Pglo Essay

Answer the following questions on another piece of paper in complete sentences using no unidentified pronouns.

No other aquatic organism like the jellyfish instills fears in ocean swimmers. Known for their graceful movements, tentacles, and stinging ability, the jellyfish is avoided at all cost. There is, however, a particular species of jellyfish Aequorea Victoria that has been very beneficial to scientists. This species of jellyfish produces green bioluminescence from small photo organs located on its umbrella. In the 1960, while working at Princeton University, the scientist Osamu Shimomura was attempting to answer the question how some organisms can produce light which led him to work on the jellyfish and the discovery of GFP. In his work with the jellyfish Aequorea Victoria, he discovered and isolated Aequorin, the bioluminescence protein.

Experiments introduce the idea that the uptake of DNA by competent bacteria can be the result for survival reasons such as need for food or evolution purposes (5). The difference between natural and artificial gene transformation in a bacteria, however, with those who are incompetent versus competent allow he or she to conduct an experiment with artificial transformation. By doing this one may be able to determine and observe the formation of biofilms or mutations by transferring a gene to another organism for beneficial or harmful purpose. This could lead to finding resistants or if a gene introduced has any effect on the bacteria. In the experiment, the pGLO plasmid contained encodes the gene for GFP as well as a gene for resistance to an antibiotic allowing a transformation to take place when adding a carbohydrate such as arabinose to the medium. Research has been found on the horizontal gene transfer on how virulence factors are acquired as well as genes resistant to antibiotics spread with microorganisms (5). By conducting a similar experiment he or she can observe similar findings or observe the results and conduct some sort of understanding of these ideas. The findings in this experiment are to show a positive result in what causes growth and the glowing when the plasmid pGLO is added to the positive as well as the difference in the medium. Understanding the way artificial

Genetic transformation is the process involving the insertion of new DNA into a bacteria cell such as E.Coli. While the inserted DNA contains a large chromosome, it also contains small circular pieces of DNA called plasmids. The injected plasmids hold specific genes as they are encoded through genetic engineering. Genetic engineering is described as “the direct manipulation of genes for practical purposes” (Urry et. al., 2016). The DNA injected directs the synthesis of proteins due to genetic expression and explains how bacteria is given the ability to resist antibiotics through the use of the pGLO plasmid. The “pGLO plasmid has been genetically engineered to carry the GFP gene which codes for the green fluorescent protein” (Biorad). This

Plasmids are the extra-chromosomal DNA in which most bacteria contain in addition to their bacterial chromosome. Plasmids can be used as vectors to transfer DNA into a host bacterium. A nonpathogenic strain of Escherichia coli (E. coli) HB101;K-12, is the host bacterium used to take up the plasmid with the imported gene to be observed. pGLO is a recombinant plasmid that consists of a gene that codes for Green Fluorescent Protein (GFP) and also contains a gene, beta-lactam antibiotic (bla), that expresses antibiotic resistance. GFP is a protein that is produced by the jellyfish Aequoria victoria and has the ability to fluoresce green when exposed to ultraviolet (UV) light. The gene for GFP can be turned on in the transformed bacterial cell in the presence of the sugar, arabinose. The bacteria that contain the beta-lactam antibiotic have the ability to create beta-lactamase, which is an enzyme that hydrolyzes the beta-lactam rings of antibiotics which penicillin derivatives (among a broad range of others) contain, deeming them incompetent. Beta-lactam antibiotics inhibit the peptidoglycan layer of the bacterial cell wall and because E.coli is a gram-negative bacteria, its peptidoglycan layer is easily susceptible to inhibition.

Green fluorescent protein was first recognized in the early 1960s by a group of researchers studying the bioluminescent properties of the Aequorea victoria jellyfish. They discovered that the jellyfish produces green bioluminescence from small photoorgans located on its umbrella. Osamu Shimomura found that in order to bioluminesce Aequorea releases calcium ions. These bind to a protein that he called aequorin, which release blue light upon calcium binding. The blue light is absorbed by green fluorescent protein, which in turn gives off the green light (Shimomura, O., Johnson, F. H. and Saiga, Y. 1962.)

Figure 3 shows the growth of three differently treated E. Coli plates. -pGLO plates are both controls. -pGLO with only LB experienced the most grow because

The plasmid was then inserted into a strain of E. coli and grown. After incubation the E. coli strain was found to fluoresce upon illumination with UV light inside of the growing cell, without the addition of A. victoria products (figure 1). Partially purified GFP protein from the bacteria was then obtained and found to posses an identical excitation and emission spectrum to the original A. victoria GFP, as seen in figure 2. This result suggested that GFP did not require additional enzymes/cofactors from A. victoria to fluoresce. Chalfie, using the same technique for GFP insertion in E. coli, inserted the gene into C. elegans under the control of the mec-7 gene, which encodes β-tubulin in C. elegans’ six touch receptors. The GFP again fluoresced when exposed to blue light, but this time the fluorescence was limited to the β-tubulin transcribed by the mec-7 gene, as seen in figure 3. Martin Chalfie, from the results obtained during his experiments was able to conclude that GFP isolated from A. victoria could fluoresce in living cells when exposed to blue light, without cofactors or enzymes form A. victoria. He also showed that gene expression could be traced using GFP, by measuring different fluorescence levels in C. elegans larvae as they

The main objective of this experiment was to attempt bacterial transformation, and alter the initial plasmid of DNA to one that expressed GFP, and was therefore able to glow under the UV light. The methodology we used to complete this process was bacterial transformation which is a widely used method where foreign DNA is introduced into a bacterium, which can then amplify, or clone the DNA. Our results were that there was growth on all of the plates except for the-pGLO plate with LB and ampicillin. The results for the fluorescence of the plates was that only the +pGLO plate with LB, ampicillin and the arabinose sugar glowed.

If Genetic transformation has the meaning of “change caused by genes” and involves the placing of a gene into an life form in order to modify the organisms characteristic; the progression of placing genes from one life form to a different is used to assist of a plasmid and the pGLO plasmid codes the gene used for GFP as well as the gene for resistance to ampicillin. It is used to manage the expression of the fluorescent protein; hence, the GFP gene is able to be switched on by adding the sugar arabinose to nutrient medium of the cell, then the bacteria will be able to glow a bright green underneath UV light when arabinose is within the nutrient agar medium. Hence, then when one micro test tube +pGLO and –pGLO are labeled and placed into a foam rack and the tubes are open and using a sterile pipet used to transfer 250 micro liters of transformation solution (CaCl2 ) in each tube, position the two tubes on ice, pick up 2-4 colonies of bacteria with a loop, submerge the loop into the +pGLO tube, repeat steps for –pGLO, put in to ice, and put plasmid DNA into the pGLO; after the pGLO’s need a heat shock by placing the cold tubes into the 42 degrees Celsius hot bath for 50 seconds and back into ice for 2 minutes, later insert the 250 micro liters of LB nutrients broth into the tube and then placing 100 micro liters into the 4 plates, each individual plate contains +pGLO LB/amp, +pGLO LB/amp/ara, -pGLO LB/amp or –pGLO LB). If bacteria that contains +pGLO plasmids is resistant to

IVF treatment must occur before PGD. Embryo development occurs on day 3 when a small hole is made in the soft shell of the embryo (Zona Pellucida) using a laser. On day 5, one or more cells are removed from each embryo (embryo biopsy). The embryos are then frozen and cells are sent to a PGD laboratory for analysis. The cells are tested from each embryo which informs them which embryos are normal. Normal embryos are thawed and transferred to the woman later.

In the scientific community, there have been many advancements in the technologies and processes used to help us understand the truth of the world around us. The invention of the electron microscope by Ernst Ruska and Max Knoll in 1933 is just an example of one of these advancements. There is another advancement that has roots over one hundred and sixty million years old, and that is the green fluorescent protein (GFP). The fluorescence from GFP molecules has made it feasible to look through a microscope at a spatial resolution higher than the diffraction limit. The green fluorescent protein was first found in the Aequorea Victoria, or the crystal jellyfish. It is a bioluminescent hydrozoan jellyfish, or hydromedusa (1). In the crystal jellyfish, there are two proteins that are responsible for its bioluminescent, aequorin and GFP. They are both located in the photo-organs of the jellyfish. The jellyfish releases calcium ions which the aequorin binds to. The process of binding gives off a blue light, which is then absorbed by the GFP. The blue light absorbed is then converted into the green light that GFP is named for (2). At the moment there is no apparent reason for the bioluminescence displayed by the Aequorea Victoria since it has not been observed being used as a defense mechanism or as a signalling mechanism. Osamu Shimomura was the first person to isolate the GFP. He started to study the bioluminescence of crystal jellyfish at Princeton University in 1960 and he and

Day one’s lab focused on bacterial transformation. This process is the way in which new DNA is introduced into a bacteria cell. In this process, the bacterial cells need to be made permeable through treatments by way of heat or by mixing with different salts. Once the bacteria cells are made permeable, the plasmid can introduce the genetic instructions used for GFP in the cells. Those that are made permeable have a much higher tendency to express the protein.

Hypothesis: If genetic transformation involves the insertion of a gene into an organism in order to change the organism’s trait where genes can be moved from one organism to another with the aid of a plasmid and pGLO plasmid encodes for a gene resistant to the antibiotic ampicillin as well as the gene GFP which allows the bacteria, E. coli, to express the glowing gene by producing the fluorescent protein if switched on after having undergone transformation by adding sugar arabinose – which is a nutrient for the cell -, then when the procedure of the pGLO transformation lab is completed as instructed in the student manual, the agar plate +pGLO Lb/ampicillin will produce colonies of bacteria that do not glow, -pGLO LB/ampicillin will not produce any bacteria, and –pGLO LB (constant) will produce a lawn of bacteria that does not glow (instead appearing as a

DNA encodes the genetic instructions for cells to carry out their daily activities. DNA can come in many forms; plasmids for example are small circular DNA molecules found in most bacterial cells. Though plasmids may not be essential for the life of bacteria, it can give cells resistance in foreign environments. For the purpose of this experiment, an ampicillin-resistant plasmid is introduced to E. coli. This is done through a process of genetic engineering called transformation. Transformation works through the uptake, incorporation, and expression of a foreign gene to alter the genetic code of a cell. Three conditions are needed for successful transformation: a host, a vector, and a technique to identify the transformed cells. E. coli is used in this experiment as the host (E. coli is commonly used in biotechnology due to its rapid rate of growth and short reproduction time). A vector mediates the transfer of foreign DNA into the host cell. Plasmids are commonly used vectors that will also be used in this experiment. The procedure of tagging is used in this experiment to differentiate the transformed cells from those that were not. The learning objectives of this experiment are to: observe the process of bacterial transformation in an experiment; and demonstrate a change in phenotype due to uptake and expression of the genes in a known plasmid.