Pglo Report

The transformation experiment successfully altered the ampicillin resistance of E. coli through the incorporation of the pGLO

In preparing for the bacterial transformation, DNA plasmid is introduced into the E. coli cells that will express newly acquired genes. Two tubes were used and labeled both as +pGLO and -pGLO. A solution of (CaCl2) was transferred 250 µl onto the two tubes. The tubes were placed on the ice. A sterile loop was then used to gather a single colony of bacteria from a starter plate. Now, that both tubes contain bacteria they were placed on the ice for 10 minutes. Four agar plates were labeled as: +pGLO LB/amp, +pGLO LB/amp/ara, +pGLO LB, -PGLO LB/amp. Heat shock was used to transfer both the +pGLO and -pGLO, at exactly 42°C. Time was observed for 50 seconds and quickly return the tubes to the ice for another 2 minutes. As the tubes, cold down they

The purpose of this experiment is to make E.Coli competent so that it can be transformed in order to become immune to ampicillin, then we would be able to determine the transformation efficiency of the culture. We determine this by preparing 4 plates of E.coli, each labeled “LB-plasmid”, “LB+plasmid”, “LB?Amp-plasmid”, and “LB/Amp+plasmid”. This meant that either should have lacked plasmid and Ampicillin, with plasmid but lacked Ampicillin, without plasmid but with Ampicillin, or were with Ampicillin and plasmid, respectively. Then we made the bacterial cells competent by adding CaCl2 to 2 vials of the colony (one with plasmids), and incubating on ice, then heat shocking, and returning to ice. Luria Broth is then added and left to sit for 5-15

The purpose of this lab is to use genetic engineering to transform E. coli bacteria by inserting the plasmid pGLO, and to then see if the bacteria was transformed by using the antibiotic, ampicillin.

Next, each tube was opened and 250 µl of transformation solution was added to each and then placed in the ice. While the tubes were on the ice, a sterile loop was used to pick up to 2-4 large bacteria colonies of bacteria from the starter plates and immerse to both +pGLO. The tubes were then placed back on the ice. Once the colonies of bacteria were well placed in the +pGLO solution, the same process was repeated for the –pGLO tube using a new sterile loop.

This pBlu lab had for purpose to present the changes of the strain of E. coli bacteria due to new genetic information being introduced into the cell. In this experiment we are freezing and heat shocking the E. Coli bacteria that is then forced to take the plasmid DNA. The E. coli then transforms the pBLu plasmid, which carries the genes coding for two identifiable phenotypes. After following the Carolina Biological steps our lab worked well and we able to see some colonies of bacteria on the plates. The x-gal plate showed a significant amount of bacteria to confirm that the pBlu plasmid took over the E. coli strain.

Spin the two tubes in a centrifuge for 5 minutes on opposite side of the centrifuge. The bacterium will collect at the bottom of the tube, so pour out the extraneous supinate. Then, add 250 microliters of buffer. The Ca2+ cation of the buffer neutralizes the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane allowing the DNA to pass through the cell wall and enter the cells. Place both tubes on ice. Then add 10 microliters of water into one tube and 10 microliters of plasmid DNA into another tube labeling the one with DNA with a + and the one with water -, and place on ice for 10 minutes.

To start the experiment two small plastic tubes were labeled negative pGLO and positive pGLO as the first step in this experiment. A pipet was then used to move 250 microliters of calcium chloride into each tube and the tubes were iced. Bacteria was added using a new sterilized loop each time to the positive and negative pGLO tubes. The loop was twisted around in the tube to ensure that the bacteria was evenly mixed into the solutions and the tubes were iced once again. Another loop was dipped into the tube containing the plasmid and it was removed when there was a visible slight film of residue. The plasmid was dipped into the tube that was labeled positive for the pGLO gene. The two tubes labeled positive pGLO and negative pGLO were iced

Transformation; this is the final step in the subcloning process, it can be described as the incorporation of DNA into a cell. This is done using the “Heat Shock” method in which the volume of the previously ligated plasmid is added to a competent cell and repeatedly incubated on ice or at 47°C or 37°C for variable times after which LB Broth is added and the mixture is shook at 37°C for 1 hour this allows the expression of antibiotic resistance and it has also been found to encourage cell recovery. Then centrifuge the solution and spread on an ampicillin plate and incubate at 37°C overnight to allow the bacteria to grow. This allows the transformation efficiency to be calculated and therefore the number or approximation of cells which took up the antibiotic resistance gene and the required gene to be subcloned (Sara Cormier, 2009).

Method and Materials So the first thing we’re going to talk about is the experimental procedure and tools used for the experiment. Both changed more than a lot by the time I was finished, my original tools were, 12- 100x15 mm agar Petri plates, 12 disinfected swabs, 2 bottles of DI water, 12- 18x150mm test tubes and a rack, 120 ml E. coli bacteria,1 sharpie, 3- 100mm beakers, 1 hot plate, 2 Celsius thermometers, 1 bag of ice, 2 incubators, cm ruler, microscope, fridge.

A transformation was performed so a vast quantity of the recombinant plasmid DNA might be obtained. There was a total of six transformations performed from four DNA ligations (excluding the fifth ligation). All six transformations were then incubated on ice for five minutes, followed by a heat shock, which came from putting the transformations in a 42-degree Celsius heat block for two minutes. Afterwards, the transformations were incubated on ice for another two minutes, then placed in a 37-degree Celsius incubator overnight, then plated. All the transformations included E.coli and luria broth, but differed in their other growth mediums. The first transformation consisted of the 1:1 molar ratio of pET-41a(+) to egfp ligation, kanamycin and

This experiment was performed to test the hypothesis if LB nutrient broth, +pGLO and -pGLO Ampicillin, and Arabinose was placed in the E. coli plates, then there will be a significant growth in the newly transformed bacteria and it will possess the ability to glow under UV light. The measurements were recorded from the bent glass tube in each glass test tube. The transformation protocol tested for the newly possessed traits in E.coli bacteria. Throughout the experiment there were many probable reasons for failure. If the pipettes and sterile loop were not thrown out in between each use, a cross contamination could cause a miscalculation in the experiment causing the data results to fail. The hypothesis that was tested was validated due to the positive results with each experiment stating that newly transformed organisms due in fact pass on traits.

Transformation in bacteria is something that could be essential for survival in a bacteria. In order to perform this transformation naturally a bacterium must considered competent, otherwise it must undergo an artificial transformation. Being a competent cell means that the bacteria can take up DNA from its environment naturally (5). Those that are not competent such as Escherichia coli that are not naturally competent can be tested with an artificial transformation, such as what we will use in this experiment. Methods used can obtain things such as chemical mutagens or radiation (1). The gene used for the transfer is the GFP gene (Green Fluorescent Protein), which gives an illuminating appearance under a UV light when conducted properly,

A sterile pipette was used to add 0.1ml of E. coli culture to the pH 3.0 tube. This was then repeated for the tubes at pH 7.0 and pH 9.The tubes were then incubated at 37oC for 48 hours. This was then repeated for saline culture of Saccharomyces cerevisiae but incubated for 72 hours at 25oC.

Genetic transformation is when an organism is given genes that it is able to express from the foreign DNA (Weedman 111). Genetic transformation is used widely today, especially in agriculture where organisms are genetically altered to meet the food demands from corns color to squash size. When transferring genes you need a competent cell which is the cell that is going to express the foreign DNA (Weedman 111). There are three ways to transfer DNA, transformation where the competent cell takes DNA from the surrounding environment, conjugation where two bacteria join and transfer DNA, and then there is transduction where virus or vector carries DNA from a foreign organism to another (hammiverse.com...na). In our experiment we used transduction, using a vector. There are three ways to make the competent cell permeable. There is projectile bombardment when a gene gun is used to coat the competent cell with DNA, there is electroporation when electric pulses are used to increase permeability and then there is heat shock when you rapidly change the temperature of the environment the competent cell is in from low to high temperatures making the permeability of the cell membrane more profound (Weedman 111). We used the heat shock method in our experiment to increase the permeability of E.coli. In the case of our experiment the competent cell is E.coli. E.coli is known for its ability of horizontal gene transfer which is the transfer of genes from a foreign organism and is not