Introduction:
a) Proteomics is the study of proteins. The central dogma of biology is DNA --> RNA --> protein. Thus, by studying proteins, scientists can see which genes are expressed in a cell and which aren't. For example, skin cells may express genes that code for proteins that make up one's skin color, while blood cells may express more genes that code for oxygen absorbing proteins. Proteomics can prove useful for studying evolutionary relationships between species. For example, if two species have similar protein expression, it is likely that they are closely related. Muscle cells contain a lot of protein, specifically proteins actin and myosin. Thus, we will be using muscle cells in our proteomics lab.
b) The purpose of this lab is to analyze the protein of different fish species to see how closely related the fish are to each other.
c) We will be utilizing many techniques in this lab. First, we will grind up the fish muscle and denature the proteins via a hot water bath. This leaves the protein exposed and stretched out so that they can be more easily analyzed. We will run the protein samples on a gel and then compare the results. Fish with similar banding patterns are closely related in the evolutionary tree and fish with very different banding patterns are more distantly related.
d) I do not know which kinds of fish we will be comparing in this experiment, so I cannot predict which will be closely related. However, I predict that the lab will go well and gel electrophoresis will work.
Monday, April 11, 2011
Monday, March 28, 2011
Comparing Mitochondrion DNA
Introduction:
a) Mitochondria are the power plants of the cell. They provide energy, or ATP, for the cell through a process called oxidative phosphorylation. Without mitochondria, our cells would not be able to perform tasks necessary to live. Unlike the rest of the cell's organelles, mitochondria have their own unique set of DNA. One theory for why this is that mitochondria were once bacteria cells that became incorporated in a symbiotic relationship with eukaryotic cells. Mitochondria and bacteria are about the same size and both have similar, simple functions.
It is known that mitochondrial DNA can be traced back through the mother of each generation. A woman's egg contains all the organelles necessary for the embryo to survive, including all the mitochondria. A man's sperm has a lot of initial energy, but lacks mitochondria (thus, they eventually die). This way, when fertilization occurs and a baby is eventually born, all of the baby's mitochondria (and the mitochondrial DNA) were originally from the mother. This knowledge proves very useful for paternity testing and tracing family lines back for centuries.
a) Mitochondria are the power plants of the cell. They provide energy, or ATP, for the cell through a process called oxidative phosphorylation. Without mitochondria, our cells would not be able to perform tasks necessary to live. Unlike the rest of the cell's organelles, mitochondria have their own unique set of DNA. One theory for why this is that mitochondria were once bacteria cells that became incorporated in a symbiotic relationship with eukaryotic cells. Mitochondria and bacteria are about the same size and both have similar, simple functions.
It is known that mitochondrial DNA can be traced back through the mother of each generation. A woman's egg contains all the organelles necessary for the embryo to survive, including all the mitochondria. A man's sperm has a lot of initial energy, but lacks mitochondria (thus, they eventually die). This way, when fertilization occurs and a baby is eventually born, all of the baby's mitochondria (and the mitochondrial DNA) were originally from the mother. This knowledge proves very useful for paternity testing and tracing family lines back for centuries.
b) The purpose of this lab is to extract our mitochondrial DNA and analyze it/compare it with other people at our lab table.
c) We will be using many similar techniques as the previous DNA extraction and "disease" testing lab. First, we will extract DNA from our cheek cells, using a saline mouthwash. Then we will concentrate the cells via centrifuge and add Chelex to the solution (Chelex binds to the ions released from the cells which inhibit PCR, thus allowing PCR to occur). The cheek cells will be lysed open via a hot water bath, releasing all the DNA. We will then add primers which specifically target a gene from the mitochondrial DNA. That way, when we run the DNA through PCR, only the mitochondrial gene will be amplified. Lastly, we will run the amplified DNA through gel electrophoresis so that we can get a visual on the DNA and compare our DNA results with the results of other people.
d) Since this lab is more of a comparison/knowledge activity than an experiment, there are no variables or controls. You could say the DNA is a variable, because obviously each person's DNA is different. However, we do not have a "control" gene to compare it to, so it makes no difference.
c) We will be using many similar techniques as the previous DNA extraction and "disease" testing lab. First, we will extract DNA from our cheek cells, using a saline mouthwash. Then we will concentrate the cells via centrifuge and add Chelex to the solution (Chelex binds to the ions released from the cells which inhibit PCR, thus allowing PCR to occur). The cheek cells will be lysed open via a hot water bath, releasing all the DNA. We will then add primers which specifically target a gene from the mitochondrial DNA. That way, when we run the DNA through PCR, only the mitochondrial gene will be amplified. Lastly, we will run the amplified DNA through gel electrophoresis so that we can get a visual on the DNA and compare our DNA results with the results of other people.
d) Since this lab is more of a comparison/knowledge activity than an experiment, there are no variables or controls. You could say the DNA is a variable, because obviously each person's DNA is different. However, we do not have a "control" gene to compare it to, so it makes no difference.
Tuesday, March 15, 2011
Testing for Genetic Diseases via DNA extraction, PCR, and gel electrophoresis
Introduction:
a) DNA testing is the extraction and analysis of the entire genome or, more commonly, a specific gene of interest. DNA testing has multiple applications. For example, it can be used for forensics, paternity testing, or, in this case, genetic disease diagnosis. Of course, we're not actually testing for a real genetic disease in class, but the principle is the same. DNA testing involves three main steps - DNA extraction, PCR, and gel electrophoresis - which will be discussed later in part c (techniques).
b) The purpose of this lab is to test ourselves for a "genetic disease". We will be utilizing all the techniques below in order to achieve this goal.
c) First, we start with DNA extraction - cheek cells will be swabbed and placed into a test tube containing a lysis buffer to help break open the cell/nuclear membranes. We will then put the cheek cells into a hot water bath of 95 degrees Celsius to further lyse open the membranes. Instagene matrix beads are used to kill DNAse, which would otherwise kill the exposed DNA. Second is PCR (polymerase chain reaction), which is used to make many copies of the DNA so that the DNA will be easier to test. The more copies, the more likely it will show up on the gel (part 3). A main ingredient of PCR is the primer, which targets the specific gene of interest. That way, we make copies of just the gene of interest (in this case, the "disease" gene), rather than the entire genome, which would be unnecessary. Third, we run the mass-produced genes on a gel. This process is called gel electrophoresis. The electric current run through the gel pulls the slightly negatively charged DNA toward the positive end - the smaller segments of DNA (in this case, the "diseased" gene is smaller than the "healthy" gene) will be able to work their way through the matrix faster and will thus travel farther toward the positive end. The larger pieces (the "healthy" gene) will be bogged down in the matrix and will not make it as far. Thus, gel electrophoresis has three potential outcomes:
1. Two bands that did not travel far --> indicates two "healthy" genes
2. Two bands that did travel far --> indicates two "diseased" genes
3. One band that did not and one band that did travel far --> indicates one "healthy" gene and one "diseased" gene.
Only outcome #2 will result in the expression of the disease, since the disease is recessive (both alleles must be recessive in order for the recessive genes to be expressed in the phenotype).
Results:
I do not have a picture of the gel results to post on this blab, but I can tell you that 3 of the people at my lab table tested positive for the "disease". In other words, each of us had 2 bands that travelled far and matched the bands of the "diseased" control gene. The fourth person's bands did not show up, so we cannot determine whether she was positive or negative. Luckily this "disease" is an intron, or else we'd all be in a bad spot!
a) DNA testing is the extraction and analysis of the entire genome or, more commonly, a specific gene of interest. DNA testing has multiple applications. For example, it can be used for forensics, paternity testing, or, in this case, genetic disease diagnosis. Of course, we're not actually testing for a real genetic disease in class, but the principle is the same. DNA testing involves three main steps - DNA extraction, PCR, and gel electrophoresis - which will be discussed later in part c (techniques).
b) The purpose of this lab is to test ourselves for a "genetic disease". We will be utilizing all the techniques below in order to achieve this goal.
c) First, we start with DNA extraction - cheek cells will be swabbed and placed into a test tube containing a lysis buffer to help break open the cell/nuclear membranes. We will then put the cheek cells into a hot water bath of 95 degrees Celsius to further lyse open the membranes. Instagene matrix beads are used to kill DNAse, which would otherwise kill the exposed DNA. Second is PCR (polymerase chain reaction), which is used to make many copies of the DNA so that the DNA will be easier to test. The more copies, the more likely it will show up on the gel (part 3). A main ingredient of PCR is the primer, which targets the specific gene of interest. That way, we make copies of just the gene of interest (in this case, the "disease" gene), rather than the entire genome, which would be unnecessary. Third, we run the mass-produced genes on a gel. This process is called gel electrophoresis. The electric current run through the gel pulls the slightly negatively charged DNA toward the positive end - the smaller segments of DNA (in this case, the "diseased" gene is smaller than the "healthy" gene) will be able to work their way through the matrix faster and will thus travel farther toward the positive end. The larger pieces (the "healthy" gene) will be bogged down in the matrix and will not make it as far. Thus, gel electrophoresis has three potential outcomes:
1. Two bands that did not travel far --> indicates two "healthy" genes
2. Two bands that did travel far --> indicates two "diseased" genes
3. One band that did not and one band that did travel far --> indicates one "healthy" gene and one "diseased" gene.
Only outcome #2 will result in the expression of the disease, since the disease is recessive (both alleles must be recessive in order for the recessive genes to be expressed in the phenotype).
Results:
I do not have a picture of the gel results to post on this blab, but I can tell you that 3 of the people at my lab table tested positive for the "disease". In other words, each of us had 2 bands that travelled far and matched the bands of the "diseased" control gene. The fourth person's bands did not show up, so we cannot determine whether she was positive or negative. Luckily this "disease" is an intron, or else we'd all be in a bad spot!
Tuesday, February 1, 2011
Testing for GM foods via DNA extraction, PCR, and gel electrophoresis
Introduction:
a) GMO stands for Genetically Modified Organism. Many GMOs are agricultural plants, which have been enhanced to be cold or drought resitant, pest resistant, bigger, stronger, healthier, etc. Genetic modification can be used to create the "perfect apple" that can withstand forces of nature and be produced in vast amounts, increasing the farmer's yield and profit.
GMOs are made by inserting a plasmid with a gene of interest (say, a gene for a redder color) into agrobacteria. Usually the plasmid is a Ti plasmid, meaning Tumor-inducing. The plant is then infected with the agrobacteria containing the Ti plasmid and the plant accepts the bacteria and allows the protein to be made from the red-color gene. The plant's fruit then becomes a beautiful red color!
GMOs can be identified by a certain method called PCR. PCR stands for Polymerase Chain Reaction and it involves making many copies of DNA so the DNA can be easier to test. DNA polymerase, a primer specific to the Ti plasmid, nucleotides, and plasmid DNA are needed for PCR to occur.
The controversy surrounding GMOs involves a conflict between technology, natural selection, and morals. Some people are greatly in favor of GMOs, saying that humans are only speeding up the process of natural selection and we have the technology so why not use it? Others say that GMOs go against nature and humans have no right to "play God". GMOs also result in a decrease in genetic variation in a population, which can have terrible consequences if a certain strain of virus were to emerge - the entire population of crops would be rapidly wiped out. An example from history - the potato famine in Ireland. Too much dependence on one type of crop is bound to result in catastrophe. But whether humans will be able to resist the possibility of creating and shaping "perfect" crops, animals, and maybe even humans is doubtful.
b) The purpose of this lab is to test grocery store produce to see if it is genetically modified. This can be done in the real world as well - scientists must varify that a farmer's crop is organic by performing similar tests as the one we will do in class. A fruit is not organic if it has been genetically modified.
c) We will be using many techniques for DNA extraction in this lab. First, we will use a mortar and pestle to grind up the produce, breaking open the cell walls. Next, a hot water bath will be used to lyse open the cell and nuclear membranes. This leaves the DNA vulnerable, so Instagene matrix beads will be added to kill the DNAse in the cells. DNAse is present in all cells to kill foreign DNA - in this case, we do not want DNAse to kill the cell's own DNA, so the DNAse must be killed first. During PCR, we will use two primers - one to target the plant DNA (green primer) and the other to target GM DNA (red primer). The green primer serves as a control - all plant cells contain plant DNA, so if the plant DNA does not show up during gel electrophoresis, then we know the lab was not successful. If the plant DNA does show up, then we know PCR worked and if there is no GM DNA that shows up, then we know that the plant is not a GM product. Gel electrophoresis is used to separate and analyze the DNA found in the cells.
d) We are testing for GM DNA (the Ti plasmid) in the plant cells. That is the variable. Plant DNA is the control (as explained above). My hypothesis is that the non-organic produce (the corn, apple, etc.) will be genetically modified. Some 85% of non-organic foods are genetically modified. But the organic foods will be all natural.
Results:
a) GMO stands for Genetically Modified Organism. Many GMOs are agricultural plants, which have been enhanced to be cold or drought resitant, pest resistant, bigger, stronger, healthier, etc. Genetic modification can be used to create the "perfect apple" that can withstand forces of nature and be produced in vast amounts, increasing the farmer's yield and profit.
GMOs are made by inserting a plasmid with a gene of interest (say, a gene for a redder color) into agrobacteria. Usually the plasmid is a Ti plasmid, meaning Tumor-inducing. The plant is then infected with the agrobacteria containing the Ti plasmid and the plant accepts the bacteria and allows the protein to be made from the red-color gene. The plant's fruit then becomes a beautiful red color!
The controversy surrounding GMOs involves a conflict between technology, natural selection, and morals. Some people are greatly in favor of GMOs, saying that humans are only speeding up the process of natural selection and we have the technology so why not use it? Others say that GMOs go against nature and humans have no right to "play God". GMOs also result in a decrease in genetic variation in a population, which can have terrible consequences if a certain strain of virus were to emerge - the entire population of crops would be rapidly wiped out. An example from history - the potato famine in Ireland. Too much dependence on one type of crop is bound to result in catastrophe. But whether humans will be able to resist the possibility of creating and shaping "perfect" crops, animals, and maybe even humans is doubtful.
GloFish - the first genetically modified pet
c) We will be using many techniques for DNA extraction in this lab. First, we will use a mortar and pestle to grind up the produce, breaking open the cell walls. Next, a hot water bath will be used to lyse open the cell and nuclear membranes. This leaves the DNA vulnerable, so Instagene matrix beads will be added to kill the DNAse in the cells. DNAse is present in all cells to kill foreign DNA - in this case, we do not want DNAse to kill the cell's own DNA, so the DNAse must be killed first. During PCR, we will use two primers - one to target the plant DNA (green primer) and the other to target GM DNA (red primer). The green primer serves as a control - all plant cells contain plant DNA, so if the plant DNA does not show up during gel electrophoresis, then we know the lab was not successful. If the plant DNA does show up, then we know PCR worked and if there is no GM DNA that shows up, then we know that the plant is not a GM product. Gel electrophoresis is used to separate and analyze the DNA found in the cells.
d) We are testing for GM DNA (the Ti plasmid) in the plant cells. That is the variable. Plant DNA is the control (as explained above). My hypothesis is that the non-organic produce (the corn, apple, etc.) will be genetically modified. Some 85% of non-organic foods are genetically modified. But the organic foods will be all natural.
Results:
According to the results, the bands of our test foods match the bands of the GM+ food - thus, all our test foods (the corn powder and the lettuce) are genetically modified.
Thursday, January 27, 2011
Glowing Bacteria: Transformation of pGLO plasmid from sea jelly to bacteria
Introduction:
a) Genetic transformation is the process in which a gene of interest is inserted into or taken up by another organism's cell (usually a bacterium), changing the organism's trait(s). Transformation can be used to transfer a insect resistant gene into crop plants or to enable bacteria to digest oil spills. In this lab, we will be transferring a sea jelly's GFP gene (Green Fluorescent Protein) contained in a pGLO plasmid into bacteria to make the bacteria glow! The pGLO plasmid also contains a gene for ampicillin resistance and a special gene regulation system which can be used to control expression of the fluorescent protein in transformed cells. The gene for GFP can be switched on by adding sugar arabinose to the bacteria's nutrient medium. The bacteria will also be grown on antibiotic plates, killing all the bacteria which did not take up the pGLO and thus do not have antibiotic resistance. This will allow us to determine which bacteria contain the pGLO plasmid.
b) The purpose of this experiment is to transfer the pGLO plasmid (which is derived professionally from a sea jelly gene) into the bacteria. Hopefully we will be able to see the bacteria glow, like a sea jelly! It is a simple way of seeing how genetically modified organisms are made in the real world of biotechnology.
c) There are many techniques we will be utilizing in this lab. Among them are proper pipetting techniques, to fill the test tubes with the Calcium Chloride solution and to put the pGLO plasmid into the tube.. We will use sterile loops to collect bacteria from a dish and put the bacteria in the solution. Heat shock is a technique used to induce transformation - the solution of CaCl2, plasmids, and bacteria is iced for about 10 minutes before put into a hot water bath and then iced again. This causes a sort of current, allowing plasmids to go in through the bacteria's pores. After heat shock, we will use pipetting techniques to transfer the solution onto a petri dish. Incubation of the dish will allow bacteria to grow faster so we can see the results!
d) My hypothesis is that the experiment will work! I think we will be successful and will be able to create glowing bacteria.
a) Genetic transformation is the process in which a gene of interest is inserted into or taken up by another organism's cell (usually a bacterium), changing the organism's trait(s). Transformation can be used to transfer a insect resistant gene into crop plants or to enable bacteria to digest oil spills. In this lab, we will be transferring a sea jelly's GFP gene (Green Fluorescent Protein) contained in a pGLO plasmid into bacteria to make the bacteria glow! The pGLO plasmid also contains a gene for ampicillin resistance and a special gene regulation system which can be used to control expression of the fluorescent protein in transformed cells. The gene for GFP can be switched on by adding sugar arabinose to the bacteria's nutrient medium. The bacteria will also be grown on antibiotic plates, killing all the bacteria which did not take up the pGLO and thus do not have antibiotic resistance. This will allow us to determine which bacteria contain the pGLO plasmid.
b) The purpose of this experiment is to transfer the pGLO plasmid (which is derived professionally from a sea jelly gene) into the bacteria. Hopefully we will be able to see the bacteria glow, like a sea jelly! It is a simple way of seeing how genetically modified organisms are made in the real world of biotechnology.
c) There are many techniques we will be utilizing in this lab. Among them are proper pipetting techniques, to fill the test tubes with the Calcium Chloride solution and to put the pGLO plasmid into the tube.. We will use sterile loops to collect bacteria from a dish and put the bacteria in the solution. Heat shock is a technique used to induce transformation - the solution of CaCl2, plasmids, and bacteria is iced for about 10 minutes before put into a hot water bath and then iced again. This causes a sort of current, allowing plasmids to go in through the bacteria's pores. After heat shock, we will use pipetting techniques to transfer the solution onto a petri dish. Incubation of the dish will allow bacteria to grow faster so we can see the results!
d) My hypothesis is that the experiment will work! I think we will be successful and will be able to create glowing bacteria.
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