Pglo Transformation Lab Report

918 Words2 Pages
bulb-icon

Recommended: Bacterial transformation molecular lab report

Abstract: Bacterial transformation involves the change of genetic composition of bacteria by altering its genetic identity. The pGLO plasmid was ingrained in the E. coli cell, which allows the modified E. coli cell to begin to code for the GFP protein gene and the beta lactamase gene (ampicillin resistance gene). After modifying the bacteria cell, the changes involved with the plasmid were tested on 4 plates, two plates containing the pGLO plasmid (+) were treated with LB nutrient media. One of the LB plates contained arabinose in it, which should fluoresce green under UV light. The other LB plate contained ampicillin. Two other plates which did not contain the pGLO plasmid (-) both had LB media growth, one plate just to show cell growth, …show more content…

Through transcription and translation, the DNA (plasmid) from one organism (jellyfish pGLO) can be expressed in another organism (E. coli). Plasmids are used for this experiment due to their small size, making it easier to enter the bacteria. Transformation of bacteria can happen by two different procedures, electro-puration, or electro-compentent. Electro-puration was the method used, it involves heat shocking the cells with calcium ions. This was the method used because it left the most amount of viable cells as compared to other methods. The plasmid pGLO contains the GFP gene which can only be activated while in the presence of arabinose. The plasmid also contains an ampicillin resistance gene, which explains why bacterial cells transformed with pGLO can survive when ampicillin is present. GFP is a protein that is found in jellyfish, and is known for emitting a green fluorescent light. In vitro, it is able to fluoresce this green color by releasing photons from being in an excited energy state. The energy was provided by a UV light at the end of this experiment. Jellyfish can fluoresce this green on their own because in vivo, GFP works with another protein that allows the GFP to fluoresce without being in an excited energy …show more content…

coli cells were grown at 37 degrees Celsius with calcium ions. These cells were then incubated in ice and placed into 4 polypropylene tubes. Each tube consisted of 25 microliters of E.coli cells. Two of the four were labeled with (+) while the other two were labeled (-). Positive was with samples that contained 100 pg/ microliter of pGLO, while negative was for the samples that did not. The 4 tubes were incubated in ice for 30 minutes, then heat shocked for 30 seconds at 42 degrees Celsius. The 4 tubes were then returned to the ice bath for 5 minutes. After the removal, .975 microliters of LB media was added to each tube at room temperature. The tubes were then shook at 225 RPM at 37 degrees Celsius for one hour. 300 microliters of cells were then extracted from the tubes and then placed in agar plates. The cells were spread across the plate and the incubated overnight at 37 degrees

Show More
Related

  • Ligation of EGFP into pET41a(+) vector transformed into E. coli cells

    3721 Words  | 8 Pages

    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.

    Read More

  • Identifying Two Unknown Species of Bacteria

    1333 Words  | 3 Pages

    The first day an unknown sample was assigned to each group of students. The first test applied was a gram stain to test for gram positive or gram-negative bacteria. The morphology of the two types of bacteria was viewed under the microscope and recorded. Then the sample was put on agar plates using the quadrant streak method for isolation. There were three agar plates; one was incubated at room temperature, the second at 30 degrees Celsius, and the third at 37 degrees Celsius. By placing each plate at a different temperature optimal growth temperature can be predicted for both species of bacteria.

    Read More

  • Biology: Arabidopsis Culture Cell and Transformation

    564 Words  | 2 Pages

    Transformation of T87 cells was done by culturing the cells in B5 medium supplemented with 1 μM 1-naphthaleneacetic acid (NAA) and 40 g L-1 sucrose. The cells were cultured for one day at 22°C with continuous illumination and shaking at 120g. Next, 10 μL of overnight cultured Agrobacterium transformed with respective vectors were added into the cell suspension and cultured for an additional two days. After co-cultivation, the cell suspension was washed thrice with 10 mL of JPL3 medium supplemented with Carbencilin (250 μg mL-1) by centrifuging at 100g for two minutes. Finally, the cells were resuspended and spread onto JPL3 selection agar plate supplemented with Carbencilin (250 μg mL-1), Kanamycin (30 μg mL-1) an...

    Read More

  • E. Coli Lab Report

    1693 Words  | 4 Pages

    Hypothesis: If a GFP gene is inserted into an E.coli cell, then the E.coli will glow in the dark.

    Read More

  • The Pros And Cons Of A Recombinant Plasmid

    1633 Words  | 4 Pages

    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).

    Read More

  • Pglo Transformation Lab Report

    1121 Words  | 3 Pages

    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,

    Read More

  • Green Fluorescent Protein Lab Report

    433 Words  | 1 Pages

    In the “Transformation with Green Fluorescent Protein (GFP)” lab there were two samples of bacteria. One of which had been exposed to the GFP (+DNA) and one that was not (-DNA). The two DNA samples were subjected to a heat shock process. Both tubes incubate on ice for 10 minutes and are then are placed in a 42* C water bath for 90 seconds. Immediately after that transfer the tubes back to the ice for 2 minutes after that the tubes are put into a 37* C water bath. The heat shock process facilitates the entry of the plasmid into the bacteria. The plasmid carries the GFP and the antibiotic resistance genes. The samples were placed on one of four petri dishes labeled; -DNA, -DNA/AMP, +DNA/AMP, and +DNA/+AMP/+IPTG. AMP or ampicillin is an antibiotic

    Read More

  • Reproductive and Therapeutic Cloning: Dolly The Sheep

    1462 Words  | 3 Pages

    New research techniques have made it possible to engineer and explore differences in the sets of chromosomes in organisms. This has been a technological revolution during the last decade. Allowing scientists to be able to explore DNA to a new extent. During the process of this research it has come apparent that foreign DNA inserted into self-replicating genetic elements such as bacteria plasmids can replicate. This breakthrough has also shown that the plasmids that have been used can also be used to change the genetic constitution of other organisms (1).

    Read More

  • Pglo Transformation Essay

    483 Words  | 1 Pages

    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

    Read More

  • Pglo Transformation Lab Report

    1183 Words  | 3 Pages

    coli HB101 strain with the pGLO plasmid. The HB101 strain is a wild strain of E. coli that lacks plasmids and it is useful for the study of bacterial transformation [10]. The pGLO plasmid is a genetically modified plasmid containing the bla gene, the gfp gene, and the arabinose repressor (araC) gene [6]. The bla gene encodes the β-lactamase enzyme, which binds to the β-lactam ring of penicillin and its derivatives providing antibiotic resistance to bacteria. The gfp gene is a gene derived from the jellyfish Aequorea victoria and encodes the green fluorescent protein, a protein that is green under UV light. The araC gene encodes a repressor protein that activates the gfp gene when arabinose is present in the medium used. In the pGLO plasmid the genes that permit arabinose catabolism have been substituted by the gfp gene [1]. The medium used was Lysogeny (or Luria) broth, a nutrient rich medium that permits faster E. coli growth [12]. The E. coli were inoculated in a CaCl2 solution, which aids in the binding of plasmids to the lipopolysaccharides (LPS) membranes of E. coli by attracting the negatively charged backbone of the plasmid and the negatively charged core of the LPS with the Ca2+ ions. A heat shock process was also used to facilitate the uptake of the pGLO into the E. coli cells

    Read More

  • Ligation Lab Report

    699 Words  | 2 Pages

    Once plasmids are digested and confirmed the next phase of making recombinant DNA is to successfully ligate the fragments. Ligation is the process of sealing sticky ends of plasmids fragments that contain the ampicillin resistant gene or the kanamycin resistant gene. Refer back to Figure 3 for a visual representation of ligation in action. Once ligase is added to the sample, a confirmation test must be done in order to prove ligation successfully occurred. One must remember that ligated plasmids will be enormous. The reasoning being is that, the fragments that were cut by the restriction enzyme where big. Therefore, these

    Read More

  • Bacterial Transformation Lab Report

    688 Words  | 2 Pages

    In this experiment, the bacteria, E Coli, was transformed with the Green Fluorescent Protein (GFP). To start, the bacteria was grown, harvested, and added to a tube with -DNA. Calcium chloride was also added to the tube to allow the cells to take up the DNA and become transformed. Half of this mixture was then placed into a tube with +DNA, which contains the Green Fluorescent Protein, and both were placed in an ice bath for 10 minutes. When time was up, the tubes were placed in 42℃ water for 90 seconds. Next, the tubes were put back in ice water for 2 minutes. Then recovery broth was added to give the bacteria more fluid to grow in and provide nutrients, and the tubes were placed into 37℃ water for 15 minutes. The purpose of placing the tubes in warm and cold water was to heat shock the mixture so the cell membranes would become permeable and the DNA could enter the cells. Once the heat shocking was complete, the -DNA and +DNA were placed on 4 petri dishes with agar. The -DNA was placed on one dish with only agar, and another dish with

    Read More

  • Lederberg And Tatum: A Genetic Study

    595 Words  | 2 Pages

    There are two type of transduction which is generalized and specialized transduction. Generalized transduction is where phages can carry any host gene while specialized transduction only specific host gene can be transferred by transducing phage (3,4). This process was discovered by Lederberg and Zinder in 1952 while they were studying the process of conjugation in Salmonella typhimurium using same method that was used in determination of E coli conjugation method. Two different strains were used which phe− trp− tyr− in one strain and the other was met− his− (3,4). There are no wild-type cell observed after the strains was plated individually, however when the two strain were combined wild-type strain become visible at about 1 in 105 frequency (3). Thus they conclude that the situation is similar to the recombination of E. coli and therefore E.coli also used transduction as their mating process. Interestingly, in 1970, Morton and Akiko Higa confirmed that transformation can also occur in E.coli using artificial method of

    Read More

  • Transformation Vs Transfection

    1703 Words  | 4 Pages

    2. Give 2 reasons why one would transform a cell with a plasmid? Give 2 reasons why one would transfect cells. (8 points)

    Read More

  • Impact of Refrigeration on Bacterial Growth in Mayonnaise

    1659 Words  | 4 Pages

    Growth is characteristic feature of life; this simply means the regulated and coordinated enlargement in biological mass over time. Bacterial growth involves division of one cell of the bacteria into two cells in a process known as binary fission. Specific conditions are required for the survival of bacteria. Most bacteria require environments that are moderate, suitable and assure optimum growth. The common conditions that need to be set include temperature, oxygen concentration, nutrients, salt concentration and pH. Different bacterial species have to be well examined to allow bacterial growth in an artificial lab condition. Optimum oxygen levels and aeration

    Read More

More about Pglo Transformation Lab Report

Open Document