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Pglo Transformation Lab Report

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Transformation of Escherichia coli. Introduction The purpose of this lab is to genetically transform the bacteria Escherichia coli using plasmids. Transformation is the process of inserting recombinant DNA into cells (Hillis, 2019, concept 13.2). Bacterial transformation is commonly used to make insulin, and to produce disease resistant crops. The purpose of this transformation is to add the gene Green Fluorescent Protein (GFP) into bacteria. Transformation is achieved by transforming the plasmid pGLO into the bacteria. pGLO has been inserted with GFP, a gene that is resistant to the antibiotic ampicillin. GFP is a gene found commonly in the species Aequorea victoria, which is a bioluminescent jellyfish. In order to isolate GFP to insert it …show more content…

In this lab, antibiotic resistance is demonstrated when pGLO DNA is able to grow on the agar plates with ampicillin. Plasmids are able to incorporate genes into their DNA because of bacterial competence. Bacterial competence is the ability of bacteria to take in any stray DNA that they encounter. This ability allows the plasmid to integrate GFP and the gene for …show more content…

When the bacteria integrates GFP into its DNA, it allows the bacteria to glow in the dark. The ability of GFP bacteria to glow in the dark is controlled by an operon. An operon is a genetic regulatory system that can be found in bacteria (Britanica, 2018, “operons”). An operon includes an operator, which acts as a switch that turns genes off and on. Operons can be inducible or repressible. The GFP operon is inducible (Mcauley, 2021, pGLO). This means that in the presence of an inducer, the operon will produce GFP. The inducer that causes GFP to form is arabinose. Repressible operons are the opposite of this. When the repressor is present, it will turn off the formation of the product. Hypothesis If E coli bacteria is added to all four agar plates, then both of the agar plates labeled +pGLO and the -pGLO plate without ampicillin would grow. Variables The independent variables in this experiment is the addition of pGLO, ampicillin, and arabinose. The dependent variables in the experiment are whether or not bacterial growth occurs, or if the bacteria grow. The constants in the experiment are the size of the agar plate, the temperature in which the agar plates incubate, and the length of time the

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chinesis. A construct of R751::Tn4351 (the physical map of R751::Tn4351 and restriction sites are shown in fig. 7) was selected for introduction into F. chinesis to discover if the introduction and insertion of the vector R751 and the transposition of T4351 into the F. chinesis chromosome by a triparental mating occurred. One parent was E. coli GJ342 which carried a helper plasmid, the second parent was E. coli HB101 which contained R751::Tn4351 and the third parent was the F. chinesis target strain. 189 colonies were isolated on LB agar plates which in passage in fresh media were able to grow in 200µgml-1 erythromycin.

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Gram positive organisms are susceptible to antibiotics such as penicillin (Introduction to Penicillins, 2012). This is because, gram positive bacteria have a thick layer of peptidoglycan, a sugar and peptide coating that gives a cell its shape and helps it stay intact (Wiley, 2004). The original antibiotic, penicillin, and the likes are used to treat infections caused by bacteria that are gram positive. Based on the scope of the experiment and the materials needed, a budget estimate of $1,000.00 would be needed to buy reagents and materials for this experiment.

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For a plasmid to be useful as a recombinant DNA vector, it must have some essential features. What

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Hypothesis: If the pGLO plasmid is inserted into E. Coli bacteria, then the surviving cells with the arabinose will glow green under UV light as they will be genotypically identical and phenotypically different. Experimental Design: In this experiment, recombinant DNA technology was used to modify the genetic properties of an organism. First a plasmid was created which consisted of ori, araC, GFP, bla and pBAD promoter. The GFP is a jellyfish gene that codes for the production of green protein; bla breaks down ampicillin which then can be used to select only the bacteria containing the bla gene by placing ampicillin in the growth medium.

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To begin, during this lab experiment, genetic transformation was successfully carried out. After observing the agar plates, it was found that only the plate with ampicillin and no pGLO plasmid did not grow any of the E.coli bacteria. All three of the other plates grew the E.coli bacteria, however it grew differently in each plate. In the control plate where the pGLO plasmid, ampicillin, and arabinose were not present, the bacteria grew in the pattern that it was spread in originally. In the two other plates, bacteria grew in colonies that eventually joined together due to prolonged time in the incubator.

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The Mut section is expected to have no growth as mutants require the amino acids leucine and valine to grow which is not provided in the minimal medium. Results Figure 2. Testing of mutant mixed with DNA, mutant bacteria and DNA on LB medium Growth was observed on the Transformed (Trsf) section and the Mutant (Mut) section but not on the DNA section. Due to human errors, the photo of our experiment was lost, but we have obtained similar results as from group1.1 and their photo is presented.

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Though this is usually beneficial, transformation can lead to bacteria developing the ability to be resistant to antibiotics by having a mutant bacteria cell that has resistance, spreading that resistance to every bacterial cell it can find. Transforming single cell organisms is also much easier than transforming multi-cellular organisms because in order to affect a whole unicellular organism, you must transform every cell whereas if you want to transform a unicellular organism, you only have to transform one cell. Also, the reason why only one plate successfully glowed is because that plate was the only one where the pGLO plasmid and arabinose were present at the same

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It could have also been helpful to show a diagram of how the cell transfers the genetic information containing antibiotic resistance characteristics.

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If scientists can investigate ways to simply measure the activity of that expression plasmid, it would simplify this entire process and diminish the need for transactivation assays

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coli genomic library facilitated the selection for genes conferring the resistance through amplification. One plasmid from the library, plasmid pXV62, provided a high level of resistance for E. coli. Deletion and nucleotide sequence analyses of bacitracin resistance revealed that a single open reading frame, designated the bacA gene, sufficiently caused antibiotic resistance. The bacA gene mapped locus deduced amino acid sequence of the bacA encoded protein suggesting an extremely hydrophobic protein of 151 amino acids with 65 percent reflecting nonpolar amino acids. E. coli cells with plasmid pXV62 have increased isoprenol kinase activity suggesting the bacA gene may confer

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A secondary sample that could have been tested, Sample 6, could contain an environment with -pGLO, Luria Bertani broth, and arabinose. This sample’s results should further illustrate the results of the lab, as due to a lack of +pGLO the bacteria should not grow, even with the arabinose sugar as a food source. Furthermore, due to a lacking of ampicillin resistance, again the bacteria should not show any growth as the cells will be considered “not transformed” and will not survive the plating. The addition of more samples would help broaden the understanding of genetic transformation and further illustrate the main point of the

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The subsequent 25+ years of research experience have demonstrated that genetic engineering may be conducted in a safe manner when an appropriate risk assessment is performed and adequate safety measures are used. Recombinant DNA technology or genetic engineering was first used to clone DNA segments in bacterial hosts in order to overexpress specific gene products

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INTRODUCTION Today, societies live in a dynamic era, where lives are governed by scientific advancements and technologies. Science has made our lives very easy and new developments are being made on a daily basis. Science and research has unveiled the secrets of life on this planet and has opened an entirely new door to DNA and genetics. This branch of science has been termed as GENETIC ENGINEERING. (www.wikipedia.org/wiki/Genetic_engineering Date accessed: 31 Jan. 2016) Genetic engineering has provided the tools that can assist the scientists in adding new traits in the DNA of an organism.

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As advances in genetic engineering and technology rapidly increase, synthetic biologists have now contrived and manipulated the genetic code of the bacterium Escherichia coli in a study published by researchers at Harvard Medical School in Boston on August 18, 2016. Scientific American’s article, “Most Extensive Reengineering of an Organism’s Genetic Code Now Complete,” poses the onset of a very palpable now and conceivable feat - remaking and altering the genetic makeup of a living organism beyond what is naturally derived from nature, opening the contingency for major breakthroughs and progress in the scientific world. To explain, researchers and synthetic biologists have now reported the most paramount modification of a bacterial genome yet, attempting to find whether such changes can be applied to engineer organisms with properties such as resistance to viral infection, and ascertaining the effects of

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To transfer genes, scientists use yeast cells instead of bacteria because yeast cells are eukaryotic and they can translate genes from other eukaryotes. To transfer DNA, a vector is used in genetic engineering is a carrier. Bacteria contain vectors called plasmids, which are small circular pieces of DNA within the bacteria. An example in medicine would be diabetes. Before genetic engineering was common, doctor’s cures diabetes with animal insulin obtained from farm animals, but that used to cause allergic reactions in some patients.

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