Pglo Lab Report

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 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.

Coli cells would engage in the pGLO plasmid and fluoresce. one funding did not support the hypothesis. One plate that contained arabinose did not no glow because the cells did not engage in the pGLO plasmid, due to the lack of ampicillin on that plate. The presence of ampicillin is a motivator for the E. Coli cells to engage in pGLO, which also contains beta-lactamase. According to the student manual, “Beta-lactamase acts against ampicillin, protecting the cell from the antibiotic’s harmful effects. Without the threat of ampicillin, the cells have no reason to take up the pGLO plasmid, and therefore the presence of arabinose has no effect on whether the cells fluoresce or not” (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

This acts as a control for the LB/AMP/+pGLO. The third plate, which contained LB/AMP/+pGLO, formed scattered colonies of bacteria. These colonies of bacteria were formed by +pGLO, which is the new gene that the bacteria took in. It is an antibiotic resistant bacteria which fights against the AMP. With the insertion of this +pGLO gene, many bacteria were able to survive the AMP and grow, forming colonies.

coli colony through the process of heat shock to observe differences in growth and bioluminescence with and without GFP and arabinose. It is expected to see no growth in the colonies that have not been treated with ampicillin and pGLO, the plasmid containing the genes for GFP and ampicillin resistance. Those that have been treated with pGLO and/or no ampicillin will grow due to the inheritance of the ampicillin resistance gene, whether or not there is arabinose present. The hypothesis for this experiment is that only the colony that has been treated with pGLO, arabinose, and UV light will be bioluminescent because all are required to glow.

In this lab, Escherichia coli is the experimental bacteria and transformed by pGlo plasmid. The pGlo plasmid from Bio-Rad contains the coding genes for Green Fluorescent Protein (GFP) and a gene resistant to the antibiotic, ampicillin (Mardigan, 2011a). Normally, the GFP is coded from a gene in the jellyfish, Aequorea Victoria. The GFP can also only produce its green fluorescent color when the chromophore is in a specific conformation. When the GFP is denatured, the chromophore loses its original shape thus losing its fluorescent capabilities.

In the transformation plates, the p-GLO plasmid was inoculated producing a different response, the traits present in the plasmid appeared as part of the E. coli response. In the transformation plates labeled as # II for LB/+pGLO/amp. , and as # III for LB/+pGLO/Amp/Ara other responses were observed: Plate # II, responded by growing the amount of colonies which means that they acquired the ß-lactamase gene and after the experiment the new colonies of E. coli expressed the antibiotic resistance trait. However, the colonies in plate # II did not glow because they did not have the arabinose C protein, which regulates the expression of the GFP gene. Finally, Plate # III expressed both genes, the antibiotic resistance, and the green fluorescence trait because the araC was inoculated as well, so in this plate, the phenotype changed and the bacterial colonies grew in number and glow in a brilliant green color under the ultraviolet

This allows for some colonies to pick up the plasmid DNA, allowing them to transform into colonies. The GFP gene is in the plasmid DNA but is not turned on because there is no AraC. The plate labeled ‘LB/Amp/Ara +DNA’ have the plasmid DNA, ampicillin, and AraC. The presence of AraC allows for the GFP to turn on, resulting in the transformed colonies to glow in

Transformed bacteria cells would be found on plates that contain pGLO, so the LB/amp +pGLO plate and the LB/amp/ara +pGLO plate. The pGLO is a plasmid that expresses the gene for resistance to ampicillin. On the plates that contain pGLO, cells take up the pGLO plasmid and will be ampicillin resistant. When cells take in a foreign plasmid, they are transformed.

One group had no growth in both +pGLO plates but yielded the expected results in both control plates. This might mean that the pGLO plasmid might have not been incorporated in the DNA of the bacteria. The other group resulted in a positive growth for +pGLO amp but negative for +pGLO amp ara thus it can be said that the pGLO was introduced to the DNA of the bacteria unlike the previous group because it became resistant to the antibiotic in the +pGLO amp plate however, improper inoculation might have been the cause of error for the other

The reason why it did not inhabit any change after the experiment was conducted is because it had no plasmids added to support the growth and the ampicillin actually killed the bacteria. The plasmid would have served as the vector to insert the gene into the E. coli. The -pGLO LB plate thrived because there was no ampicillin present. While the +pGLO LB/amp/ara plate ended up glowing under the UV light because the GFP was only able to show with the presence of the arabinose, because it is what activates the operon. The +pglo LB/amp plate contained a few colonies but it did not glow under the UV light due to the absence of the

After completing the experiment and recording all the results, we were able to analyze in what medium the bacterium, E. coli, was able to grow. The fact that some bacteria were able to grow in the presence of ampicillin, suggests that they were capable of uptaking the gene from the plasmid, pUC18. These bacteria which included LB/AMPLUX and LB/AMPC experienced a colonial growth as shown in pictures two and five. The reason why these bacteria experienced a colonial growth is that only some of the bacteria inside each spot plate became resistant to the antibiotic. There was an error in the experiment since LB/AMPNP was not supposed to experience any growth.

In this lab, we demonstrated that a plasmid could be inserted into the DNA of bacteria in order to alter the physical characteristics of the bacteria. We used biotechnology to manipulate the genetic code of the bacteria so that it would glow when exposed to a UV light. After a couple of days when the glow of the bacteria started to fade, it became apparent that the operon system that enabled the pGLO gene to take affect was stopping.

Coli. To be able to activate the GFP gene the presence of the sugar arabinose is required. In order to make the E. Coli grow, the presence of ampicillin, an antibiotic, and pGLO is present, however, if ampicillin is present without pGLO it will stop the growth of the E. Coli because the pGLO would not be present to stop the antibiotic. In the experiment E. Coli was tested with the different proteins and plasmids, testing if the plate would glow or grow. If the E. Coli was tested with ampicillin, pGLO, and arabinose the plate would both glow and grow because of the proteins present, however, if the E. Coli was tested with ampicillin and pGLO the plate would neither glow or grow because the proteins were not