...E.coli Transformation Brooke Rowlett November 26, 2013 BioL 230 Lab Introduction In this experiment we attempted to transform e.coli using a green fluorescent protein plasmid. This green fluorescent protein is naturally found within the bioluminescent jellyfish. The protein can be expressed in both prokaryotic and eukaryotic cells. Normally whenever bacterial cells contain this protein are exposed to long wave UV radiation, they emit a green light. Bacterial cells acquire the glowing ability through the transformation of the bacteria by a plasmid containing the GFP gene. Transformation is the process in which bacteria take up exogenous DNA to acquire to traits (Witucki). The GFP would be our evidence to see whether or not the transformation was successful. Within this experiment E.coli was supposed to be transformed to have the ability of antibiotic resistance. In the experiment E.coli was first transferred into both DNA- and DNA+ microcentrifuge tubes, and then manipulated into competency so that the plasmid can enter into the cells. E.coli is not naturally competent and therefore we tried to manipulate it by exposing it to abrupt heat and cold treatments and through the treatment of the metal cations of chloride salts. The samples were then transferred to Ampicillin positive and negative plates to test for growth. After incubation, if there was any growth on the plates, the plate was placed under a UV light to look for the green light from the protein. We hypothesized...
Words: 1047 - Pages: 5
...The Transformation of Escherichia coli With pGalTM OBJECTIVE: To develop an understanding of bacterial transformation by plasmid DNA, and determine the transformation efficiency of a bacterial sample. INTRODUCTION: Transformation is an important biological mechanism for generating genetic diversity in the natural world. It is also one of the most used tools used in modern biotechnology. It has allowed for a lot of genetic engineering advances. Scientists can design and build DNA containing genes they want and then transfer these genes into bacterial cells that they can get to express those genes. This is a very basic technique that is used on a daily basis in a molecular biological laboratory. This is based on the natural function of a plasmid: to transfer genetic information vital to the survival of the bacteria. Transformation is an example of horizontal gene transfer. There are three mechanisms for horizontal gene transfer in bacteria. Conjugation is the transfer of DNA from one cell to another through conjugation pili. Transduction is the transfer of genes between bacterial cells using a bacteriophage (a type of virus). Transformation is the ability of cells to take up freely floating DNA found in the environment. Bacterial cells that are able to take up free-floating DNA from the environment are called competent cells. Bacteria are not always competent. When growth conditions are optimal, most bacteria cannot uptake DNA. In most cases, in response to some stimuli...
Words: 863 - Pages: 4
...Greene Gardens Question 1: If you were Sam Greene, how would you respond to the first reports of contaminated spinach? Be specific as to the actions you would take. The first step would take is calling the Food and Drug Administration and I will ask them the follow questions: - Where did the contaminated spinach come from? From which farm? Which processor? Which area/ region? - Arrange a meeting with stakeholders about the E. coli outbreak. This meeting will be arranged for worried stakeholders that don’t know If there companies supply contaminated spinach. Question 2: What factors would you consider and how would you make your decision? To whom are your primary obligations? My second action would be considering contacting an independent consultant for food safety like Fresh Express. And ask for an audit at the Greene Garden company and ask specific ally if they would check the spinach. Question 3. How would you respond to this new information? Be specific as to the actions you would take. I would respond to this information by contacting GRT Salads. Ask if they still want your spinach. Contact owners of the surrounding areas of the harvest fields. 4. What actions would you take regarding the spinach products you market through Tossed Fresh? I would call Tossed Fresh for any information about the E. coli. Check if the transport of the spinach is contaminated. Is the transport proper? And ask Tossed Fresh if their packing machine is contaminated with E. coli...
Words: 1444 - Pages: 6
...E.coli is a bacteria that is often associated with cows. Recent studies have shown that outbreaks of E.coli are the result of diets in cows containing corn and starches. (www.ansc.purdue.edu). In the past couple of years cattle diets have been changed to be more cost effective and now contain mostly corn and corn-byproducts. This change in diet has led to many severe effects in the health of the animals including in increase of E.coli. This E.coli strain is the third most deadly bacterial toxin, coming in behind the pathogens that cause tetanus and botulism. So when not handled properly handled E.coli can cause serious health problems or even death in the people that eat beef and other meats. E. coli contamination is responsible for more than...
Words: 256 - Pages: 2
...evolution. Are we really descendents of apes? From a scientific perspective this is conceivable. The outbreak of Malaria is tolerated in most countries, but in Africa it is a widespread pandemic. This is an example of genetic variation. I believe 100 percent of us are genetic. We are created through genetic variation and the inheritance of variation. Natural selection is how we can cope with environmental changes. This is why certain individuals can deal with extreme temperatures better than others. The ability for each individual to go longer without water during times of extreme heat is an example. The first benefit of E. coli is that its presence reduces the chance of pathogens from colonizing in the intestines and causing illness. E.coli lives in our intestines and they produce important vitamins such as vitamin K and B complex vitamins. Vitamin K aids in the absorption of calcium into our bones and is necessary for effective blood clotting. Vitamin...
Words: 535 - Pages: 3
...is possible that the bacteria produces some acidic products, but not enough to overwhelm the phosphate buffer in the broth to turn it red. D. What metabolic end product does the VP test for? The end product we are testing for in the VP test is acetoin or acetyl methyl carbinol. I tested E.coli and S. epidermidis by preparing two tubes with incubated broth of each organism and added 12 drops of Barrit’s A Reagent to each tube. After mixing gently, I added 4 drops of Barrit’s Reagent B to each tube, shook gently and allowed to stand for 30 minutes. E. Why do you need to be careful not to jostle the VP tube while waiting for the results to show? It is important not to move or shake the tubes because the Reagent A and B cause the acetoin to produce a maroon band that rises to the top of the broth indicating a positive VP result. If the tube was shaken it would stir the band into the broth and appear to be a negative result. F. Which of the organisms, if any, fermented glucose? E. coli and S.epidermidis both fermented glucose. The broth in both tubes turned red, indicating a pH at or below 4.4. G. Which of the organisms, if any, produced measurable acidic byproducts? E.coli and s. epidermidis both produced measurable acidic byproducts. H. What is the cellular role of catalase? The role of...
Words: 453 - Pages: 2
...The objectives of the experiments performed in the past three weeks were to induce mutagenesis in the E.coli K-12 strain W3110 through short ultra violet light (UV). This particular strain of E.coli lacked photolyase (phr) and the uvr system, which are non-mutagenic repair mechanisms. A survival curve was constructed based off various irradiation times and number of viable cells obtained. Mutant strains were then isolated and purified on MacConkey (MAC) plates to rule out false positives. During week 7, the generation time of the potential Lac+ and lac- strands of E.coli were also determined. The lac operon was also induced via IPTG to determine if β-galactosidase induction is affected by mutations. During week 8, we determined if β-galactosidase...
Words: 1276 - Pages: 6
...penetrate the outer membrane of Gram- bacteria. It inhibits bacteria from making cell walls and leads to cell lysis and is a broad spectrum drug. All bacteria that we tested, Gram -and Gram + were resistant to this drug. E.Coli was resistant to this drug, its Zone of Inhibition was 0mm. S. Aureus was resistant to this drug, its Zone of Inhibition was 8mm. P.Aeruginosa was resistant to this bacteria, its Zone of Inhibition was 0mm. S.marcescens was resistant to this drug, its Zone of Inhibition was 0mm. 3. Cephalothin’s mode of action is to prevent cell wall synthesis and is narrow spectrum. E.coli was resistant to this drug. Its Zone of Inhibition was 11mm. S. aureus was sensitive to this drug. Its Zone of Inhibition was 34mm. P.aeruginosa was resistant to this drug. Its Zone of Inhibition was 0mm. S. marcescens was resistant to this drug. Its Zone of Inhibition was 0mm. Gram + bacteria: S.aureus was sensitive to this drug. Gram – bacteria: E.coli, P.aeruginosa, and S.marcescens were resistant to this drug. 4. Erythromycin’s mode of action is to inhibit protein synthesis by binding to 50s ribosomal subunits. This drug can be bactericidal or bacteriostatic depending on organism and drug concentration and is narrow spectrum. Gram - E.coli was resistant to this drug, its Zone of Inhibition was 8mm. Gram + S.aureus was sensitive to this drug, its Zone of Inhibition was 24mm. Gram - P.aeruginosa was resistant to...
Words: 906 - Pages: 4
...Assignment 1 for MCB 3020L Spring 2014 (due 2/24 or 2/26) Instructions: * Your answers have to be typed and double spaced. * Put the date, your name and your TA’s name on the cover page of this assignment. * “Give a detailed explanation” means don’t just give a one sentence answer. * Don’t forget to include your calculations and references! Late assignments are not accepted for grading. Questions: 1) Assume that you measure the OD of two E.coli cultures: culture A and culture B. The spectrophotometer gives you a reading of 2.2 for culture A and 0.02 for culture B. What would you do to determine the concentration of culture A and B and why? Give a detailed explanation! For culture A the OD reading is 2.2. This must never exceed 1, so a dilution is required. For culture B the OD reading is 0.2. You would calculate the reading by the factor 1x10^8 cells/mol of E.Coli 2) The OD of an E.coli culture is 0.46. You transfer 60µl of this culture into 5.94ml of medium. You continue this same dilution one more time. You transfer 25 µl of your last dilution onto an LA plate. After 24 hours of incubation, you observe 12 colonies on this last plate. A) Did you expect this result? If not, name at least three reasons for this result giving a detailed explanation for each reason. B) What would you do to prove your theory and why? A) 3) A friend of yours asks...
Words: 605 - Pages: 3
...ASSIGNMENT 1: PROPERTIES OF DNA/RNA Introduction: The polymerase chain reaction is an innovative technology, which amplifies a single piece of DNA across several orders of magnitude. The end result is the creation of thousands to millions of copies of a particular DNA sequence. PCR is closely patterned after the natural principle of DNA replication. It is a three-step process, referred to as a cycle, that is repeated a specified number of times. One PCR cycle consists of the following steps: * Denaturation * Annealing * Extension This process takes place in a thermal cycles, usually between 30 and 40 cycles. In the initial step, heat (usually hotter than 90 degrees Celsius) separates double-stranded DNA into two single strands. This process is called "denaturation." Denaturation is possible because the hydrogen bonds linking the bases to one another are weak. The hydrogen bonds break at high temperatures, whereas the bonds between deoxyribose and phosphates, which are stronger covalent bonds, remain intact. The goal of PCR is not to replicate the entire strand of DNA but to replicate a target sequence of approximately 100-600 base pairs unique to the organism being studied. Targeting the sequence is achieved by using primers. They are specific for the target region of the organism. Two primers are included in the PCR, one for each of the complementary single DNA strands that was produced during denaturation. The primers that anneal or in other...
Words: 2325 - Pages: 10
...Introduction: The purpose of this experiment was to successfully transform pGLO plasmid into E.Coli cells. In the first segment of this laboratory exercise, one had to carry out a restriction digest. Restriction digestion is the process of cutting DNA molecules into smaller pieces with special enzymes such as, BamH1 and EcoR1. One had to determine which of the two plasmids, A or B, were pGLO or pWEB. A plasmid is a small circular DNA strand in the cytoplasm of a bacterium (Isite, 2013). In order to determine that, one had to use BamH1 and EcoR1 on two tubes each one with plasmid A and the other with plasmid B to observe the cuts made by each enzyme. The hypothesis for this segment of the laboratory exercise states that pGLO was plasmid B, and pWEB was plasmid A. PGLO is a plasmid which contains green fluorescent protein (GFP). GFP is found in the Aquarius Victoria jelly fish. These jelly fish have a bioluminescent protein that emits blue light. GFP converts the blue light into green light, and that is why these jelly fish emit green light. The pGLO was inserted into E.coli by using transformation. Transformation is the process of transferring genetic material between microbial cells (Tu 2008). Bacterial cells need to be in a state of competency prior to transformation (Isite 2013). Some bacteria naturally achieve this stage when nutrients and oxygen are low (Isite 2013). In the laboratory, bacteria were artificially induced with calcium chloride to be competent for transformation...
Words: 1647 - Pages: 7
...labile toxin and heat stable toxins. Enterotoxins such as these, act on the intestinal mucosa and cause abnormal net fluid secretion. Moreover, Clostridium perfringes, Bacillus cereus, and some strains of Salmonella typhimurium deem to have similar enterotoxins. Enterotoxin activity has been found in some strains of other bacteria, but their role in producing illness is unclear. b. CFI and CFII are examples of colonization factors which have been established to be present in many enterogenic E.coli strains that aids them in the colonization of the small intestine. c. Various enteropathogenic E.coli strains established Adhesive factors. These, perhaps are required for producing illness. d. Cytotoxins like those produced by strains of Shigella and invasive and non-invasive E.coli may be responsible in at least a fragment for dysentery caused by shigella dysenteriae and probably other shigella and enteroinvasive E.coli and for haemorrhagic colitis and haemolytic uremic syndrome caused by E.coli 0157:H7. Many other enteric organisms have been found to produce cytotoxins but their relevance to disease is unknown. e. Causes of vomiting are believed to be cause by Neurotoxins present in Staphylococcus aureus and Bacilus cereus. f. A factor necessary for celluar invasion is the presence of lipopolysaccharide in the cell wall as found in Shigella species. Organisms deprived of this factor are incapable of invading cells in tissue culture....
Words: 259 - Pages: 2
...millions of customers over the past 22 years with high-quality, fresh food in a fast paced environment; giving them an experience to remember. However, in the past 2 years we have experienced to E.coli outbreaks that have affected numerous customers. In 2015 55 people were infected, in 11 states and 21 of them were hospitalized. Then in 2016, another E.coli outbreak infected 5 people in 3 states in which 1 person were hospitalized. (CNBC.COM) Given the recent issue, Chipotle has lost trust in its customers, as well a decline in sales since the outbreaks. In order to restore trust and regain top contender status, there must be a way to attract our customers. What is our objective? The main objective for Chipotle is to regain our customers trust. There are loyal customers who remained loyal, during the outbreak; but there are just a many customers who retreated and favored place such as, Chick-fil-a; Burger King; Fire House Subs; McDonalds. We want those customers back! While we understand the road will not be easy, we are willing to do what it takes to once again serve food with integrity. Who is the target customer? Originally the target market was those young, health conscious customers who were looking for healthier and fresh fast food options. Now that there has been 2 E.coli out breaks the original target market has been tarnished, and we must redirect focus on those customer who we lost their faith in us and our mission. What is the message? Because Chipotle...
Words: 780 - Pages: 4
...Sciences (JU), Ph. D in Environmental Science (Hokkaido University, Japan) Department of Environmental Science and Management North South University, Dhaka, Bangladesh ABSTRACT Comparative examination of different samples of portable water sources of water in Dhaka city was carried out with a view to assess the different sources of water and determine the water quality of the different sources. The sources of water examined are MUM drinking water, NSU drinking water, NSU tap water, distilled water, Pepsi and waste water. Many parameters were taken in consideration to test the water including physical conditions such as smell, color, turbidity and chemical conditions such as pH, DO, E.coli, TDS and NaCl present in the samples. Finally, a comparative analysis was done to assess the water quality of each samples based on the results from the experiment done. INTRODUCTION Importance of Water: With two thirds of the earth's surface covered by water and the human body consisting of 75 percent of it, it is evidently clear that water is one of the prime elements responsible for life on earth. Water circulates through the land just as it does through the human body, transporting, dissolving, replenishing nutrients and organic matter, while carrying away waste material. Further in the body, it regulates the activities of fluids, tissues, cells, lymph, blood and glandular...
Words: 2926 - Pages: 12
...Calvin G. Prince Mat-144 02/07/2016 Instructor Brent Bauman Mission Project: Part C Statistical Study I have a friend who returned from a mission trip in one of the villages in Sierra Leone. On their mission trip they collected randomly selected data from 100 households around the village, pertaining to the village’s well water supply. My friend’s mission trip participants included an aspiring woman who planned to major in biology, so she was put in charge of analyzing the water samples. My friend’s missionary group spent several weeks digging wells around the village that produced clean water, but they weren’t sure that the villagers were going to drink the water. At my friend’s request and because I had recently taken a math class, along with planning my own mission trip, I was asked to analyze their data to ensure the quality of the villager’s water that was from wells, and this is the results of my analyzation. In analyzing the data on the use of the mean or median as a better measure of central tendency, the data itself reveals that there are variables or outliners on the 2nd and 3rd columns of the data. These outliners have skewed the data to where the statistical data doesn’t represent the true mean value of the data that is presented. If this view tries to give way to the true meaning of the mean, then that would give rise to the disproportionate number increase on one side that would represent a very real uptick in the numbers that are being skewed. That is what...
Words: 676 - Pages: 3