Confirmation of Bacterial Transformation and DNA as the Inducing Material
Confirmation of Transformation and DNA as the Inducing Material
Introduction
The experiments in this report are a recreation the famous Griffith and Avery experiments which discovered the transformation process that bacteria can undertake and that DNA is the genetic material, respectively (Griffth, 1928. Avery 1944). Bacteria can incorporate foreign DNA found in their medium into their genomes in a process known as transformation. This process can be accelerated in CaCl solution at colder temperatures. The first part of this report deals with confirming that transformation does occur following a standard procedure, and the second of the report uses a similar procedure to confirm that DNA is the particle that induces transformation.
Methods
In the following procedures Escherichia coli was used:
(Part 1) For the first set of experiments a solution of half live ampicillin sensitive strain and half of a heat killed ampicillin resistant strain was mixed and incubated at 37°C for forty five minutes. The resulting fluid was spread onto a prewarmed LB and a prewarmed LB Amp100 medium, respectively. In addition six controls were set up which shared a LB and a LB Amp100 medium. These controls were given their own area on the media and streak plated. The controls consisted of the live ampicillin sensitive strain, the dead ampicillin resistant strain and the live ampicillin resistant strain plated onto each medium. Aseptic technique was used in treating all solutions, media and transfers between.
(Part 2) In the second set of experiments 1.0µL of kanamycin sensitive bacteria was added to two test tubes, both subjected to the same treatment. The bacteria in the...
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...colonies for live ampicillin sensitive cells grown on LB Amp100 media with only the buffer.
The colonies found on media treated with both the live ampicillin sensitive and heat killed resistant bacteria generated colonies with one exception: The treatment which used DNAse. This treatment showed the same result as the ones without heat killed bacteria: no growth was observed indicating that transformation could not occur without DNA. In conclusion this confirms that DNA is the hereditary medium which can transform bacteria, as discovered by Avery in 1944.
References
Avery, O. T., MacLeod, C. M. & McCarty, M. (1944). Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types. The Journal of Experimental Medicine, 79, 137-158.
Griffith F. (1928). The Significance of Pneumococcal Types. Epidermiology & Infection, 27(2), 113-159.
The first day an unknown sample was assigned to each group of students. The first test applied was a gram stain to test for gram positive or gram-negative bacteria. The morphology of the two types of bacteria was viewed under the microscope and recorded. Then the sample was put on agar plates using the quadrant streak method for isolation. There were three agar plates; one was incubated at room temperature, the second at 30 degrees Celsius, and the third at 37 degrees Celsius. By placing each plate at a different temperature optimal growth temperature can be predicted for both species of bacteria.
The purpose of this study is to identify an unknown bacterium from a mixed culture, by conducting different biochemical tests. Bacteria are an integral part of our ecosystem. They can be found anywhere and identifying them becomes crucial to understanding their characteristics and their effects on other living things, especially humans. Biochemical testing helps us identify the microorganism present with great accuracy. The tests used in this experiment are rudimentary but are fundamental starting points for tests used in medical labs and helps students attain a better understanding of how tests are conducted in a real lab setting. The first step in this process is to use gram-staining technique to narrow down the unknown bacteria into one of the two big domains; gram-negative and gram-positive. Once the gram type is identified, biochemical tests are conducted to narrow down the specific bacterial species. These biochemical tests are process of elimination that relies on the bacteria’s ability to breakdown certain kinds of food sources, their respiratory abilities and other biochemical conditions found in nature.
pBK-CMV is a plasmid vector 4518 in size, it also contains a multiple coding site (polylinker) that has recognition sequences for many restriction endonucleases. cDNA molecule CHI-1, which is 600bp, has been previously inserted. pUC19 is a cloning vector developed by….. in …….at….(REF). This vector is 2686bp in size and contains a 54 base pair (bp) polylinker containing 13 specific restriction sites, Xba1 and EcoR1 inclusive. It makes a good cloning vector as it is small in size, this makes it easier to be taken up by its host during transformation and allows for a faster replication time (Green, 2015). It contains an origin of replication pMB1 which is essential to be able to replicate. pMB1 has a high copy number allowing for multiple copies to be made (REF hcn pmb1). The pUC19 plasmid vector contains an ampicillin resistance gene, the host containing this plasmid will survive in the presence of ampicillin allowing for the selection of transformed host bacteria. The polylinker of pUC19 is contained within a lacz’ gene allowing us to distinguish between recombinant pUC19 and non-recombinant pUC19 through a process call insertional inactivation (Green, 2015).
ABSTRACT: Water samples from local ponds and lakes and snow runoff were collected and tested for coliform as well as Escherichia coli. Humans as well as animals come into contact with these areas, some are used for recreational activities such as swimming and some are a source of drinking water for both animals and humans The main goal of this experiment was to see which lakes, snow run off and ponds tested positive for coliform or Escherichia coli and to come up with some reasoning as to why. It was found that the more remote pond with less contact contained the most Escherichia coli. However, another lake that many swim in and use as their drinking water indeed tested positive for a small amount of Escherichia coli. The two samples from the snow showed negative results for both coliform and Escherichia coli and the two more public ponds that aren’t as commonly used as a source of human drinking water but animal drinking water tested in the higher range for coliforms but in the little to no Escherichia coli range. It was concluded that the remote pond should be avoided as it’s not a safe source of drinking water for humans or animals. Other than that, the the other ponds are likely to be safe from Escherichia coli, but coliforms are a risk factor.
In this experiment the heat shock method will be used to deliver a vector (plasmid) of GFP to transform and grow E. coli bacteria. Four plates containing Luria Bertani (LB) broth and either –pGLO and +pGLO will have E. coli bacteria added to it. The plate containing –pGLO (no pGLO) and LB will show growth as ampicillin will be present killing bacteria but no glowing because no arabinose will be present for glowing to be activated, the same result will be seen in the plate containing +pGLO, LB and ampicillin. The plate with –pGLO, LB and ampicillin will show no growth and no glowing as no arabinose is present for glowing to be activated
Abstract: Bacterial transformation involves the change of genetic composition of bacteria by altering its genetic identity. The pGLO plasmid was ingrained in the E. coli cell, which allows the modified E. coli cell to begin to code for the GFP protein gene and the beta lactamase gene (ampicillin resistance gene). After modifying the bacteria cell, the changes involved with the plasmid were tested on 4 plates, two plates containing the pGLO plasmid (+) were treated with LB nutrient media. One of the LB plates contained arabinose in it, which should fluoresce green under UV light. The other LB plate contained ampicillin. Two other plates which did not contain the pGLO plasmid (-) both had LB media growth, one plate just to show cell growth,
Background Information and Research: Inserting a gene from the Aequorea victoria jelly fish into the DNA of rabbits, pigs, and mice genetically modifies them to glow-in-the-dark. The production of specific genes are coded by genes. This particular type of jelly fish naturally glows in the dark because a gene coded for a green fluorescent protein (GFP). The goal of genetically modifying organisms is to have the modified organism produce a protein that has been coded by the inserted gene thus causing the modified organism to express the new trait. Genetically modifying organisms is important because it has had health benefits in the development of vaccines. E.coli is a rod-shaped bacteria that is a part of the Escherichia genus and is commonly found in the intestines. When demonstrating how to genetically modify an organism, E.coli bacteria is commonly used because it is a simple organism whose process for protein production, gene expression, is the same as a complex organisms’ process. In this experiment, a GFP was inserted into E.coli as well as a gene that causes E.coli’s resistance to ampicillin. Half of the agar plates that the bacteria was growing on had ampicillin. Ampicillin kills E.coli, so the successfully modified bacteria will have been grown on those ampicillin plates. Plasmids contain genes that are resistant to antibiotic ampicillin; scientists have used plasmids in the manipulation of genes. Plasmids were used because it is resistant to the ampicillin used, so if the bacteria was
With the earliest recordings coming from the Fifth Century B.C., streptococcus pyogenes, and more frequently, its symptoms have been prevalent among doctors and historians for hundreds of years. The first mentioning of streptococcus pyogenes is to be credited to Hippocrates, in which he describes the relative symptoms of the flesh-eating bacteria in its early stages. Then depicted by Billroth in 1874, patients carrying erysipelas were determined to have this certain bacterial infection. In 1883, the chain-forming bacteria were isolated by Fehleisen; and in the following year, Rosenbach applied the S. pyogenes name. Further advances in hemolytic and non-hemolytic studies were made by Lancefield in the 1930’s, in which the alpha, beta, and gamma subgroups of the hemolytic structures – detailed and defined by Schottmueller and Brown - were divided into serotypes.
182 people became infected, and 29 died (most of which were older men or cigarette smokers). Although this organism was named in the 70’s, retrospective studies have shown cases since 1943. GRAPH Diseases: L. pneumophila has a very wide range of effects. Healthy individuals usually go through an asymptomatic seroconversion, while less healthy people may undergo Pontiac Fever or Legionnaires’ Disease (LD). In 1968, employees at the county health department in Pontiac, Michigan came down with a fever, but the responsible pathogen was not identified at the time.
The purpose of this experiment is to identify an unknown insert DNA by using plasmid DNA as a vector to duplicate the unknown insert DNA. The bacteria will then be transformed by having it take in the plasmid DNA, which will allow us to identify our unknown insert as either the cat gene or the kan gene.
This biotechnology lab analyzes the effect of transferring genetic information through the alternation of bacterial gene in E. coli (Spilios, 2014). This alteration occurs through plasmid DNA transcribing the new genetic components into RNA, which will translate into an amino acid (Sadava et al., 2014). This newly transcribed amino acid is an enzyme that will give the transformed E. coli cells an antibiotic resistance, Beta-lactamase (Greenfield et al., 2009). The plasmid DNA of interest will be altered to become more resilient to the antibiotic ampicillin, since beta-lactamase could decompose the ampicillin. In addition to plasmid DNA, the bacteria contain other important features such as reporter gene. This reporter gene will act as an aid when observing the effect of the alteration, since this particular gene can be distinguished when a plasmid with foreign DNA is transferred from one to another (Spilios, 2014). Moreover, the reporter gene being used in this lab, Green Fluorescent Protein, is to determine gene resistance to ampicillin. GFP would be useful in this experiment, since it would glow when arabinose operon is present. Ampicillin is a derivative of penicillin that inhibits bacterial growth by interfering with the synthesis of bacterial cell walls. Since E. coli is gram negative, and ampicillin kills the gram-negative bacteria by synthesizing with the cell wall, E. coli should perish under no transformation. However, the ampicillin resistance gene is the enzyme Beta-lactamase, which is secreted by transformed cells into the surrounding medium where it destroys ampicillin (Dörr, 2010). In order to resist ampicillins, E.coli utilizes pGLO plasmid to protect the cell from ampicillin’s invasion. There are four components to...
Streptococcus pneumoniae is a Gram-positive and fast-growing bacteria which inhabit upper respiratory tract in humans. Moreover, it is an aerotolerant anaerobe and usually causes respiratory diseases including pneumonia, otitis media, meningitis, peritonitis, paranasal sinusitis, septic arthritis, and osteomyelitis (Todar, 2003). According to Tettelin et al., more than 3 million of children die from meningitis or pneumonia worldwide (2001). S.pneumoniae has an enzyme known as autolysin that is responsible for disintegration and disruption of epithelial cells. Furthermore, S.pneumoniae has many essential virulence factors like capsule which is made up of polysaccharides that avoids complement C3b opsonization of cells by phagocytes. Many vaccines contain different capsular antigens which were isolated from various strains (Todar, 2003). There are plenty of S.pneumoniae strains that developed resistance to most popular antibiotics like macrolides, fluoroquinolones, and penicillin since 1990 (Tettelin et al., 2001). Antibiotic resistance was developed by the gene mutation and selection processes that, as a consequence, lead to the formation of penicillin-binding proteins, etc. (Todar, 2003).
S. pyogenes is a bacterium that permeates our society. Today it is commonly known as the cause of “Strep. throat,” or Streptococcal pharyngitis. Modern medicine has caused the eradication of most of its advanced infections, while this most common form of infection still thrives. It is very contagious, and pyogenes travels quickly through places where bacteria flourish, such as schools and health institutions. The body cannot fight this bacterium very well without help, and S. pyogenes was a common cause of death until the introduction of antibiotics in the twentieth century. It has a number of ways to subdue the immune system, but it is almost completely vulnerable to penicillin, even after decades of exposure. While generally no more than a nuisance, this bacterium continues to be an interesting topic of discussion. (6,3,2)
Antibiotics have been vital tools in the fight against bacterial infections, however their effectiveness has waned in recent times due to the advent of antibiotic resistant strains of bacteria. According to a review by P, the uses of antibiotics, as well as influences from the environment have allowed such bacterial strains to respond to changes in their environment rapidly, and so develop resistance. This acquired ability can have serious and broad implications in the medical field, evident in a study by O into the resistance of intestinal Staphylococcus aureus.
On the other hand, cells that have resistance from the start or acquire it later may survive. At the same time, when antibiotics attack disease-causing bacteria, they also attack benign bacteria. This process eliminates drug-susceptible bacteria and favors bacteria that are resistant. Two things happen, populations of non-resistant and harmless bacteria are diminished, and because of the reduction of competition from these harmless and/or susceptible bacteria, resistant forms of disease-causing bacteria proliferate. As the resistant forms of the bacteria proliferate, there is more opportunity for genetic or chromosomal mutation (spontaneous DNA mutation (1)) or transformation, that comes about either through a form of microbial sex (1) or through the transference of plasmids, small circles of DNA (1), which allow bacteria to interchange genes with ease.