Genetic Transformation of E. coli to Green Fluorescent Protein
Introduction
Genetic transformation is the process a cell undergoes to uptake a piece of foreign DNA from a different organism (Aldridge 2012). The process first started by a group of men named Herbert Boyer, Paul Berg, and Stanley Cohen during the 1970s (Aldridge 2012). As a result of their work, vaccines, medicine, and insulin became available (Aldridge 2012). This lab involves E. coli and a green fluorescent protein (GFP) that will glow green if the transformation works. GFP is supposed to act like a marker that glows green when detected and shows where the protein is made (Fletcher 2003). There are three different kinds of genetic transformations, but we only used heat shock in this experiment. This lab used heat shock treatment, which caused a sudden increase in temperature so that the permeability of the cell membrane will increase (Weedman 2013). Once the E. coli undergoes heat shock, the bacteria is transformed with arabinose and should glow green. When pGLO acts like a vector, it transfers one gene from one organism to another (Weedman 2013), making the E. coli bacteria in this experiment glow. The hypothesis was the binding of the sugar arabinose to the pGLO from the heat shock treatment, that will cause a genetic transformation and the E. coli to glow green. The presence of the ampicillin acts as a resistance in the pGLO vector. This will be used to prove whether the hypothesis was correct or not because it blocks the transformed cells. If the bacterium does glow, then the transformation using heat was a success and the E.coli will make a new protein. Genetic transformation is very important and can be found in everyday transformations such as medicines...
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...lts. Thankfully we followed all the steps correctly with caution and came out with the correct results.
Literature Cited
1. Fletcher L. Marker. 2003 Systems. Genetics. Ed. Richard Robinson. Vol. 3. New York: Macmillan Reference USA, Gale Virtual Reference Library. P 15-18
2. Aldridge S. 2012 "Genetic Engineering." Biotechnology Vol. 1 Detroit: Gale. Virtual Reference Library 431-433. P 431-433
3. Weedman D. 2013. Life 102 Attributes of Living Systems Laboratory Manual. 7th edition.
Minneapolis: Bluedoor, LLC.
4. Buzzle.com: Benefits of Genetic Engineering [Internet]. [cited 2014 April 28] Available from: http://www.buzzle.com/articles/benefits-of-genetic-engineering.html
5. Biology-Online.org. Genetic Engeneering Advantages and Disadvantages. [Internet]. 2006. [cited 2014 April 28]. http://www.biology-online.org/2/13_genetic_engineering.htm
Once the recombinant plasmid was obtained, it was then inserted into E. coli cells through transformation. From a successful transformation, we expected the bacterial cells to translate the inserted EGFP sequence into its protein form. The bacteria cultures were plated on petri dishes containing growth supplement, Luria Broth (LB), an antibiotic: Kanamycin, and IPTG which induced the fluorescence property within successfully transformed bacterial colonies. Different variants of the petri dishes were also included as control and unknown.
Recombinant DNA technology: Sub cloning of cDNA molecule CIH-1 into plasmid vector pUC19, transformation of XLI-Blue Ecoli & restriction mapping.
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
A recombinant plasmid are created by first using an enzyme that can identify and isolate specifically which gene that need to be cut. They are call restriction enzymes or restriction endonucleases, and more than 100 of these enzymes have been isolated. After the human gene (gene of interest) that codes for the desire trait is located on the chromosome restriction enzyme does it job, by cutting out the gene from the DNA. Now, the two ends of the human gene will be those that will link up with the open ends of the plasmid. An enzyme, DNA ligase, is used to couple each end of the gene to the open ends of the plasmid; this thus restores the circular DNA molecule with the human gene. Now the plasmid, with the human gene, is reinserted into the bacteria. They are then cultured and produced in large quantities of identical bacteria carrying the human gene. Now, these bacteria produce the human protein coded for by the spliced human gene. The protein is then isolated and purified and are ready to be injected into patients (crop, etc.) (Gish 1998).
Coli can in fact undergo mitosis on plates containing ampicillin, and show fluorescent qualities on a plate containing arabinose. These results logically follows from the fact that the plasmid inserted possesses qualities that allow for ampicillin to be broken down, and therefore not harm the E. Coli, meaning growth on plates containing ampicillin is proof of genetic transformation. Similarly, transformation of the E. Coli is also evident on the plate containing arabinose, as there was not only growth, but clear fluorescence under the blacklight, as the plasmid also codes for that expression. It is clear that those results are a result of the plasmid, as the plates treated with the +pGLO solution can be compared to those with the -pGLO solution, in which there was no growth on any plate except for the LB broth plate. Growth on the LB plate indicates that the E. Coli is healthy, and capable of mitosis in certain conditions, but lack of growth on the other plates points to it still being wild type. Therefore, it is clear that the +pGLO E. Coli have adopted new genes that allow for new functions that wild type E. Coli are incapable of, in addition to showing that the genes were transcripted and translated in S phase of mitosis, as daughter cells possess similar qualities, as shown by their ability to subsequently grow and divide. This In further examining the plates treated with the +pGLO solution,
[7] Klug, W., Cummings, M., Spencer, C., Palladino M. (2012) Concepts of Genetics: Tenth Edition. Pearson's Education, Inc.
Genetic transformation is a process that modifies bacteria, by introducing new genetic material. In our lab we introduced the pre-engineered pGLO into the E. Coli HB101 K-12 bacteria. This pGLO plasmid consists of the gene for the green fluorescent protein (GFP), the ampicillin resistance gene that inactivates the ampicillin in the LB media and the araC gene that indirectly controls the arabinose digestion enzymes [Fig 1].
Hypothesis: If a GFP gene is inserted into an E.coli cell, then the E.coli will glow in the dark.
In this experiment the bacteria E. Coli will be genetically transformed into a competent by going through a process called Heat shock. Heat shock is when you take a bacterial cell and have sudden increase in temperature which increases the permeability of the plasma membrane this causes the cell to take up the DNA from the surrounding medium. (Lab Manual) There are several other methods of genetic transformation but in this lab those will not matter. In this specific experiment pGLO will be the medium around the E. Coli. Genetic transformation is the active up take of foreign DNA in a bacterial cell. (PubMed) For genetic transformation to occur we have to have a medium that contains a different DNA than the thing we are trying to genetically transform. The medium in this experiment will be the plasmid pGLO. This pGLO plasmid is a vector, which transfers a gene from one organism to another. (Lab Manual) The plasmid contains the GFP (green fluorescent protein) gene which makes the bacteria glow in the presence of a sugar called arabinose. The pGLO also contains a gene for resistance to the antibiotic ampicillin. Ampicillin will be used to see if the bacteria lived because the ampicillin is an
All the bacteria on the petri dish took in the plasmid and are therefore alive. The gene did not glow because Arabinose was not present and they could not turn on the operon. LB/ Amp /Ara with pglo glowed because the arabinose turned on the operon that controlled glowing. The bacteria is now making arabinose, causing them to glow. Also, all the bacteria on the petri dish were resistant to Ampicillin. Some of the bacteria colonies that did not glow had metabolized, or used up, all the arabinose. Bacterial transformation has shown great promise and progress in medicine and agriculture. It helps with research of using Insulin to treat diabetics, creating, and inserting cell hosts. Botanists use bacterial transformation to experiment with plants to resist colder temperatures, playing with the ripening process, and their ability to form a resistance to pests. However, these are always contradicted with statements challenging the ethical and moral issues that have to be unravelled with much thought before the further use of bacterial transformation. This process has opened doors for a healthier future and new, easier ways to create life saving
This encyclopedia was extremely helpful. In not knowing all of the exact terms and basic knowledge of genetic engineering, it helped inform any reader of all this and more. The pages that had information on genetics and genetic engineering, had detailed definitions and descriptions for all the terms and ideas. Instead of focusing more towards the future of genetic engineering, it gave numerous facts about the technology and accomplishments of today. In addition to basic knowledge information, history, diagrams, and background information was provided. Including genetic testing, genes and their formation, and genetic background. The encyclopedia gave easy, organized, and accessible information to use.
The scientific process of genetic engineering is very complex and much more difficult than it would seem. First, an organism with the desired trait is located and selected. Cellular DNA is extracted from this organism to transplant the desired trait into the new organism. Gene cloning follows, with the locating and copying of the desired trait. The new gene(s), called a transgene is delivered into cells of the recipient organism, or trans...
Taylor, J., Loney, B. R., Bobadilla, L., Loacono, W.G., & McGue, M. (2003). Genetic and
Genetic Engineering is the deliberate alteration of an organism's genetic information (Lee 1). The outcome scientists refer to as successful entitles the living thing’s ability to produce new substances or perform new functions (Lee 1). In the early 1970’s, direct manipulation of the genetic material deoxyribonucleic acid (DNA) became possible and led to the rapid advancement of modern biotechnology (Lee 1).
Lemaux, P.G. (2006). Introduction to genetic modification. Agricultural Biotechnology in California Series, 8178. Retrieved from http://ucanr.org/freepubs/docs/8178.pdf