In the nucleus, the process of transcription creates an mRNA strand using DNA as a template. This mRNA is then used as a template for translation at the ribosome, which builds protein strands based on the information in the mRNA. Each gene codes for a specific protein that affects a cells function in a variety of ways. Not all genes are expressed in every cell, however. The variable of gene expression is what allows an organism to have different cell types, such as blood cells, skin cells, lung cells, etc.
Scientists determine and study differences in gene expression using a microarray. Using cDNA made from mRNA, scientists are able to see only the genes that are expressed in each cell, rather than the entire genome, which is filled with many non-coding regions that are useless for this research. This type of experiment is often done to compare cancerous cells of a certain type to healthy cells of that type.
There is no single cause of cancer in human beings. As is evident on a microarray, there are many different genes that change in expression level when a cell becomes cancerous. In this lab, we will look at six genes and compare them between healthy lung cells and cancerous lung cells. The microarray simulation will tell us which genes are active in each cell, and how active they are. Genes that have the same level of expression in both cells would not be of interest to us, but genes that are off in one cell and on in another (or vice versa) could help us determine a cause for the cancer and, potentially, a cure. The six genes we will look at are C4BPA, ODC1, FGG, HBG1, SIAT9, and CYP24. These are genes with wildly different functions, any of which could be a reason for the cancer.
Monday, November 15, 2010
Sunday, October 31, 2010
CSI: Lafayette-- Restriction Enzymes, Gel Electrophoresis, and One Lengthy Prison Sentence
Only the most talented criminals are capable of committing a crime without leaving a single source of DNA at the crime scene. DNA, the molecular name tag, is found in hair, blood, skin cells, saliva, semen, etc. Just one droplet of blood or a couple cells could help police identify who committed a crime. Science has made it much harder to be a bad guy.
DNA profiling is done using Restriction Fragment Length Polymorphism (RFLP). The key to this technique is the restriction enzymes. Such enzymes are found in bacteria, where they are an intrinsic defense against bacteriophage viruses. When the bacterial cell detects foreign DNA from a virus, restriction enzymes cut the viral DNA at a specific, palindromic site, destroying the DNA. There are usually several of these sites at different locations on the DNA, varying from person to person. This is where RFLP comes into play. When restriction enzymes are used on human DNA they cut in different places, resulting in fragments of different sizes. This is how we differentiate between suspects.
In our experiment, we will use restriction enzymes and gel electrophoresis to determine who committed a crime, comparing the five suspects' DNA to DNA recovered from the crime scene. The restriction enzyme we are using is from the bacteria E. coli. Because all the suspects have different DNA, the enzyme will digest the genes at different locations, creating fragments of different lengths (in base pairs).
In order to actually see the differences in length, we use a process called gel electrophoresis. The agarose gel we will use is a matrix through which DNA can travel. Electricity is added to the gel, and the current is stabilized by a buffer. The DNA starts on the negative end, but because it has a negative charge, it slowly migrates to the positive side. Smaller fragments will travel further because they can more easily squeeze through the matrix. Conversely, larger DNA fragments (more base pairs) will not be able to travel as far. After running the gel, we will stain the DNA so it can be visualized
We will run the DNA samples of all suspects, as well as the DNA from the crime scene. One of the suspect's DNA profiles should match the profile of the crime scene DNA, and that will let us know who is guilty. There will also be a marker that serves as a control to make sure the gel worked correctly.
So, the only question is, WHO DUNNIT?
DNA profiling is done using Restriction Fragment Length Polymorphism (RFLP). The key to this technique is the restriction enzymes. Such enzymes are found in bacteria, where they are an intrinsic defense against bacteriophage viruses. When the bacterial cell detects foreign DNA from a virus, restriction enzymes cut the viral DNA at a specific, palindromic site, destroying the DNA. There are usually several of these sites at different locations on the DNA, varying from person to person. This is where RFLP comes into play. When restriction enzymes are used on human DNA they cut in different places, resulting in fragments of different sizes. This is how we differentiate between suspects.
In our experiment, we will use restriction enzymes and gel electrophoresis to determine who committed a crime, comparing the five suspects' DNA to DNA recovered from the crime scene. The restriction enzyme we are using is from the bacteria E. coli. Because all the suspects have different DNA, the enzyme will digest the genes at different locations, creating fragments of different lengths (in base pairs).
In order to actually see the differences in length, we use a process called gel electrophoresis. The agarose gel we will use is a matrix through which DNA can travel. Electricity is added to the gel, and the current is stabilized by a buffer. The DNA starts on the negative end, but because it has a negative charge, it slowly migrates to the positive side. Smaller fragments will travel further because they can more easily squeeze through the matrix. Conversely, larger DNA fragments (more base pairs) will not be able to travel as far. After running the gel, we will stain the DNA so it can be visualized
We will run the DNA samples of all suspects, as well as the DNA from the crime scene. One of the suspect's DNA profiles should match the profile of the crime scene DNA, and that will let us know who is guilty. There will also be a marker that serves as a control to make sure the gel worked correctly.
So, the only question is, WHO DUNNIT?
Tuesday, October 5, 2010
Using Cellobiase to Break Down Artificial Substrate, Mimicing Degradation of Cellobiose to Glucose for Creation of Ethanol
For years, scientists have researched alternative sources of renewable energy that could eventually replace the dwindling supplies of oil and other natural resources used for feul. One field in which there is great promise is biofeuls, or feuls extracted from natural biomass. Certain biological oils and alcohol feuls such as ethanol could be the future because they have a cyclical, renewable energy process with little to no pollution. Therefore, we could potentially create an infinite supply of energy that is less toxic to the environment.
To harness the energy in biological products, enzymes are necessary to break the mass down to its smallest components. Enzymes, usually proteins, bind to specific molecules called substrate and catalyze the reactions. Our lab will use the enzyme cellobiase, which simplifies the two-glucose molecule cellobiose to a single glucose molecule that can be harnessed for feul. Cellobiose is a simpler form of the sugar cellulose, which is found in the cell wall of plants, such as corn.
As I have already said, scientists have obvious reasons for being interested in biofeuls. Glucose, specifically, can be converted to ethanol by fermentation. Ethanol is already being used as an energy source, but researchers are currently trying to perfect it. We are doing this lab to further our knowledge of enzymes and observe the degradation of cellobiose (artificial substrate) to glucose, which can be used to make ethanol.
This lab is actually very simple. Rather than use actual cellobiose, which would break down into glucose that is invisible without a microscope, we will use an artificial substrate called p-Nitrophenyl glucopyranoside. We will add this substrate to a test tube with cellobiase, causing the substrate to break down into glucose and p-Nitrophenol.
On the side, we will set up four beakers filled with a strong base. At different times, we will pour out some of our concoction from the test tube into each beaker. The base has two effects on our solution: first, it will kill the cellobiase, thereby ending the reaction immediately; also, the strong base will react with the p-Nitrophenol, turning it yellow. This allows us to have a visible indicator of how much glucose is being produced, and that the reaction was even successful at all.
Because we are adding base at different times, we would expect the shade of yellow to be stronger the longer the reaction is occurring. The more the artificial substrate is broken down, the more glucose we will have. The variables in this lab are the different timepoints that we add the base, affecting the amount of reaction that can to occur. For our control, we will have one tube of artificial substrate in which we do not add cellobiase, so no reaction should occur.
Conclusion:
Due to contamination by the previous period, our lab was unsuccessful. Our tube of substrate had been mixed with enzyme, so the reaction had already been completed before we began the experiment. All of our tubes showed the same dark shade of yellow because all the substrate had already been broken down. Also, our control sample was yellow even though we never added enzyme.
To harness the energy in biological products, enzymes are necessary to break the mass down to its smallest components. Enzymes, usually proteins, bind to specific molecules called substrate and catalyze the reactions. Our lab will use the enzyme cellobiase, which simplifies the two-glucose molecule cellobiose to a single glucose molecule that can be harnessed for feul. Cellobiose is a simpler form of the sugar cellulose, which is found in the cell wall of plants, such as corn.
As I have already said, scientists have obvious reasons for being interested in biofeuls. Glucose, specifically, can be converted to ethanol by fermentation. Ethanol is already being used as an energy source, but researchers are currently trying to perfect it. We are doing this lab to further our knowledge of enzymes and observe the degradation of cellobiose (artificial substrate) to glucose, which can be used to make ethanol.
This lab is actually very simple. Rather than use actual cellobiose, which would break down into glucose that is invisible without a microscope, we will use an artificial substrate called p-Nitrophenyl glucopyranoside. We will add this substrate to a test tube with cellobiase, causing the substrate to break down into glucose and p-Nitrophenol.
On the side, we will set up four beakers filled with a strong base. At different times, we will pour out some of our concoction from the test tube into each beaker. The base has two effects on our solution: first, it will kill the cellobiase, thereby ending the reaction immediately; also, the strong base will react with the p-Nitrophenol, turning it yellow. This allows us to have a visible indicator of how much glucose is being produced, and that the reaction was even successful at all.
Because we are adding base at different times, we would expect the shade of yellow to be stronger the longer the reaction is occurring. The more the artificial substrate is broken down, the more glucose we will have. The variables in this lab are the different timepoints that we add the base, affecting the amount of reaction that can to occur. For our control, we will have one tube of artificial substrate in which we do not add cellobiase, so no reaction should occur.
Conclusion:
Due to contamination by the previous period, our lab was unsuccessful. Our tube of substrate had been mixed with enzyme, so the reaction had already been completed before we began the experiment. All of our tubes showed the same dark shade of yellow because all the substrate had already been broken down. Also, our control sample was yellow even though we never added enzyme.
Wednesday, September 15, 2010
DNA Precipitation Intro
DNA, the molecule of life, carries the genetic information from generation to generation. Deoxyribonucleic acid, found in all living organisms, is a double helix containing millions of pairs of nucleotide bases, each base being connected to a deoxyribose sugar and a phosphate group. The four bases are adenine, guanine, thymine, and cytosine; adenine pairs with thymine and thymine pairs with guanine. In short, DNA makes us who we are. It determines everything from eye color to skin color to body type to susceptibility to certain diseases. Certain sequences of DNA that code for a specific trait are called genes. Differences in the base pairs in genes, often occurring because of mutation, are what account for diversity among organisms.
In humans, DNA is stored in the nucleus of all cells. Every cell has a complete set of DNA, but only certain genes are expressed in each cell. What DNA (genes) really does is code for certain proteins in the cell. DNA is first transcribed into messenger RNA, and this mRNA travels out of the nucleus to the ribosomes where the genetic message is read and the correct proteins are built.
In this lab we will attempt to precipitate DNA out of solution, extract it, and put in in a necklace. Visualizing the genetic molecule in this way could be very beneficial to scientist's in the real world. Isolating DNA allows biologists to study and compare between individuals or different species. The sequencing of DNA has been an extremely important development of the last twenty years and will have even more significant benefits in the future when we are more knowledgeable.
The precipitation of DNA is actually a very simple process. First, we will chew on the insides of our cheeks to loosen the cells. Then we will swish a saline solution around in our mouths to extract the cheek cells. The isotonic solution has a concentration that is favorable to the cells. In order to get to the DNA we will add a lysis buffer that breaks open the cell membrane by dissolving the phospholipids. To further isolate the DNA, we will break down the histone proteins DNA is wrapped with using an enzyme called protease. Protease will also destroy the DNAse in the cytoplasm that would otherwise dismantle our DNA. We will speed this reaction up by placing the test tubes in a hot water bath. If our DNA was in just a water solution the DNA, which is negatively charged and polar, would interact with the polar H2O molecules. The added salt binds with the DNA, making it non-polar. We complete the lab by adding cold ethanol, which has a lower freezing point than water. This allows us to get the solution to extremely low temperatures, stimulating precipitation.
Finally, we will have a beautiful necklace containing the very molecules that give us life and make us who we are.
In humans, DNA is stored in the nucleus of all cells. Every cell has a complete set of DNA, but only certain genes are expressed in each cell. What DNA (genes) really does is code for certain proteins in the cell. DNA is first transcribed into messenger RNA, and this mRNA travels out of the nucleus to the ribosomes where the genetic message is read and the correct proteins are built.
In this lab we will attempt to precipitate DNA out of solution, extract it, and put in in a necklace. Visualizing the genetic molecule in this way could be very beneficial to scientist's in the real world. Isolating DNA allows biologists to study and compare between individuals or different species. The sequencing of DNA has been an extremely important development of the last twenty years and will have even more significant benefits in the future when we are more knowledgeable.
The precipitation of DNA is actually a very simple process. First, we will chew on the insides of our cheeks to loosen the cells. Then we will swish a saline solution around in our mouths to extract the cheek cells. The isotonic solution has a concentration that is favorable to the cells. In order to get to the DNA we will add a lysis buffer that breaks open the cell membrane by dissolving the phospholipids. To further isolate the DNA, we will break down the histone proteins DNA is wrapped with using an enzyme called protease. Protease will also destroy the DNAse in the cytoplasm that would otherwise dismantle our DNA. We will speed this reaction up by placing the test tubes in a hot water bath. If our DNA was in just a water solution the DNA, which is negatively charged and polar, would interact with the polar H2O molecules. The added salt binds with the DNA, making it non-polar. We complete the lab by adding cold ethanol, which has a lower freezing point than water. This allows us to get the solution to extremely low temperatures, stimulating precipitation.
Finally, we will have a beautiful necklace containing the very molecules that give us life and make us who we are.
Tuesday, September 7, 2010
Yogurt Lab Discussion
Because I was starting this lab with little introduction, I was a little confused throughout the experiment. Therefore, I am not that confident in the results.
In tube 1 (just milk), the milk was sour but the texture was normal. Airborne bacteria probably contaminated the milk and then divided during inoculation. The milk was spoiled, but these particular bacteria were not yogurt-ness bacteria, so the texture did not change.
In tube 2 (milk and yogurt), the substance was thicker and had a smell like yogurt. I was unsure whether this was because the milk actually turned into yogurt or because yogurt was added. However, I am assuming that the yogurt really did change the composition of the milk because the whole sample changed even though we only added a tiny amount of yogurt. The bacteria in the yogurt must have created the original yogurt, as well as altering our milk sample.
As we expected, tube 3 (yogurt, milk, and ampicillin) was absolutely unaffected by the experiment. The smell and consistency were normal, as was the pH, because all the bacteria that could have altered the milk were killed by the ampicillin.
Like tube 1, tube 6 (milk and E. coli) also resulted in a sour smell with normal consistency. The smell of this sample was overwhelmingly sour and made me want to throw up. E. coli seems to spoil milk but not turn it into yogurt.
Our pH readings may have been a little skewed, but it is clear that every tube subjected to bacteria had a lower pH than the tube with ampicillin. Tube 1 had the lowest pH (5), and tubes 2 and 6 had a pH of 6. These numbers don't seem to make sense because the milk with lowest pH should curdle as casein proteins denature. Tube 2, however, was the only one that curdled.
There were a few sources of error that may have affected the results of the experiment. First of all, if the inoculating loops touched any non-sterile surfaces, the tubes could be contaminated with outside bacteria. Also, the colors on the pH scale are so similar that someone could conclude a strip shows a pH of 5 when it is really closer to 7. Finally, we may have used the vortex for too long, which potentially could kill the bacteria by breaking open the sturdy cell walls.
Despite some confusion, I believe our results turned out fairly accurate. Most importantly, we had a great time in our first lab of the year.
In tube 1 (just milk), the milk was sour but the texture was normal. Airborne bacteria probably contaminated the milk and then divided during inoculation. The milk was spoiled, but these particular bacteria were not yogurt-ness bacteria, so the texture did not change.
In tube 2 (milk and yogurt), the substance was thicker and had a smell like yogurt. I was unsure whether this was because the milk actually turned into yogurt or because yogurt was added. However, I am assuming that the yogurt really did change the composition of the milk because the whole sample changed even though we only added a tiny amount of yogurt. The bacteria in the yogurt must have created the original yogurt, as well as altering our milk sample.
As we expected, tube 3 (yogurt, milk, and ampicillin) was absolutely unaffected by the experiment. The smell and consistency were normal, as was the pH, because all the bacteria that could have altered the milk were killed by the ampicillin.
Like tube 1, tube 6 (milk and E. coli) also resulted in a sour smell with normal consistency. The smell of this sample was overwhelmingly sour and made me want to throw up. E. coli seems to spoil milk but not turn it into yogurt.
Our pH readings may have been a little skewed, but it is clear that every tube subjected to bacteria had a lower pH than the tube with ampicillin. Tube 1 had the lowest pH (5), and tubes 2 and 6 had a pH of 6. These numbers don't seem to make sense because the milk with lowest pH should curdle as casein proteins denature. Tube 2, however, was the only one that curdled.
There were a few sources of error that may have affected the results of the experiment. First of all, if the inoculating loops touched any non-sterile surfaces, the tubes could be contaminated with outside bacteria. Also, the colors on the pH scale are so similar that someone could conclude a strip shows a pH of 5 when it is really closer to 7. Finally, we may have used the vortex for too long, which potentially could kill the bacteria by breaking open the sturdy cell walls.
Despite some confusion, I believe our results turned out fairly accurate. Most importantly, we had a great time in our first lab of the year.
Monday, September 6, 2010
Yogurt Lab Intro
It was not until a little over a hundred years ago that scientists discovered bacteria were capable of causing disease. Bacteria had been identified in sick people much earlier, but it took many years of experiments and guesswork to prove that bacteria were the actual infecting agents. German physician Robert Koch developed a series of tests to prove anthrax was caused by bacteria, but his method can be used to prove that any microbe causes a specific disease. These tests, termed "Koch's postulates," are as follows:
1. The microbe is found in organisms with the disease but is not found in healthy organisms.
2. The microbe is isolated from the diseased subject and grown in culture.
3. The cultured microbe causes disease when introduced to a healthy organism.
4. The microbe is again isolated from the host and shown to be identical to the original.
Bacteria have several characteristics that make them effective disease-causing agents. Most importantly, they are tiny prokaryotic cells much smaller than the eukaryotic cells that make up animals and other living organisms, allowing them to infiltrate a host. Also, the thick cell walls of bacteria make them very resilient.
Using milk as a model test subject, we will use apply Koch's postulates to determine whether microbes in yogurt cause milk to thicken and turn into yogurt. By adding yogurt to milk and inoculating, we will see if the milk turns into a substance similar to the original yogurt.
We will use four test tubes for our experiment: a negative control with milk only; a positive control with milk and yogurt; one with milk, yogurt, and ampicillin; and one with milk and E. coli. The tube with just milk is a control because it shows that milk will not turn into yogurt just by fermenting for a day. The tube with yogurt and ampicillin is a similar control because ampicillin kills all the bacteria in the yogurt, so the milk should not be spoiled. The E. coli tube should spoil the milk, but it won't necessarily turn the milk into yogurt. For the procedure, we will transfer the bacteria using sterile inoculating loops. Then, we will let the tubes sit in a hot water bath overnight to stimulate bacterial fission.
I predict that the positive control (milk and yogurt) is the only tube where the milk will turn into yogurt. The yogurt itself was once milk and, therefore, must have the yogurt-making bacteria in it.
1. The microbe is found in organisms with the disease but is not found in healthy organisms.
2. The microbe is isolated from the diseased subject and grown in culture.
3. The cultured microbe causes disease when introduced to a healthy organism.
4. The microbe is again isolated from the host and shown to be identical to the original.
Bacteria have several characteristics that make them effective disease-causing agents. Most importantly, they are tiny prokaryotic cells much smaller than the eukaryotic cells that make up animals and other living organisms, allowing them to infiltrate a host. Also, the thick cell walls of bacteria make them very resilient.
Using milk as a model test subject, we will use apply Koch's postulates to determine whether microbes in yogurt cause milk to thicken and turn into yogurt. By adding yogurt to milk and inoculating, we will see if the milk turns into a substance similar to the original yogurt.
We will use four test tubes for our experiment: a negative control with milk only; a positive control with milk and yogurt; one with milk, yogurt, and ampicillin; and one with milk and E. coli. The tube with just milk is a control because it shows that milk will not turn into yogurt just by fermenting for a day. The tube with yogurt and ampicillin is a similar control because ampicillin kills all the bacteria in the yogurt, so the milk should not be spoiled. The E. coli tube should spoil the milk, but it won't necessarily turn the milk into yogurt. For the procedure, we will transfer the bacteria using sterile inoculating loops. Then, we will let the tubes sit in a hot water bath overnight to stimulate bacterial fission.
I predict that the positive control (milk and yogurt) is the only tube where the milk will turn into yogurt. The yogurt itself was once milk and, therefore, must have the yogurt-making bacteria in it.
Friday, September 3, 2010
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