Pglo Lab Report

The pGLO plasmid is engineered to express green fluorescent protein (GFP) in the presence of the sugar arabinose as well as the ampicillin resistance gene β-lactamase (bla) (Brown, 2011). Original E. coli HB101 do not have ampicillin resistance or the GFP gene allowing them to glow under UV light. In this experiment, we transformed E. coli HB101 with the pGLO plasmid by heat shock to make the bacterial cells competent, allowing the plasmid to enter the cell (Brown, 2011). The mixture of bacteria with pGLO plasmid were given recovery time after heat shock, then spread on LB/amp and LB/amp/ara agar plates. The bacteria mixture with no plasmid added were spread on LB and LB/amp agar plates and all four plates were incubated at 37°C for

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

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

The plasmid pGLO contains an antibiotic-resistance gene, ampR, and the GFP gene is regulated by the control region of the ara operon. Ampicillin is an antibiotic that kills E. coli, so if E. coli, so if E. coli cells contain the ampicillin-resistance gene, the cells can survive exposure to ampicillin since the ampicillin-resistance gene encodes an enzyme that inactivates the antibiotic. Thus, transformed E. coli cells containing ampicillin-resistance plasmids can easily be selected simply growing the bacteria in the presence of ampicillin-only the transformed cells survive. The ara control region regulates GFP expression by the addition of arabinose, so the GFP gene can be turned on and

In the pGLO Bacterial Transformation lab, Escherichia coli is transformed with a gene encoding green fluorescent protein by inserting a plasmid containing the GFP gene, beta-lactamase, and arabinose into the bacterium. Successfully transformed bacteria will grow in the presence of ampicillin and glow a bright green color under ultraviolet light. The sugar arabinose is responsible for switching on the GFP gene in the transformed cells, without it, the gene will not be expressed.

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.

The pGLO plasmid will transform the E. coli bacteria with a gene called GFP that codes for the Green Fluorescent Protein in the genetic code. GFP was discovered in the jellyfish, Aequorea victoria as a green fluorescent light emitted from the jellyfish. It was typically seen in the dark upon its activation and since then has been used in studies relating to genetic transformation. (Chalfie and Tu 1994) The majority of the studies test the many different factors that are required in the transformation of pGLO which will determine the functionality of GFP in the E.coli bacterium. The first experiment in transforming GFP and E. coli was completed in 1994 by Chalfie and was further refined the same year. The experiment proved the importance of using restriction enzymes, and DNA ligase in the process of transforming GFP to identify arabinose as the primary activator, and to identify the ampicillin

To start this laboratory, it must first label two transformation tubes of different color; one with – DNA and the other with + DNA. Then, using a P-1000 micropipette adds 250 ul of the ice cold transformation solution (CaCl2) into each tube and places both tubes on ice. Using a disposable sterile inoculating loop to transfer colonies of E. coli bacteria from the starter plate to the +DNA tube. Immerse the loop in the CaCl2 solution in the transformation tube and spin the loop until all bacteria is incorporated into the transformation solution. Repeat this step with the – DNA tube. Using a P-10 pipette, transfer 10 ul of pGLO directly into the + DNA tube. Tap the tube lightly with a finger to mix. Place both tubes onto ice, and incubate for 10 minutes.

Discussion The expected result from this experiment, was that the plates containing plasmid DNA, GFP, and IPTG would grow and exhibit a florescent glow. All the plates containing plasmid DNA would grow and those that contained no plasmid would not grow. The plasmid DNA is able to be absorbed only if the bacteria is competent, which is a result of the heat shock (Bacteria Transformation and Selection, 2017). The head shock opens the pores of the membrane allowing the passage of plasma.

When trying to transfer several specific plasmids into a new host DNA, you need a way to check and see if the desired plasmids were combined to the DNA, that’s where there pGLO test comes in. By using an ultraviolet light, a person is able to immediately see if the new plasmids have been accepted into the bacteria by whether or not a bright green fluorescent light is emitting from the bacteria. So the process in how pGLO is expressed would be the plasmid itself is extracted from the A. Victoria, modified and inserted into bacteria with the three genes. The beta lactamase then comes first, to give it a resistant to the ampicillin antibiotics that would initially kill it. This then allowed the araC operon to take the arabinose and allow

These are properties E.coli normal does not have so control bacteria should look white and die in the presence of ampicillin. The ampicillin marker was irrelevant in this experiment due an error of not placing the experiments ‘s data in ampicillin. In the end, data on how the bacterium with plasmids would react with ampicillin is unknown.

coli cells, using a green fluorescent protein (GFP), to make them glow. GFP is what causes the bioluminescent jellyfish, Aequarea victoria, to glow in the dark (Cramerie and Kitts 1996). Another piece of valuable information is that E. coli is killed off in the presence of ampicillin. Therefore, we used the plasmid pGLO, which contains the green fluorescent protein as well as an ampicillin resistance. Additionally, the sugar, arabinose, was needed to allow the GFP to glow ( Cramerie and Kitts 1996). I hypothesise that the addition of ampicillin and arabinose will allow us to see how much of the E. coli was transformed by killing off the not transformed E. coli, while enabling the transformed cells to glow. Assuming the transformation is successful, the transformed E. coli will continue to grow and glow, regardless of the presence of ampicillin. The significance of this experiment is that it helps us better understand genetic transformation. Once we have a good grip on the topic, we can then use it to further human medicine. For example, humans that have diabetes cannot produce enough of their own insulin. Instead, they rely on insulin being pumped into their bodies. This insulin used to come from pigs- which was rejected by many people’s bodies. Because of genetic transformation, we are able to produce mass amounts of a person’s own insulin by transferring DNA from them into the insulin of a pig, to create something the human’s body could

If the pGLO plasmid is inserted into competent Escherichia coli cells, then the transformed bacteria will be resistant to ampicillin and will glow green under UV light. If samples of DNA are cut using certain restriction enxymes and separated using gel electrophoresis, then the smaller the DNA fragment cut, the greater the distance it will travel in the gel.

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.

During this study, plasmids with ampicillin resistance (pAMP) known as pUC18, were manipulated due to their stability in extreme environments. The pUC18 plasmid pAMP gene codes for the production of beta-lactamase, an enzyme utilized in the breakdown of ampicillin. Once E. coli obtains this gene, it is able to survive in an environment that is ampicillin-rich. Lux plasmids were the second type used to provide bioluminescence in the cultures of E. coli grown. The lux plasmid contains genetic material which codes for luciferase, an enzyme which catalyzes bioluminescence, a light-emitting reaction. This is considered the marker where transformation efficiency can be analyzed through the light emissions.