed with the use of healthy genes.
To move genes from one organism to the other, the gene must be isolated and added to a plasmid. Plasmids are a small circular pice of DNA in addition to the chromosome. They allow bacteria to share genes and help them adapt to their environment more rapidly. To force the bacteria to accept the DNA, the plasmids and bacteria are subjected to heat shock, or a rapid raising and lowering of temperature. This triggers the bacteria's' instincts to absorb new DNA, a survival skill.
This lab involves taking the Green Fluorescent Protein gene from jellyfish and inserting it into bacteria, which will cause them to become bioluminescent.
A DNA chip (also known as a gene chip, genome chip, or microarray) is used in the study of genes and how they affect cell functions. It is a solid glass slide containing a pattern of spots, each of which is a copy of a certain gene. To compare, for example, cancerous and noncancerous tissue, scientists label each with a different colored dye and apply them to the DNA chip. Each labeled gene attaches to its compliment on the chip; the brighter the resulting color, the stronger the gene is expressed in that specific tissue. A computer is used to see which genes are expressed in cancerous tissue (marked in one color), which in healthy tissue (marked in another), and both (a mix of the two colors). Genes expressed in the cancerous tissue but not in the healthy tissue could be important and require further examination.
Procedure
Obtain miniature "microarray"
Add 20µl of each sample of cDNA, incubated at 70°, to each spot of the microarray
Add 20µl of hybridization solution to each sample
Observe results
Results
Samples 3 and 6 were blue. Those genes are expressed only in healthy cells.
Sample 2 was purple, meaning it is expressed in both healthy and cancerous cells.
Sample 4 was purple. It is not expressed at all in lung cells.
Samples 1 and 5 were pink, meaning they are expressed only in cancer cells. Sample 1 was more vivid, meaning it is more likely to be an important factor than sample 5.
DNA is essentially the blueprint to create a person, and as we are all unique, so our DNA is unique as well. This has a great number of uses in proving relatedness of individual organisms, identifying species, and studying ancient peoples. DNA is also particularly important in the field of forensic science. Although human beings are 99.9% the same, that 0.1% can be crucial in telling one individual from another. DNA evidence can be used either to incarcerate a man, or to set him free.
Restriction Enzymes and DNA Analysis
DNA analysis is carried out through Restriction Fragment Length Polymorphism, or RFLP. Restriction enzymes are normally used within bacteria to protect them from invading viruses, cutting a virus's DNA at the recognition sequence, or one specific point in DNA that the restriction enzyme is made to cut. If there are multiple recognition sequences, the restriction enzyme will cut at multiple points. However, restriction enzymes are not limited to the bacteria in which they originate. In the RFLP process, these "scissors" snip DNA into fragments, the resulting sequence unique to each individual because of his or her specific DNA.When compared to DNA evidence obtained from a victim or crime scene, the cut DNA patterns can be compared to determine from which suspect the DNA originated.
Agarose Gel Electrophoresis
However, if the restriction enzyme is not aided in any way, the cut DNA will sit in an invisible tangle, undecipherable by forensic scientists. To give the DNA color and place it in the aforementioned patterns, the process of agarose gel electrophoresis is necessary. In electrophoresis, cut DNA fragments are loaded into an agarose gel slab, which is placed in a chamber containing a conductive buffer solution and two dyes, bromophenol blue and xylene cyanol. Electrodes are attached to the chamber, and direct current is passed through these electrodes, polarizing the it. Because DNA is negative, it will be drawn towards the positive pole in the field.The gel is filled with holes, which filter the DNA fragments according to the size of their base pairs. Smaller pairs will slip easily down through the gel, while larger ones will become stuck and take more time. Fragments of the same size ultimately stay together, forming the bands seen on a completed DNA test. While the DNA is filtering down through the agarose gel maze, so are the bromophenol blue and xylene cyanol dyes. These group around the bands of DNA, making them visible to the naked eye.
Procedure
Restriction Enzyme
Pipet 10 µl of restriction enzyme into each of the 6 micro test tubes containing 10 µl DNA from a suspect. Flick/tap the tubes to mix the contents.
Incubate for 45 minutes at 37°C. This is the temperature of the human body, and will help the restriction enzyme thrive.
Agarose Gel Electrophoresis
(Note: I was absent during this part of the lab, so I am paraphrasing the instructions in the lab packet.)
Place tubes in a centrifuge to bring all the liquid to the bottom
Add 5 µl of "loading dye" into each tube. The dye helps to make the samples denser, so they sink into the wells in the gel.
Place the agarose gel in the in the electrophoresis apratus, and cover it with 1x TAE buffer.
Load 10 µl DNA size marker and 20 µl of each of the DNA samples into separate wells, using a separate tip each time to prevent contamination.
Turn on the electrophoresis apparatus and let it run for 30 minutes.
Visualization of DNA Fragments
Add 120 ml of 100x Fast Blast DNA stain into a staining tray
Stain the gels for 2 minutes
Rinse with warm water
Results
Because the restriction enzymes are made to cut at one specific sequence in DNA, the patterns formed by the DNA of different individuals are all unique. It is fairly simple to tell which one of the six samples is identical to the sample taken from the crime scene.
Ethanol is a sustainable alternative to the oil that we use today. However, it can be difficult to break down the cellulose contained in plant cell walls, and for this reason an enzyme is needed.
Enzymes are used to speed up chemical reactions. They do this by positioning the reactant in a manner that reduces the activation energy, or the energy needed to make the reaction occur. Because they are not actually consumed in the reaction, they can be used multiple times. Reactions can also be sped up by increasing the temperature at which they occur.
Producing biofuels will become an increasingly important part of our lives in the future. Because there is no one perfect solution, multiple sources such as wind and solar power should accompany biofuels. It is crucial that we develop alternative fuel sources before our finite oil reserves are drained and we are left with no fuel whatsoever.
Procedure
Part 1
1. Measure 500 µl of stop solution into five cuvettes. The stop solution will be used to stop a sample of the reaction at a certain time, allowing us to see how much product has been created in that amount of time.
2. Pipet 2 ml of 1.5 mM substrate, or reactant, into a tube labeled "enzyme reaction". In this case the substrate is cellulose, found in plant cell walls.
3. Add 1 ml of cellobiase, an enzyme, to the tube. Cellobiase will speed up the reaction time, creating product faster.
4. At 1, 2, 4, 6, and 8 minutes, remove 500 µl from the reaction and add it to the appropriate cuvette. The stop solution in the cuvettes will halt the reaction, and dye the p-nitrophenol in the reaction yellow. The deeper the yellow, the more product has been formed.
Part 2
Repeat steps above, substituting ground mushroom for the cellulose in step 2. The mushroom is a potential natural fuel source.
Results
In each vial, the yellow was significantly more vivid. This proves that the reaction continued over the observed timeframe, creating more and more ethanol (the product). Because of the added cellubiase, the product was formed much faster than it would be if left to react without an enzyme.
The greatest chance for error is in grinding up the mushrooms with the mortar and pestle. It is possible to measure either too much or too little mushroom, and the mortar may be contaminated with chemicals from past work.
Deoxyribonucleic Acid, also known as DNA, is the "building blocks" for life. It is shaped like a "twisted ladder". The ladder's vertical supports are composed by a sugar and phosphate, with the rungs formed by bases- adenine, guanine, thymine, and cytosine (or A, G, T, and C). A will always pair with T, and G with C. These pairs arrange themselves in such a manner that they form a chemical message, which dictates how an individual is made. DNA is what makes species unique. As each individual is different, our DNA is different as well. By extracting DNA from cells, one can map and sequence DNA, clone and compare it, or even test for a predisposition to certain genetic diseases or traits.
Procedure
Chew on cheeks to loosen cells- it is not necessary to draw blood.
Rinse with saline solution to keep the cells from bursting open prematurely.
After spitting saline solution and saliva into a vial, add lysis buffer to the sample to burst the cell. Lysis buffer works on a cell the same way detergent works on a stain- it dissolves the walls, allowing the contents to flow freely.
Add protease (an enzyme) to break down proteins. DNA is wrapped around proteins like thread around a spool, so destroying the protein allows it to unravel. Protease will also kill DNase. DNase exists in the cytoplasm to kill foreign DNA from viruses, but because of this, the cell's own DNA is confined to the nucleus. If the DNase wasn't destroyed by the protease, it would kill the DNA before we had the chance to isolate it.
Add salt. DNA is negatively charged, so it would dissolve in the solution. Adding salt will neutralize it, making it hydrophobic (water-fearing) instead of hydrophilic (water-loving). This will also cause the DNA to clump together, rather than being spread out in the solution.
Place the vial in a hot-water bath. This will speed up the reaction that breaks the cell membranes.
Add cold ethanol to precipitate the DNA, as well as keep it from dissolving. Other molecules in the sample will not precipitate, and therefore not be visible.
Bacteria are single-celled prokaryotes which have existed for billions of years. They are relatively simple in comparison to other life forms, and are most likely the most abundant (one colony can contain billions of individuals) and diverse. Differences range from shape to grouping to physical composition of the organism.
The most well-known bacteria are pathogens, or disease-causing microorganisms. In the 19th century, scientists were beginning to gain knowledge of the existence of bacteria and their relation to illness, but their knowledge was not specific enough until the German physician Robert Koch devised a series of tests to identify bacteria's role in disease. Koch's Postulates, as they came to be known, state the following:
1. The pathogen must be found in all organisms suffering from a disease, but no others.
2. One must isolate the pathogen and culture it separately, free of contact from other bacteria or host organisms
3. When introduced to a healthy subject, the cultured bacteria must cause the same disease as the bacteria taken from the original, ill subject
4. The bacteria must once again be isolated from its host and identified as the same pathogen in step 1.
Because it is often dangerous to work with pathogens, researchers may use a safe alternative. There are "good bacteria" which people use to perform many daily functions, including the making of foods such as cheese, pickles, and yogurt.
Yogurt bacteria works roughly the same way on milk as a pathogen works on living organisms- the milk is infected and "consumed" by the bacteria. Like any other living thing, of course, bacteria require food. Yogurt bacteria consume lactose from milk, and create lactic acid as a by-product. It is this lactic acid which curdles the milk, changing it into yogurt. However, yogurt bacteria requires certain conditions in order to thrive. The milk to be used must be pasteurized- not boiled- to kill other bacteria which would spoil the milk before the yogurt bacteria completed its process. After pasteurization, the yogurt bacteria is introduced. After about 24 hours, the bacteria has consumed all the lactose, replacing it with lactic acid. This acid lowers the pH of the yogurt, which kills any spoilage bacteria surviving the pasteurization, as well as preventing the growth of new spoilage bacteria. Therefore, yogurt does not become simply spoiled milk, and is void of its rancid smell and taste.
Procedure
1. Obtain four vials of milk:
Only milk- the negative control. The negative control is used to test what happens to the milk when it is left untouched.
Milk with yogurt- the positive control. This proves that there is something in yogurt that helps it create more yogurt from milk.
Milk, yogurt, and ampicillin. Ampicillin is an antibiotic, which will kill all bacteria in the sample. If there is no yogurt in the vial with ampicillin, it will prove that bacteria do cause the change into yogurt.
Milk and E. coli. The sample containing E. coli will answer the question of whether all bacteria can create yogurt, or if there is a bacteria specific to the process.
2. Put vials in the Vortex to thoroughly mix their contents.
3. Leave the vials in the cold water bath overnight to allow the bacteria to work.
Results/Observations
Discussion
The negative control formed a small lump of curdled milk floating near the top of the vial, suspended in a clear liquid. It smelled slightly old, but not too pungent.
The positive control was liquid on the top fourth, but the bottom three-quarters had coagulated into yogurt. (We're fairly certain it was yogurt, it had the smell and consistency of yogurt although none of us tasted it.)
The vial containing ampicillin resembled the negative control; because the ampicillin killed the yogurt-producing bacteria, there were no traces of yogurt. My group had hypothesized that the ampicillin would kill off spoilage bacteria, but neglected the fact that it would kill the yogurt-producing bacteria as well.
The vial containing milk and E. coli appeared the same as the first and third vials. It also had a smell which is best described as "rancid".
Fortunately, my group didn't encounter any errors in our work (as far as we know), although there were multiple opportunities, such as cross-contamination between vials.
