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The main objective of this lab was to identify unknown plasmids by observation of their genotype and phenotype. We observed the plasmids phenotype by using it to transform bacteria. When the plasmid is in a bacterial host the antibiotic resistance gene can be expressed and impart resistance to the host. We also analyzed the genotype of the unknown plasmid by performing a variety of molecular genetic techniques such as predigestion, electrophoresis in arose gel, staining with ethidium bromide, and finally a photograph of the gel, in order to compare the plasmid to a standard, to in turn determine the plasmids actual size and the sizes of its digestion fragments. By performing these experiments we found that our unknown plasmid in the blue tube was resistant to p KAN. We also found that the unknown plasmid when compared to a standard closely matched the size of digestion fragments, of p KAN, therefore our unknown plasmid was p KAN.
Introduction There is growing concern that the control of infectious diseases is threatened by the upward trend in the numbers of bacteria that are resistant to multiple antibiotics in the medical armamentarium. Resistance costs money and human lives. Resistant infections are associated with increased morbidity, prolonged hospital stays, greater direct and indirect costs, prolonged periods during which individuals are infectious, and greater opportunities for the spread of infection to other individuals (2). In many developing countries, the availability and use of antibiotics are poorly controlled, which results in a high rate of resistance, particularly to the older antibiotics (2).
The procedure used in our interpretation of Bacterial Transformation, is one that has a larger impact than our simple usage for finding unknowns when compared to known's. This importance lies mainly in the medical field and more specifically in gene therapy. Using procedures, like this, Many human genes have been cloned in Escherichia coli or in yeast (4). This has made it possible - for the first time - to produce unlimited amounts of human proteins in vitro. Cultured cells (E. coli, yeast, mammalian cells) transformed with the human gene are being used to manufacture: insulin for diabetics, human growth hormone (GH), erythropoietin (EPO) for treating anemia, granulocyte-macrophage colony-stimulating factor (GM-CSF) for stimulating the bone marrow after a bone marrow transplant, adenosine deaminase (ADA) for treating some forms of severe combined immunodeficiency (SCID), angio statin and endo statin for trials as anti-cancer drugs, the list goes on and on (4).
For example GM-CSF is important for stimulating bone marrow (4). Therefore it can be said that without the procedure of transformation GM-CSF would have never come to exist, along with hundreds of other treatments that are helping Hundreds of Thousands today. The procedure of transformation cannot be completed without an organism such as a vector known as a plasmid (1). Plasmids are molecules of DNA that are found in bacteria separate from the bacterial chromosome. They are small (a few thousand base pairs) usually carry only one or a few genes that convey resistance to such antibiotics such as kanamycin, tetracycline, and ampicillin (1). They are also circular and have a single origin of replication.
Due to their circular nature, plasmids may exist in a number of different physical states: super coiled, relaxed circular (nicked), and linear (1). The plasmid that is used (produced) in the laboratory is the relaxed control plasmid which is nicked from manipulation of its DNA in the lab, most likely through detergents. These nicks of the plasmids DNA produce an opening by which the DNA uncoils allowing access for the replicating enzymes. Antibiotic resistance genes are located on the plasmid and not on the bacterial chromosome that the plasmid is transforming (1). This is due to the fact that, antibiotic resistance requires relatively large amounts of the enzymes to nutreulize the antibiotics.
By being located on the plasmid the respective genes are copied more frequently then they would be on the genomic DNA of the bacteria. When the bacteria recieves a plasmid transfer it becomes immune to that particular plasmid al antibiotic. So in perspective of our experiment, this is exactly what we saw occur (1). To find the genotype of an unknown plasmid it is best to run tests using Agarose gel electrophoresis.
These fragments can be visualized by subjecting the digestion mixtures to electrophoresis in an agarose gel, because of its negatively-charged phosphate groups, DNA migrates toward the positive electrode (anode) when a direct current is applied (3). The smaller the fragment, the farther it migrates in the gel. The phenotype of an unknown plasmid can be discovered through the treatment of E. coli with the mixture of relegated molecules, which will produce some colonies that are able to grow in the presence of both ampicillin and kanamycin (1). Restriction enzymes are DNA-cutting enzymes found in bacteria (and harvested from them for use). Because they cut within the molecule, they are often called restriction endonuclease's.
Restriction endonuclease's, attack and cleave or digest, internal regions of foriegn DNA (4). While leaving the host DNA intact. A restriction endonuclease from Haemophillius influenzae (Hindi II) cleaves DNA in a predictable fashion, cutting at specific recognizable sites (1). This particular enzyme, after it cuts the DNA, produces sticky ends.
These are called "sticky ends" because they are able to form base pairs with any DNA molecule that contains the complementary sticky end (4). Another example of this is the restriction endonuclease enzyme, Bacillus amyloliquefaciens H, (Bam HI) (1). Also note that some restriction endonuclease enzymes recognize four base sequences, and others sequences of six bases. Both of the enzymes above cut the circular DNA, producing a linear form of DNA, each enzyme cuts it at one unique site that may be present at different positions on the DNA (1). This now digested DNA can be loaded into an agarose gel and electropheresised to seperate by size, stained with ethidium bromide and then viewed (1).
As stated before the plasmid can take on many different physical states. Such as super coiled, relaxed circular (nicked), and linear. The plasmid DNA itself, a double helix, is wound around proteins to produce a compact structure (1). Adding these coils to the coiled DNA helix produces a super coiled DNA molecule. Super coiled DNA migrates through the agarose gel at a faster rate than linear DNA of the same size, because the super coiled DNA is compact (1). During DNA replication, enzymes (Topoisomerase) introduce a nick into the one strand of the DNA helix, and rotate the strand to release the torsional strain that holds the molecule in the super coil (1).
The relaxed section of the DNA uncoils, allowing access for the replicating enzymes. These nicks produce the relaxed, opened, circular structure. Nicking occurs commonly when DNA is manipulated in the lab. When a plasmid breaks or is cut at one place on the molecule, through both strands, the circular form then becomes the linear form of DNA (1). The most common mechanism used in a research laboratory to transfer naked DNA is, transformation. Bacterial transformation, deals with the modification of the genotype of the bacteria due to the addition of DNA from another source; in this case the source was a plasmid (1).
One of the found critical steps, in most recombinant DNA work involves E. coli as the host. Mandel and High found that E. coli cells become more susceptible (competent) to transformation when suspended in cold calcium chloride and subjected to a brief heat shock at 42 degrees celsius (1).
There are several critical aspects to bacterial transformation, the first is selecting the right plasmid. The plasmid should contain such qualities as a high copy number within cells, a presence of antibiotic resistance gene should be located, the size should not be too large, should be a limited number of cut sites, and finally it should be easy to transfer this plasmid to the host bacterium (1). The next aspect is selecting a host bacterium that is easy to transform, easy to culture, has a short generation time, and is known to be sensitive to antibiotics, which our selection for the experiment was E. coli DH 5 a (1).
The last important aspect is the culturing media. It is important to select a general medium that the bacterial strain can grow on, that can be used as a liquid or a solid, and can have antibiotics added (1). Our medium for our bacterial transformation was LB. Bacterial transformation is used for a myriad of reasons.
More recently it is used in Genetic cloning, which is then used for medical purposes, such as producing new antibiotics, new treatments in gene therapy, and much much more (4). The antibiotic resistance that was being used in our experiment was p KAN and p AMP. p KAN plasmid has a kanamycin resistant gene (1). This plasmid must be in a host bacterial cell in order to be expressed. Bacteria without this particular plasmid cannot be expected to grow on an LB/KAN medium because the bacteria is sensitive to kanamycin, but with the plasmid the bacteria is resistant or immune and can therefore replicate (1). p AMP, in essence works similarly when comparing it to p KAN.
The difference being that, bacteria is sensitive (in this case) to ampicillin, and in order to grow on a LB/AMP medium the bacteria must have the p AMP plasmid (1). A gel electrophoresis test was done to visualize the fragment sizes of the digested DNA and in turn determine the size of our unknown plasmid. Gel electrophoresis is a process by which DNA in a solvent is injected into multiple wells within the agarose gel medium. The electrophoresis machine is then turned on, where it takes advantage of the fact that DNA is an electrically charged molecule (3).
Thus, pulling the smaller fragments farthest from the origin in the gel, and leaving the larger fragments closer to the wells. After the fragments are seperated a staining step can make them visible. Ethidium bromide or methylene blue are the most frequently used reagents for staining. The DNA ethidium bromide complex strongly absorbs UV light and re-emits visible light (3). Allowing the visibility of bands, or populations, of DNA molecules of the same size. As seen on our pictures electrophoresis its obvious that the double digested DNA allowed for two cuts to be made, thus crea...
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Research essay sample on Dna Gel Electrophoresis Research
