E. coli or Escherichia coli is a prokaryotic cell found the in lower digestive track of mammals and other warm blooded animals. E. coli is an easy bacteria to work with as it doubles quickly and is relatively easy to grow; millions of cells can be grown in several hours (Biotechnology Learning Hub 2014). E. coli is an ideal bacterium in the lab because it does not require its temperature to be too hot, too cold, or too precise (Biotechnology Learning Hub 2014). A general warm temperature is perfect for this bacterium. E. coli is also easy to care for it does not need a specific type of nutrient, in a lab setting it can be feed any agar, making the bacteria over all cheaper to care for. Strains of E. coli can also operate in aerobic or anaerobic environments (Koh et al. 2007).
The plasmid pGLO contains a gene which produces GFP, which glows green under florescent light. However the florescence is only seen or expressed in the presence of arabinose, as the GFP will react with the arabinose (Froger and Hall 2007). This engineered plasmid ring of DNA originally comes from Aequorea victoria, a common jellyfish. When pGLO integrated the organism also inherits the ability to resist the antibacterial
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ampicillin. The bacteria that happens to accept the overall beneficial plasmid it can resist the ampicillin solution in its environment and thrive. Heat shock treatment is used to insert the foreign DNA in to the E. coli cells. Heat shock uses temperature to alter the permeability of a cells membrane, making it more competent so it will have a higher chance of accepting foreign DNA (Biotechnology Learning Hub 2014). Then the cells are quickly cooled in an ice bath so they close quickly trapping any DNA that was successfully accepted in to the cell. If heat shock is successful the plasmids will start to code for the desired trait. Significance: The purpose of this experiment is to show how single cell organisms can be easily changed if the cell is competent. It will also highlight how successfully integrated plasmids can affect the cell and what proteins are produced by the DNA, and how easy it is to change the genetics of cell and the future generations of that cell. The experiment also demonstrates how artificial selection would work in a lab environment, and how the addition of a gene or a mutation can protect an organism from adverse conditions in the environment. This lab also illustrates the change in DNA in a visual sense. Molecular genetics is a very small scale science that cannot be seen by the human eye, however by illuminating the bacteria with the GFP it will produce the very small process can be clearly seen before and after the genetic modification. The bacteria will reproduce quickly and the successful bacteria will be clearly seen by the human eye, even after generations upon generations of bacteria. Genetic modification is critical to a species adaptation, and genetic modification in a lab shows scientists how to change the composition of an organism for better or for worse. Hypothesis: The lab’s purpose is to show how the right conditions can change the genome of a particular bacterium; E. coli. The experiment will test different conditions, LB –pGLO, LB/amp –pGLO, LB/amp +pGLO, and LB/amp/ara +pGLO. These conditions will test what bacteria are transformed and what bacteria are not depending on the conditions they are placed in. The petri plate with the +pGLO LB/amp/ara will allow for the E. coli who have accepted the plasmid to glow and express the GFP. The E. coli will only express GFP if they are exposed to the arabinose sugar, and the bacteria also have to be exposed to the pGLO and transformed. Also the bacteria that are exposed to the ampicillin, but are not exposed to the pGLO and thus untransformed will perish. Other plates may grow, but not express the protein. Method and Materials: In order to begin the experiment all materials must be retrieved. Before the bacteria and pGLO is handled, vinyl glove must be worn as a live organism is used (Weedmen 2014). With the beaker obtained earlier, fill the beaker with small ice, pebble ice or crushed ice is preferred. With a fine pen label the micro centrifuge tubes; one +pGLO and the other –pGLO. With the micro pipette, carefully add 250μl of transformation solution in each micro centrifuge tube, then close the lids and place them completely in the ice in the beaker. While the broth is on ice, obtain a colony of E. coli bacteria from the prepared petri dish with a sterile inoculating loop. With the colony on the loop pick up one tube and twirl the colony in the solution. Do the same with a new loop and acquire a new colony and twirl into the other tube. With another sterile inoculating loop, dip the loop into the pGLO plasmid solution and when there is a thin layer or thin film stretches across the loop, twirl the loop in the +pGLO micro centrifuge tube, and only do this for the +pGLO tube. Once complete put both the tubes back on ice for 10 minutes. After the micro centrifuge tubes have sat on ice for 10 minutes transfer them to the floating rack and bathe them in a 42 degree Celsius water bath for 50 seconds, once complete immediately transfer them into the ice for 2 more minutes. Once complete add 250 μl of nutrient broth in both tubes, then transfer them to the rack at room temperature for 10 minutes. When the 10 minutes is up flick the contents to remix the solution. Then with the micro pipette transfer 100 μl of the +pGLO solution into LB/amp +pGLO and the LB/amp/ara +pGLO plates. Then place 100 μl –pGLO solution into the LB –pGLO and LB/amp –pGLO plates. Then, with a new inoculating loop for each plate, clam-shell the plates and spread the materials quickly across in light zigzags without pressing into the agar, with as little air being exposed to the experiment as possible. Dispose of the used inoculating loop in in the autoclave bag. Stack the petri plate’s upside-down and tape together, label with name. The plates are then placed in an incubator at 37 degrees Celsius then they are transferred into the refrigerator for 6 days. Afterwards view plates under UV light to view results. Results: Over the course of a week the bacteria grew and reproduced in a controlled environment, and when the results were checked under an ultraviolent light. The bacteria transformed with the pGLO and were in the arabinose and ampicillin petri plate glowed under the light. The other plates did not glow at all. Figure 1 displays the LB –pGLO plate. The plate was the control in the experiment, it contained the grow substrate and also had no pGLO added to the bacteria. The plate had a lawn of E. coli across the entire plate. Under the UV the bacteria do not glow. Figure 2 displays the LB and ampicillin –pGLO plate. The plate contained the growth substrate and the antibacterial ampicillin. On this plate there were no colonies of bacteria, as they were not exposed to pGLO which would have given the bacteria a resistance to the drug. Figure 3 displays the LB and ampicillin +pGLO plate. The plate had the growth substrate and ampicillin. This plate contained many colonies of bacteria that were transformed with the pGLO and they were able to resist the ampicillin infused environment. Figure 4 displays the LB, ampicillin, and arabinose +pGLO plate. The plate contains the growth substrate, ampicillin, and arabinose creating the perfect environment for the bacteria to glow. The bacteria that transformed were able to resist the ampicillin and due to the arabinose the bacterial colonies within the plate were able to glow like in Figure 5. Discussion: This experiment tested different conditions, LB –pGLO, LB/amp –pGLO, LB/amp +pGLO, and LB/amp/ara +pGLO. The growth of the bacteria would change depending upon what bacteria were transformed and what bacteria were not, depending on the conditions they grew in. The petri plate with the +pGLO LB/amp/ara will allow for the E. coli who have accepted the plasmid to glow and express the GFP. The Hypothesis was proven as the bacteria glowed green under the ultraviolent light as seen in figure 5. The E. coli were able to express GFP because they were exposed to the arabinose sugar, and the bacteria also were exposed to the pGLO and transformed. Also the bacteria that were exposed to the ampicillin, but were not exposed to the pGLO perished. Other plates grew, but not express the protein under ultraviolent light, Figure 3 and Figure 1. And experiment similar in nature to the one tested is involving a very popular and easy pet.
Much like how in the lab the bacteria were transformed to express the gene GFP, the same gene was used to make zebra danios, and several other species in to a Glofish. Like the E. coli, the Glofish will glow under ultraviolent light. The way the DNA is inserted into the fish cells differs from the E. coli experiment because the fish eggs were more delicate, but the concept of the experiment is the same. The fish are not bioluminescent, but florescent. They don’t just glow normally, a specific light must be over them for the GFP to be expressed. This genetic modification to the fish is a huge business and will not end due to their popularity, the business will likely only grow
larger. Transforming the E. coli fell under the science of genetic modification, and in this field many organisms have been genetically modified. Of all the genetically modified organisms, corn is the most popular. It’s been modified to resist drought, resist insects, have a higher yield, etc. Some rice has been modified to produce vitamin C, something not normally made by the plant. This is a very common science in today’s world, and is unlikely to stop regardless of the environmental impact. Throughout the modern world many species of plants have been changed to the will of society. Throughout the experiment there were many mistakes and improvements that could have altered the experiment in many ways. The way the E. coli was placed on the petri plate and how everything was made, there was lots of exposure to air and possibly other bacteria species. Also, the bacteria were not all able to accept the plasmid, there were many that did not, and that could be due to a multitude of reasons, it’s possible the heat shock did not have a high success rate compared to the original bacteria population. Another issue might have been the conditions the bacteria grew in, the ideal condition was not consistently kept, so the bacteria reproduced at slower rates. Also, the bacteria were not constantly watched and they might have been tampered with. Though, through the mistakes the experiment was still a success as the bacteria that could glow, did glow under the ultraviolent light.
Enhanced green fluorescent protein (EGFP) was originally isolated from a bioluminescent jellyfish called Aequorea victoria. As suggested by the name, this protein fluoresces green when exposed to light in the ultraviolet range. The ultimate goal of the following experiment was to successfully create a pET41a(+)/EGFP recombinant plasmid that was transformed into live E. coli cells. The success of this transformation could be evaluated based on whether EGFP’s fluorescence properties were displayed by the colony in question. The protein’s fluorescence properties “triggered the widespread and growing use of GFP as a reporter for gene expression and protein localization in a broad variety of organisms” (Ormo, et. al., 1996). Although EGFP and GFP differ for a few amino acids that make EGFP’s fluorescence mildly stronger, the basic principle that such a protein allows for the evaluation of transformation success remains intact.
Figure 2 shows the results of the electrophoresis. Lanes 5 and 7 indicate the fragments obtained when the plasmids are digested with both restriction enzymes, indicating the approximate fragment size for the hlyA gene, the pK184 plasmid and the pBluescript plasmid. This is useful for identifying the recombinant DNA needed for this experiment
Therefore colonies containing the non-recombinant pUC19 plasmid have a functional lacz’ gene appear blue on the agar and colonies containing recombinant pUC19 would have a non-functional lacz’ gene due to insertional inactivation and appear white on the growing medium.
To begin the lab, the variable treatment was prepared as the Loggerlite probe, used to later measure oxygen consumption, warmed up for approximately 10 minutes. To prepare the variable treatment, 200ml of Sodium and Ammo-lock water was measured in a container and a pre-prepared “tea bag” of tobacco was steeped in the room temperature treated water until a light yellow color was visible. After preparing the tobacco solution the preparation for the live goldfish began as two beakers were filled with 100 ml of treated water. Each beaker was weighed before addi...
Ligation one was a 1:1 molar ratio pET-41a (+) vector: egfp insert that used 50ng NotI/NcoI cut pET-41a (+) DNA, 7ng egfp insert DNA, 1uL of DNA ligase, and the proper quantity of water to dilute 10x ligase buffer to a 1x final concentration. Ligation two was a 1:3 molar ratio pET-41a (+) vector: egfp insert made up of 50ng NcoI/NotI cut pET-41a (+), 21ng egfp insert DNA, 1uL of DNA ligase, and the proper quantity of water to dilute 10x ligase buffer to a 1x final concentration. Water was sterilized and deionized. The remaining three ligation samples served as controls. Ligation three contained 57ng uncut pET-41a (+)/EGFP recombinant plasmid DNA and sterile water. Ligation 4 was a negative control that consisted of only sterile water. Ligation five lacks DNA ligase but has the same properties of the 1:3 molar ratio pET-41a (+)/EGFP vector.
al. (1994) explain that a complementary DNA for GFP produces a fluorescent product when expressed in E. coli cells as the expression of GFP can be used to monitor gene expression and protein localization in living things. 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 or +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 fish is modified to carry a growth hormone gene from the Pacific chinook salmon and DNA from the eel-like ocean pout. This allows the salmon to grow faster as the hormone is kept active all the time.
Escherichia coli is a member of the family Enterobacteriaceae. It is a bacterium with a cell wall that has many components. Escherichia coli can live without oxygen which means that it is a facultative anaerobe. It is also capable of fermenting lactose under anaerobic conditions, and in the absence of alternative electron acceptors. There are effects and various factors that limit its growth rate. Its morphology consists of a rod-shaped gram negative bacteria that is commonly found in soil, water, vegetation, human intestines, as well as the intestines of animals. Its presence can be good or bad.
An example of bioluminescence is a firefly. The production of light in bioluminescent animals is caused by converting chemical energy to light energy (Bioluminescence, 1 of 1). In a firefly, oxygen, luciferin, luciferase (an enzyme), and ATP combine in the light organ in a chemical reaction that creates cold light (Johnson, 42). This bright, blinking light helps the male firefly attract female fireflies as a possible mate. Other examples of bioluminescent organisms are fungi, earthworms, jellyfish, fish, and other sea creatures (Berthold Technologies, 1 of 2).
There are numerous types of bacteria that can be found in every environment. Each bacterium has different morphology which includes shape, texture and pigment production. These bacteria also have different food requirements which are important in being able to identify a microorganism. Microorganisms are a diverse group containing all bacteria a single cell prokaryotic organism that is found in every type of environment, archea single cell microorganism that lacks nuclei and almost all microorganisms are protozoa a unicellular eukaryotic organism. By identifying the causative agent of a bacterium within an individual, an antibiotic can be developed to prevent health issues. Microorganisms are also used to make certain food products for human consumption. An example of this would be the production of yogurt. It has probiotics that help with digestive abnormalities amongst other things. Probiotics are microorganisms that are consumed to provide health benefits in the body. Probiotics work by replacing the disturbed microbe with ones that are useful to digest. With the methods that wer...
U.S. Food And Drug Administration (2013, July 23). An overview of Atlantic salmon, its natural history, aquaculture, and genetic engineering. Retrieved May 1, 2014, from http://www.fda.gov/AdvisoryCommittees/CommitteesMeetingMaterials/VeterinaryMedicineAdvisoryCommittee/ucm222635.htm
Household bacteria have always been a problem. Millions of people each year get some sort of sickness from bacteria in their kitchen, bathroom, living room, etc. What if there were bacteria in your kitchen that could be deadly? This bacterium is called Escherichia coli. According to Oregon health Authority: “By one estimate, 10,000 to 20,000 E coli. Infections occur in the United States each year.” Many of us use disinfectants such as Clorox and Bleach every day but are these disinfe...
Over the past few years there has been several cases of food contaminated with Escherichia Coli and Salmonella, mostly from organic food manufacturers. For a better understanding of this issue it is necessary to go back to the basis for organic agriculture. Which is essentially about the nutrients needed for the soil and its direct relation to the quality of the product. The more nutrients are added to the substrate the better the quality of that product. Farmers replace synthetic fertilizers and chemical pesticides for natural alternate methods, using fertilizers based on decomposed organic matter like Humus and Compost. Among the active components found in this products are animal feces which contain harmful parasites that could be transmitted to the plants. Several studies support this statement; an evaluation from the University of Minnesota revealed that “the percentages of E.coli–positive samples in conventional and organic produce (on farms in Minnesota), were 1.6 and 9.7%, respectively” (Mukherjee, Speh, Dyck, Diez-Gonzalez,
E. coli are bacteria that can cause an infection in various parts of your body, including your intestines. E. coli bacteria normally live in the intestines of people and animals. Most types of E. coli do not cause infections, but some produce a poison (toxin) that can cause diarrhea. Depending on the toxin, this can cause mild or severe diarrhea.
Paavo Nurmi is considered by some to be the greatest runner of all time. He was known as "The King of Runners" or the "Flying Finn". Famous all over the world, Nurmi became an unending source of national pride for the newly independent Finland. Paavo Nurmi was driven by love of running. He had a burning will to succeed in life, and racing was his way to gain recognition from his fellow men and to fulfil the high standards he had set for himself. Martti Jukola, a famous Finnish sports journalist, wrote in 1935: "There was something inhumanly stern and cruel about him, but he conquered the world by pure means: with a will that had supernatural power." At three Olympic Games from 1920 to 1928 Nurmi won a total of nine gold and three silver medals.