Amir Ahemedin
Ms.Buckley
Genetics
11/06/15
Transformation of E.coli Lab
Purpose The purpose of this lab is to genetically engineer the E.coli strain by introducing two genes, the green fluorescent protein gene (GFP) and the ampicillin resistant gene (AMP). Then observe whether or not the E.Coli strain would take up these genes and become fluorescent.
Background Information In this lab, bacterial transformation was one of the three processes that occurred when genetic material is introduced to a bacterial cell. Bacterial transformation is important because it allows for the cloning and movement of DNA between strains. This transformation usually occurs within plasmids, which are closed circular molecules made up of double stranded DNA. The function of the plasmid is to provide bacteria with genetic advantages such as antibiotic resistance. In this lab, the plasmids provided the ampicillin resistance and the fluorescence. If the bacterial cells are grown in the presence of the antibiotic ampicillin then only the cells that took up the plasmid have the resistance gene. As a result the resistance gene will have to keep the plasmid and the GFP gene. The other thing that plasmids
…show more content…
Many scientist today cut portions of DNA with restriction enzymes and include a piece of new genetic material into the bacterial cells. 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. In order for this to happen,one would treat the bacterial cells with Calcium Chloride (CaCl2). The bacteria used in this lab was E.Coli and it was an ideal bacteria because it can be easily grown on agar.
Hypothesis
The transformed E.Coli with the ampicillin resistance gene will be able to grow in the ampicillin plates and it would have a green glowing color.
For a plasmid to be useful as a recombinant DNA vector, it must have some essential features. What
The purpose of this lab report is to employ a myriad of skills, tools and, methods learned throughout this semester to perform the appropriate tests for the identification of the assigned unknown bacteria. Add more background information here!!! The most important tools and techniques used during this identification include aseptic technique, microscopic examination and, the use of selective and differential media. Aseptic technique is an important tool for microbiologists. It is imperative that aseptic technique is maintained throughout the length of any test to avoid any cross-contamination that may lead to inaccurate results.
Results Continued: The purpose of this experiment was to show the transformation of E. Coli with pGLO. We made four different plates each with different additives to compare them to one another, and be able to track the transformation. Initially we had predicted that only one of the four plates would glow. The plate with the plasmid (+pGLO)/LB/amp/ara was the one we said would grow and glow because it contained all the necessary tools to do so.
The hypothesis for this experiment was that transformed bacterial cells would grow on ampicillin plates and glow green when exposed to UV light. The rationale for this hypothesis was because the plasmid would code for ampicillin resistance and a green fluorescent protein, we would have the outcome explained in the hypothesis. 4. Our predictions were that for the standard protocol plates with agar and ampicillin, there would be growth and the colonies would glow under UV light. On the modified protocol on plates with agar and ampicillin where we changed the time of the heat shock, we expected less growth but the colonies would still glow.
R plasmids are responsible for carrying the gene for resistance to antibiotics e.g. ampicillin, which are normally used in the lab. The normal function of a plasmid is to transport genetic information essential to the survival of the bacteria. (Barnnet, 1995). The plasmid can work as vectors for introducing strange DNA. Restriction enzymes are normally used cut foreign DNA and placed it into the plasmid vectors.
Figure 3. Testing of transformed and mutant bacteria on minimal medium Growth was observed on the Transformed (Trsf) section and not on the Mutant (Mut)
Transformation was successful in the plates where the bacteria consumed the pGLO plasmid. In the first plate that the bacterium was plated on it included the LB broth and of ampicillin antibiotic (amp), 2 colonies were present. The second plate of bacteria was grown with the presence of LB broth, ampicillin, arabinose sugar (ara), and 22 colonies were observed. But a green fluorescent glow of the colonies was only present in plate 2. Plates 3 and 4 were the control plates.
These microorganisms are used to teach us how multicellular organisms came to be and how they can survive today. These small, microscopic organisms are so unique that the identification of them is paramount in the advancements of science. Knowing the chemical makeup, the shape, and the biochemical processes is important in identifying these organisms to understand how they survive and where. A number of tests can be ran on an unknown bacteria to determine their ideal
Introduction Our world is composed of many bacteria’s’ that can either help or destroy us. Therefore, its’s imperative to learn and study them. The purpose of the lab was to put into action the methods that have been learned in the laboratory to determine our unknown bacteria. Bacteria’s can have different features, shapes, and or arrangements that help microbiologist determined their role in our life (whether they are good or bad for humans).
Afterwards, with the use of Glimmer 3.02 and BLAST, gene prediction and annotation were completed with another plasmid, pHN122-1, as a reference. To confirm the role of the gene that caused polymyxin resistance and contained mcr-1, the gene and its sequence were place into a cloning vector pUC18 that yielded pUC18-mcr-1. With this yield and electroporation, it was used to transform an E.coli strain, revealing its ability to confer colistin resistance. Q3D:
It could have also been helpful to show a diagram of how the cell transfers the genetic information containing antibiotic resistance characteristics.
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.
Escherichia Coli 0157: H7 This paper will specialize on a specific type of bacterial foodborne illness caused by the bacteria Escherichia Coli. E. coli was discovered by Theodore von Escherich in 1885. E.coli is a natural found bacteria that lies throughout the intestinal tract of warm blooded animals and comes in many forms only one of which is deadly. This form is E. coli 0157:H7 which can be caused by direct exposure to fecal matter to kill this rouge
INTRODUCTION: In this experiment I was testing for antimicrobial sensitivity of Staphylococcus epidermidis by using the Kirby-Bauer Diffusion test. The three antibiotics utilized in this lab were: gentamicin, novobiocin, and penicillin. I determined the effectiveness of the antibiotic by observing and measuring the zone of inhibition for each antibiotic.
Extracts Inhibition zone (in cm) Strains 1. 5C 1.3 E.coli 2. 1A 1.5 E.coli 3. 3E 1.9 E.coli 4. 4E 2 E.coli 5.