Pglo Transformation Essay

In order to find transformation efficiency, the total number of colonies on the plate is divided by the total amount (µg) of DNA spread on the plate. All the factors that must be taken into account while finding the transformation efficiency include; the total amount (µg) of plasmid DNA used, the total volume (µl) of cell suspension prepared, the fraction of DNA spread on the plate and finally the total amount (µg) of DNA present on the plate. In the LB/AMPC plate the transformation efficiency was 8.2 x 103 colonies per µg of plasmid DNA while the LB/AMPlux plate the transformation efficiency was 1.07 x 104 colonies per µg of plasmid DNA. The transformation efficiency for the LBC and LBlux plates were not taken into account, even though they too transformed because they were not in a restrictive environment where they needed to express their ampicillin resistance genes in order to

This is important in the medical field because identification of unknown bacteria can help treat a patient by knowing the contributing source of a disease. Also knowledge of different bacteria helped others make antibiotics used today. This lab was completed by using the methods learned thus far in identification of bacteria.

I looked at he pGLO through a uv light. Then I put my loop into the pGLO plasmid DNA tube and made sure that the ring was covered with he solution, then mixed it into the +pGLO. I then place the tubes back on ice for 10 minutes and I pushed them under the ice. While my tubes were sitting in the ice I begin Ito label my LB nutrient Agar plates on the bottom. When the 10 minutes is up for the tubes in the ice, I removed them from the ice and put them into a film holder and placed them into a heated water bath for 50 seconds.The settings for the water should be 42 degrees Celsius. When the 50 seconds are up, I placed both tubes back on the ice and had them sit in there for 2 minutes. After the 2 minutes I I removed

E. coli HB101 was transformed with pGLO plasmid then grown on media containing ampicillin and/or arabinose and on medium containing neither (Brown, 2011). This is done for selection of transformed cells since not all cells are expected to take up the plasmid (Brown, 2011). We also expect roughly the same CFU on any plate(s) receiving samples from the same microcentrifuge tube, since they are getting the exact same

The color of the bacteria was a whitish color and the colony size is similar both before and after the transformation. The best way to do it is to compare the control of the experimental plates. Cells that were typically not treated with the plasmid could not grow on ampicillin, although cells that were treated with the plasmid can grow on the LB/AMP plate. The plasmid would have to confer resistance to ampicillin. Moving on, the GFP gene is what is glowing in the plate because it was activated by the sugar arabinose. The sugar arabinose and the plasmid DNA are also needed to be present because that is what initially turns on the GFP gene which makes the bacteria glow. Organisms can also turn on and off particular genes for camouflage reasons. An organism would benefit from turning on and off certain

There were two tubes used in this process: the tube that contained the primary culture and the tube that contained the nutrient agar where the unknown bacteria would grow. First, the inoculating loop was flamed. After removing the caps of both the test tubes, they were flamed to prevent contamination of the unknown bacteria. The inoculating loop was cooled for a few seconds and was then placed into the test tube containing the bacteria. The inoculating loop with the bacteria was placed into the nutrient agar test tube for cultivation. Before the test tubes were capped, they were flamed once again. Also, isolation of the unknown bacteria had to completed. Nutrient agar was placed in the petri dish, and was left to gel for a few minutes. After the agar gelled, the inoculating loop was used to acquire bacteria and streak the unknown onto the plate for

How does the addition of pGLO plasmid to a solution containing E. coli bacteria affect the growth and characteristics of the bacteria? Genetic transformation is the incorporation of foreign DNA into an organism to potentially change the organism’s trait. Plasmids are small circular DNA that replicate separately from the bacterial chromosome. In nature, these plasmids can be transferred between bacteria allowing for the sharing of beneficial genes. Due to this characteristic, plasmids allow for genetic manipulation and can be moved between bacteria easily. The pGLO plasmid utilized in this experiment encodes the gene for Green Fluorescent Protein (GFP), which under the right conditions can produce a glow. The gene regulation system present in the pGLO plasmid requires

Crystal violet stains the cell of the bacteria and is placed over the heat fixed slide for one minute then rinsed with H2O. The mordant that binds the stain is gram’s iodine and is recommended to be present on the slide for one minute. Alcohol is used as the decolorizer and is dripped over the recently rinsed slide until the slide runs slightly blue. If the bacterium retains the crystal violet it is due to the thicker peptidoglycan in the cell wall (gram-positive). The bacteria will then be stained with the counterstain safranin for 45 seconds. When crystal violet is not retained in the cell wall of the bacterium it will stain pink (gram-negative). A gram stain was immediately performed with the recently inoculated streak plate. An inoculating loop was used to gather some of the bacteria from an isolated colony. The loop was placed on a new slide and the transferred bacterium to the slide. The slide was dried, heat-fixed, and the gram stain process just described was completed. After microscopic analysis, it was determined the first unknown bacteria was a gram-positive cocci. The second unknown was determined to be gram-negative cocci. With this information a set group of test decided and performed based on the Unknown Bacteria Flow Chart as provided in the lab manual (Pg.) these test and the results are shown in record on the following pages. These are separated for the gram positive and the gram negative

After gaining some knowledge about bacteria, we were giving an investigating bacteria growth lab to do. Our objective was to observe the conditions required for bacteria to grow and to test the effectiveness of substances that may be antibacterial, disinfecting, and or sanitizing. My group and I began our procedure by gathering all the bacteria by swabbing our necks and mouths. After this, we inoculated the culture by rubbing the bacteria on the agar, a nutrient rich gel made from sea kelp, on the bottom side of the container where we grow bacteria, the Petridish. We hoped for the results to come back with little or even no colonies and an immense zone of inhibition around the tiny circle cut out of filter paper covered in toothpaste, Neosporin, and Chlorhexidine Gluconate 4% Solution.

Based on the experiment results and the data obtained, this experiment showed that the predicted results were accurate. It is possible to produce a bacterial transformation of the genes of the bacteria E. Coli by the inoculation of a p-GLO plasmid. The bacterial transformation produced a change in the bacterial genome showing a change in the phenotype of the bacteria, by acquiring the traits of the genes introduced into the plasmid. In this experiment, each of the four plates had LB broth for nutrients and a bacterial suspension of E. Coli to study its reaction in the presence of the antibiotic ampicillin and the p-Glow plasmid. In the control plates, labeled as # I for LB/-pGLO, and # II for LB/-pGLO/

The detection of unknown bacteria number 63 began with examining macroscopic and microscopic morphology. Macroscopic morphology of unknown bacteria was done by a streak for isolation of bacteria on a plate with nutrient agar, which allowed the bacteria to ingest the nutrients it needed to survive and reproduce to form pure colonies. After growth, the colonies appeared to be mucoid, beige in color with smooth edges and no elevation. Another macroscopic test was done in a tube with semi-solid agar to test for motility and the result of this test

After several biochemical tests, Unknown Bacteria #30 was identified as Staphylococcus aureus. After growing the bacteria on Nutrient Agar to ensure a pure sample, it was Gram stained to determine morphology and arrangement. It was observed to be a Gram positive staphylococci. Then, the bacteria was inoculated onto a Mannitol Salt Agar plate. After incubation, it was observed to have bacterial growth and the agar was yellow in color. According to the lab manual (2), MSA contains 7.5% NaCl and phenol red, a pH indicator. Due to the salt content, MSA is selective for salt-tolerant bacteria and the phenol red allows MSA to differentiate for mannitol fermentation. Mannitol fermentation is indicated by a yellow color change, which is the result of acidic byproducts changing the pH of the agar. The results showed that the bacteria was both salt-tolerant and able to ferment mannitol.

Then, we placed 100 µl of the E. coli cells into four Eppendorf tubes each and labelled them A, B, C and D. After filling them up, we placed them into an ice box. After this, we needed to perform the transformation process by mixing the E. coli cells with different concentrations of plasmid DNA. In test tube A, we added 5 µl of distilled water to the E. coli cells. In test tube B, we added 2.5 µl of undiluted plasmid DNA to the E. coli cells. In test tube C, we added 1.0 µl of undiluted plasmid DNA to the E. coli cells. Lastly, in test tube D, we added 1.0 µl of plasmid DNA diluted 1 in 10 to the E. coli cells. Then, we used a P100 pipette and mixed all the liquids in all the Eppendorf tubes by pipetting the solution up and down. Then, we left the Eppendorf tubes along with its contents in the ice box for twenty minutes. After twenty minutes, we labelled the base of the four agar plates to match with the appropriate plasmid DNA concentration about to be plated. We also wrote our initials, the date, and the table number on the base of the agar plate. We then gently lifted up the cover of the agar plate with just enough space to allow us to plate each tube of cells onto four different agar plates – one agar plate to one plasmid DNA concentration. We used a spreader to spread the cells evenly over the surface of the agar, being careful not let the spreader touch

Bacterial transformation is the process of moving genes from a living thing to another with the help of a plasmid.The plasmid is able to help replicate the chromosomes by themselves; laboratories use these to aid in gene multiplication. Bacterial transformation is relevant in everyday lives due to the fact that almost all plasmids carry a bacterial origin of replication and an antibiotic resistance gene(“Addgene: Protocol - How to Do a Bacterial

A fresh pipette was used to transfer 0.5ml of broth culture of E. coli, to be inoculated, into a tube of molten agar previously boiled to drive off oxygen. The tube was then rolled to distribute the bacteria and allowed time for the agar to harden. The tube was incubated at 37oC for 48 hours. The procedure was repeated for broth cultures of the bacterium Clostridium sporogenes and B. Subtilis.