Plasmid Transformation Lab

What was expected that the strains of E. coli that did not have a resistance to ampicillin would not grow. The transformed strain also changed to a blue color when the X-gal was present in plate. The transformed cells also grew because they were free of the ampicillin because they possessed the amp gene that they used as an shield against the ampicillin antibiotic. The transformed cell who turned blue did so because the gene converted the sugar to a blue color but also contained the amp gene to ensure that they grew even when the ampicillin was present. The growth of the colonies on the plates

Cells are rinsed with chilled PBS, scraped into chilled PBS, and then centrifuged at 200 × g for 4 min. Discard the supernatant. The cells are now ready for lysis or storing at -80 ℃ for future use.

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

70µL of competent E.coli are added to both test tubes; pUC18 and Lux (Alberte et al., 2012). Both test tubes are then tapped and placed back into the ice bath for 15 minutes. While waiting, another test tube is obtained, filled with 35µL of competent cells and labeled NP for no plasmid. A water bath is preheated to 37 degrees Celsius and all three labeled test tubes are inserted into the bath for five minutes (Alberte et al., 2012). Using a sterile pipet 300µL of nutrient broth are inserted into both the control and Lux test tubes and 150µL are inserted to the no plasmid test tube to increase bacterial growth. All three test tubes are then incubated at 37 degrees for 45 minutes. Six agar plates are obtained and labeled to correspond each test tube, three of the plates contain ampicillin. A pipet is used to remove 130µl from each test tube containing a plasmid and insert it into the corresponding agar plate. For this, a cell spreader is first

mids. The miniprep from the previous step was done in order to use the isolated plasmids for the

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.

Before the pGLO plasmid was added to the +pGLO microtube, the pGLO plasmid was observed under a UV light. It was determined that the pGLO plasmid did not glow when placed under the UV light. Following this observation, a single loopful of +pGLO plasmid was added to only the +pGLO microtube and mixed thoroughly. After returning the +pGLO microtube to the ice, there was a ten minute wait

The field of biotechnology involves the concept of genetic engineering, altering the DNA/genetic material of an organism using information from a different one. The process in which bacteria can obtain this manipulated genetic information from another source is called genetic transformation. The goal of this experiment was to genetically transform Escherichia coli bacteria’s DNA by inserting the vector pGLO plasmid which codes for ampicillin resistance as well as the green fluorescent protein, GFP. For the experiment, the E. coli bacteria were separated into two groups; control and

How do you insert the plasmid inside the bacteria? What process do you use? (1/2 pt)

In our hypothesis we stated that only the container containing all of the components +pGLO, LB broth, ampicillin, and arabinose would be the one that genetically transformed. In order for the bacteria to grow at a rapid pace all it needed was LB broth but when you added ampicillin, an antibiotic, it killed off all of the bacteria. +pGLO has the gene to resist the antibiotic so when that was added it was allowed to grow but there was no sugar to turn on the glowing protein. Finally, after arabinose, a sugar, was added it turned on the switch located in the +pGLO for the fluorescence and enabled to grow and glow.

If a gene that codes for Green Fluorescent Protein transforms bacteria and GFP glows when transformation occurs, then when two micro test tubes have 250 microliters of transformation solution and places in an ice bath, then 2-4 bacteria colonies are added to each tube with a sterile loop; then a plasmid (pGLO) is added to one of the tubes, incubated in ice for 10 minutes, then heat shocked for 50 seconds at 42 degrees Celsius, then back into 9ice for two minutes; then LB nutrient broth is added to both tubes (250 microliters) and set out at room temperature for 10 minutes. Then, 100 microliters of each solution in the tube are added to four

Transformation is the directed modification of a genome by the external application of DNA from a cell of different genotype (Griffiths and Miller).Bacterial transformation is the easiest type of transformation to create and study due to the single cellularity of bacteria and its

The two control plates LB AMP-PGLO and LB AMP+PGLO (refer to Figure 3.0 and Figure 4.0) include a nutrient causing growth and the antibiotic, ampicillin, with or without the plasmid DNA. The purpose of the two is to provide a determination of the LB AMP ARA+PGLO and whether or not, the plasmid has an effect with the sugar. In addition, the two controls show that plasmid, pGLO, can affect the growth of bacteria with the consideration of ampicillin. As well, the two control plates prove that the results of the LB AMP ARA+PGLO and the LB-PGLO plate can be reliable knowing that they have been under the same conditions of the incubator as the control plates. Green Fluorescent Protein acts as the biological molecule for the fluorescence.

In +LB/amp 1:1 plate there were about 200 cells, +plasmid/variable has 150 and there was no cell growth in –LB/amp 1:1 plate. There were about 100 colonies in +LB/amp 1:10 and about 50 colonies in +plasmid/variable plate. The plate that is labeled with +LB/amp 1:100 got about 28 colonies, whereas in +plasmid and + variable plate, no growth is shown. There were approximately 9 colonies in LB/amp 1:1000 +plasmid and + variable, but no growth in 1:1000 +LB/amp plate. Also, there were no growths on both of LB/amp 1:10,000 plates (Table2). This experiment is consistent with our

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.