Compare this answer with what you observed when the light was shined onto the pGREEN plasmid. We poured 16 LB/amp plates. The aim of this experiment was to transform the bacteria E. coli with a recombinant pGLO plasmid that has the GFP gene next to the arabinose operon promoter. Bacillus subtilis was used for positive control and Escherichia coli for negative control for endospore, Aseptic technique was initiated at the beginning of this experiment by cleaning the work surface with disinfected wipes. Figure out how you would calculate transformation efficiency (i.e. Activity 4: Transformation of E. coli using green fluorescent protein We will use aspects of aseptic technique during our bacterial transformation labs to prevent our plates and bacterial samples from becoming contaminated. The labels include our group number, the date, if it was -plasmid with ampicillin, kan and nothing. May 31, 2023. This results in a brilliant green glow when the bacteria are viewed under a UV light source. Then observe whether or not the E.Coli strain would take up these genes and become fluorescent. This means you should always open sterile equipment carefully, never leave it sitting open on your lab bench, and never set it down on a surface that is not sterile (for example, on the lab bench or on your lab notebook). Wash hands before leaving lab. Ampicillin is an antibiotic and works by preventing E.coli from constructing cell walls, thereby killing the bacteria. Only one plate has transformants. number of colonies/ microliter of plasmid used). Retrieved February, 2016 from University, http://www.genome.ou.edu/protocol_book/protocol_adxF.html. Wipe down lab bench with bleach solution at the end of lab. Hosted by Michael Barbaro. The results showed that the LB/amp/araC +pGLO produce a lot of colony and most, The cells that undergo transformation can activate the GFP protein using a sugar called arabinose that is added to the cells' growth medium ("pGLO Transformation,"). Although that one plate was inconclusive our other plates did go as should. The same happened with the other three petri dishes except they were labeled with a +plasmid rather than -plasmid. 6) From the first and second assays, you could expect there to be more resistance in the second assay, due to the extended period of time the bacteria had to manipulate and take in the given plasmids. One gene codes for a green fluorescent protein (GFP) and the other codes for ampicillin resistance. That could have been due to the lack of precision while streaking, or perhaps there was no bacteria on our streaker to begin with. The mobilization of pSS-2 from onestrain of E. coli, An endospore is a dormant of a bacterial cell. Eschenchia coil is the full genus and species name. It is a non-reproductive structure that ensures survival of a bacterium through stressful environmental conditions. Changes in number of genes or chromosomes, 52. We then removed the glass beads by quickly dumping them into a bucket filled with bleach water. Using an antibiotic in the nutrient plate and an antibiotic resistance gene in the plasmid accomplishes our two goals of giving an advantage to cells that have a plasmid so the plasmid is retained and of having a marker so we know our cells contain new DNA. What specific observation led to this. A mass of cells was then transferred into the - tube and suspended multiple times before being placed back within the ice. The second plate of bacteria was grown with the presence of LB broth, ampicillin, arabinose sugar (ara), and 22 colonies were observed. Students who have allergies to antibiotics such as penicilllin, ampicillin, kanamycin or tetracycine should not participate in this experiment. The pGLO plasmid that is being transformed into these cells contains genes that can give colonies of bacteria the ability of antibiotic resistance and a green fluorescent glow. Two of the harmless cells together made a harmful impact. Lab report on the transformation of E. coli using pGLO plasmid DNA. When ready, have the students come up to you one at a time and dispense the plasmid DNA directly into the tubes they have labeled with a + to indicate that these tubes have the plasmid added. in fact kill the mice, they then concluded that the nonvirulent strain had transformed into a virulent strain, through the passing of genetic material. This concerns a selective medium that increases the initiation of endospore production. There are a lot of factors that could have attributed to the inconclusive growth within the regular LB plate, had it gone correctly there should have been no complications with the growth and the E.coli would have grown normally given that it was ideal growing conditions for such bacteria. We uncapped the vial cap of the slant culture of. The genes were successfully inserted, yielding a ampicillin resistant, fluorescent E. Coli strand. We saw a great amount of growth on a petri dish that shouldve had none and none of a petri dish that shouldve had a great amount of growth. Which one is it? Transformation of Escherichia Coli (E. Coli) - UK Essays PDF Transformation of the bacterium E - APS Home We then proceeded to place both tubes on ice. Plates 3 and 4 were the control plates. E.coli colonies are generally viewed as a collection of bacterium with non-cell autonomous signaling rather . Allow materials to stand in bleach solution for 20 minutes or more. [See citations for resource on our methods], Results/Data: Include pictures, data tables, / LB Ampicillin Plates / LB Kanamycin Plates / LB Plates /. Reece, J. The purpose of this lab was to insert genes that would make E. coli resistant to ampicillin and to glow. We first used a bottle of LB agar to pour 18 LB plates. All plates did not have a lawn or colonies, Both positive and negative KAN plates were clean, the other had lawns, Colonies on the ampicillin plates, lawns on the LB plates and clean plates on KAN. Having a way to measure transformation efficiency helps in discussing results or in comparing transformations that were not done at the same time. Rapid Colony Transformation of E -Coli with Plasmid DNA Lab Report We used another bottle to pour 2 LB plates and 16 LB/amp plates. It is important to develop new antibiotics that the bacteria is not yet resistant to. The film you may see is most likely condensation. There is no consistent report as to how much heat . When the inoculating loop was placed in the tube it was spun around to insure we dislodge the cell mass. The S strand evidently killed the mice and the R strand did not. In this experiment plasmids, are inserted into a host E. coli cell. Retrieved February, 2016 from University. Binary Fission: Prokaryotic Cell Division, 38. Understand that DNA can be transferred to another organism and therefore change the observable characteristics of that organism. By the end of the lab activity and analysis you will understand one method of biotechnology (transformation) that scientists use to genetically modify organisms. There was no fluorescence visible when the UV light was shined on the plasmid in the tube because it is not the plasmid that reacts to the light but the protein produced when the gene in the plasmid is translated. pGLO Transformation - Brian McCauley The goal of genetic transformation in this experiment is for the bacteria Escherichia coli to obtain an antibiotic resistance to ampicillin, which can be physically observed when the bacteria expresses the reporter gene Green Fluorescent Protein (GFP) because the transformed bacteria will glow green under UV light when in the presence of arabinose. The LB w/ Amp (-) plate, however, was predicted to not have any signs of bacterial growth as the non-transformed bacteria wouldnt have undergone transformation to become resistant to ampicillin - a vital trait for the bacteria to have grown successfully on the ampicillin plates. Holding the tube up to a light verified that the cell mass did indeed fall off into the tube. We used a sterile transfer pipette to add 150 L of ice-cold calcium chloride to both tubes, then we placed each on ice. Carroll, Chowdhury, Fox, Rodriguez, Thomas. Levels of Organization of Living Things, 10. Bacteria, which often grow in the same environment as molds and fungi, evolved to make proteins that inactivate the toxins produced by these other organisms. Tn 4351 was originally isolated from bacteroides fragilis [30] . This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After this is done, you put both tubes in ice and then put bacteria in both tubes. Remove pGREEN plasmid DNA from freezer just before class begins. Our overall outcome was achieved, as we got to see an actual transformation of bacteria occur, thus proving the theory of McCarthy and MacLeod (GNN, n.d.) (. During this lab, you will be creating your own genetically modified organism by adding the green fluorescent protein gene to E. coli bacteria. We now know that the colony with the ampicillin resistance had the greatest growth. Immediately after the 9- second water bath we returned the tubes to the ice for another minute. For more audio journalism and storytelling, download New York Times Audio, a new iOS app available for news subscribers. We then dragged the inoculating loop across an area of E. coli culture where there was obvious growth. Much like Griffiths experiment, we conducted our own transformation lab using E.coli samples and DNA for the genetic expression of fluorescence and ampicillin resistance. Our world is composed of many bacterias that can either help or destroy us. Bacterial Transformation Lab - MHCC Biology 112: Biology for Health In a typical transformation, billions of bacteria are treated to make them competent and then exposed to plasmid DNA. to obtain transformation of E. coli. We immersed the loopful of plasmid DNA into the cell suspension to mix the DNA with the cells. Arabinose is a type of sugar that can be added to the plates when they are poured. Tn 4351carries two antibiotic resistance gene. Immerse all disposable pipets, tubes, and loops that have come in contact with bacteria in 10% bleach solution for at least 20 minutes before draining, rinsing and disposing of in the trash. (Introduction to Bacteria), contained in a single, circular chain of Deoxyribonucleic acid that is not enclosed with a nuc, information required for the growth, development, and reproduction of an or. LAB REPORT NAME: ARTIFICIAL TRANSFORMATION OF E. COLI SECTION: 1. Bacterial transformation is a really easy way to transform due to the fact that it is single- cell. Clean up: Place used loops etc in the bacterial waste container. Antibiotic resistance is passed during replication. E. Coli Transformation Lab Report - 506 Words - Internet Public Library The other gene codes for resistance in tetracycline and is expressed in aerobically grpwn E. coli, but not in anaerobically grpwn E. coli or in bacteroides. Resistance to an antibiotic is known as a selectable marker because we can select for cells that contain it. Send any friend a story As a subscriber, you have 10 gift articles to give . Lab report #2 - Bacterial Transformation: pGlo Lab Report - Studocu 1.13: Transformation - Biology LibreTexts Supporters of GMOs believe that the benefits such as increasing the available food supply, increasing the nutritional content of foods, and the production of medicine outweigh the risks. Following sterilization, pour off liquid, put dishes in plastic bag, and throw away in regular trash. From the growth on the NSM, I smeared it aseptically to a wet slide. On the other hand, the plate containing pGLO and no amp added (LB), will grow colonies across the surface of the agar, Strains of E. coil are important in the digestive tract, but others may cause problems in urinary and intestinal tracts. We predicted that the non-transformed bacteria would grow successfully on the LB (-) plate and the transformed bacteria would grow successfully on the LB w/ Amp (+) and LB (+) plate. Opponents of the technology claim that GMOs pose a health risk to humans as well as potential environmental risks. INTRODUCTION: Transformation is a process whereby the genetic materials of a cell are altered by introducing DNA (exogenous DNA) from the surrounding environment through the cell membrane of the organism. PDF Rapid Colony Transformation of E. coli with Plasmid DNA Bacterial Transformation and Competent Cells-A Brief - US 2) The live bacteria was mixed in with the non-living bacteria, thus prompting the live bacteria to absorb the dead. Bacteria that are able to easily take up DNA from the environment are called "competent". The source of the GFP gene is the bioluminescent jellyfish Aequorea victoria. We should have kept the plates in a more cleaner environment. The diagram of a bacterial cell offers readers a comparison of bacterial chromosomes with that of plasmids. Immediately after, we suspended the cells by pipetting the liquid in and out with a sterile transfer pipette, then returned the +plasmid tube to the ice. As we held them in the water we spun them around or agitated them. Have available enough squirt bottles with 10% bleach for every group to access. Remove LB and LB/Amp plates from refrigerator and let warm to room temperature. Additionally, we were surprised to find that our - plasmid LB Ampicillin plate had a lawn while our + plasmid LB ampicillin plate (which was supposed to have a lawn) did not. (Take DNA from environment and integrate it into their own chromosome), the transfer of DNA mediated by conjugal plasmids or conjugal transposons; requires cell to cell contact but can occur between distantly related bacteria or even bacteria and eukaryotic cells; can transfer long fragments of DNA), (phage doesnt replicate immediately-dormant). Hence, the natural function of a plasmid is to transfer genetic information vital to the survival of the bacteria. Electrophoresis Student Input. It could have also been helpful to show a diagram of how the cell transfers the genetic information containing antibiotic resistance characteristics. Typically, fewer than 1 in 1000 bacteria will acquire the plasmid (Figure 5). Collect tips and tubes in a plastic bag and discard in the normal garbage. We then used the inoculating tube to streak E. coli onto the agar plates, using proper technique, quickly replacing the lid. After both tubes were in the ice we placed colonies of E. coli into the + tube with an inoculating loop. Heat shock the bacteria by rapidly heating and then cooling them. There seems to be an abnormally high amounts of bacteria growth in the ampicillin plate. Transformation Lab Report - Labnotebook Although the E. coli strain used in these experiments has been rendered non-pathogenic, it is important to teach the students good sterile technique and safe disposal of bacteria. Many colonies were found where the plate was streaked with the E. coli, The plate had a couple colonies of transformed bacteria. LAB REPORT NAME: ARTIFICIAL TRANSFORMATION OF E. COLI - Chegg The only way this transformation can take place is if the bacteria is competent and is grown to the right stage in which they are dividing the most. This plasmid contains several important pieces: Bacteria that are transformed with this plasmid will have two new traits: they will fluoresce green under UV light and they will be resistant to the antibiotic ampicillin. The first protocol for artificial transformation of E. coli was published by Mandel and Higa in 1970 [3]. In this process, the bacteria probably took in DNA from the dead cells, therefore transforming them into deadly cells. Heat Shock: The cell +DNA suspension is briefly incubated at 42C and then returned to 0C. The bacteria were not transformed and no growth was present in either. (PDF) Plasmid DNA transformation in Escherichia Coli - ResearchGate This can be beneficial to our final product as we learned about the nature of bacteria. The results from these three experiments are described as a, b, and c. Something has gone wrong with each of these transformations. Then begin to stir the petri dishes for 2 and a half minutes. PDF Big Genetics and Information Transfer 3 - College Board