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
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3. Weedman D. 2013. Life 102 Attributes of Living Systems Laboratory Manual. 7th edition.
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Ligation of EGFP into pET41a(+) vector transformed into E. coli cells followed by PCR amplification of extracted DNA plasmid for success evaluation along with gel electrophoresis at each step.
The ligation was expected to make four combinations. The original pBK-CMV and CIH-1 fragments would region to make a non-recombinant pBK-CMV/CIH-1 plasmid. The original pUC19 fragments would rejoin to make a non-recombinant pUC19 plasmid. The larger fragment of pBK-CMV and the small 27bp fragment of pUC19 or the desired recombinant vector, CIH-1 fragment and the larger 2659bp pUC19 fragment. As pBK-CMV does not contain the ampicillin gene then transformed Ecoli containing these would not to survive on the Agar leaving only pUC19 recombinants and non-recombinants.
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.
The purpose of our experiment was to test whether or not the Wisconsin Fast Plants, or Brassica rapa, followed the Mendelian genetics and its law of inheritance. First, after we crossed the heterozygous F1 generation, we created an F2 generation which we used to analyze. After analyzing our results, we conducted a chi-square test for for both the F1 and F2 generations to test their “goodness of fit”. For the F1 generation we calculated an x2 value of 6.97, which was greater than the value on the chi-square table at a p-value of 0.05 and 1 degree of freedom (6.97 > 3.84). This meant that we had to reject our hypothesis that stated there would be no difference between the observed and expected values. This showed us that the F1
Another weakness being not effectively utilizing the heat shock method of genetic transformation, the transformation solution of calcium chloride could not have gotten cold enough when sitting on the ice bath for certain periods of time. This lowers the effectiveness of heat shock genetic transformation as the plasma membrane of the cells do not become permeable enough to be able to take up the foreign DNA that it is being exposed
Examining the Crosses Between Drosophila Fruit Flies Introduction The major topic of this experiment was to examine two different crosses between Drosophila fruit flies and to determine how many flies of each phenotype were produced. Phenotype refers to an individual’s appearance, where as genotype refers to an individual’s genes. The basic law of genetics that was examined in this lab was formulated by a man often times called the “father of genetics,” Gregor Mendel. He determined that individuals have two alternate forms of a gene, referred to as two alleles.
-Reilly Philip. Is It In Your Genes. Cold Spring Harbor Laboratory Press. 2004: 223-228. Print
The two modes of analysis that will be used to identify an unknown insert piece of DNA would be plating the transformation cells onto LA plates that have either ampicillin or chloramphenicol and PCR. We will use the PCR thermocycler to denature the restriction enzymes that were specifically used to assimilate the vector DNA. It is important to use the PCR thermocycler because denaturation of the restriction enzyme will prevent the restriction enzyme from cutting the vector DNA, after the insert DNA has assimilated to the vector DNA. After the addition of specific primers that complement the base pair to its corresponding target strand, PCR will be used. Subsequently, Taq polymerase will be used to determine whether the insert DNA has been properly assimilated to the vector DNA. Within this specific situation, the target strand will be the insert DNA. After we let the PCR thermocycler run for approximately 2 ½ hours, we will then put our PCR products in the gel and run the gel to completion. After the gel has run to completion, we will then take a photograph of the gel using the UV transilluminator with the assistance of our TA. If the insert DNA was properly assimilated to the vector DNA, then our corresponding gel photo would have one band. After the cells have been transformed, we would g...
Lewis, Ricki, (2014), Human Genetics, 11th Edition, Chapter 15 Changing Allele Frequencies, pp 293. [VitalSource Bookshelf Online]. Retrieved from
In order to figure out the genes responsible, there are several other factors that must be determined. These factors include the number of genes involved, if each gene is x-linked or autosomal, if the mutant or wild-type allele for each is dominant, and if genes are linked or on different chromosomes. Proposed crosses include reciprocal crosses between the pure-breeding mutants of strains A and B with the wild-type will help determine if the genes or sex-linked or autosomal, in addition to which alleles are dominant (8). Another proposed cross includes complementation crosses between pure-breading mutants from strains A and B to determine if one or two genes are involved (8). Furthermore, testcrosses between F1 progeny and pure-breeding recessive mutants from strains A and B, which will help determine if genes are linked on the chromosome or if they assort independently (8). These proposed crosses are shown in the attached
The idea of the project was to experiment breeding Drosophila Melanogaster (fruit fly) to figure out if certain genes of that species were sex linked or not (autosomal). A mono-hybrid cross and di-hybrid cross was performed. For the mono-hybrid cross, white eyed female and red eyed male were placed in one vial for them to reproduce. For the di-hybrid cross, red eyed and normal winged flies and sepia eyed and vestigial winged flies were placed in their vial to reproduce. In the mono-hybrid cross the results expected were within a 1:1:1:1 ratio. Expected results similar to the expected desired null hypothesis proposed with what the F1 parental generation breeds. The potential results would have had to have been within the ratios of 9:3:3:1. The results were clear and allowed the null hypothesis to be correct. The white eyed gene in the fruit flies is sex linked. Sepia eyes and vestigial wings are not sex linked and are examples of independent assortment.
2). As a result, this scientific experiment changed the relationship of humankind and nature by foreseeing the modification of DNA of bacteria, yeast, plants, and animals to discover new medicines and to provide solutions for inherited diseases (Le Vine, 1999, p. 2).
12) Line 242, 2 or 3 alleles > 2 or 3 copies? 13) Table 2. It may be useful to have a likelihood ratio for each model. 14) Line 267, fenotype -> phenotype? 15) The introduction is human/livestock orientated, but the remainder of the paper is presented in livestock terminology. Some discussion about the relevance of the results to human populations would be useful.
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