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Essay: E. Coli Bacteria Genome Transformation

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The major objective of this experiment was to observe if the integration of pGLO, as a plasmid vector, into the genome of Escherichia Coli, would result in bacteria that glows. It was based off of three environmental factors; LB nutrient broth, the antibiotic ampicillin, and arabinose sugar. The transformation vector used included the use of two genes: AMPR and GFP. AMPR is a gene that is under control of a constitutive promoter, which supports antibiotic resistance. GFP is a gene that causes cells to fluoresce when expressed. The Escherichia Coli colonies grew in the presence of the antibiotic ampicillin and arabinose sugar but only glowed in the arabinose sugar sample.


Genetic transformation or in simpler terms: change caused by genes; is the insertion of one of more gene(s) into an organism in order to modify the organism’s traits. The process involves cutting out genes from a human, animal, or plant DNA and transferring it into bacteria. Genetic transformation is a biotechnology procedure used in many areas such as agriculture and medicine. In agriculture, it can be used to genetically transform the plants to have resistances towards pests, drought, and more. In medicine, it can be used to produce proteins that individuals with certain conditions are not able to produce on their own to combat the disease, for example the hormone insulin is produced using this method (Bio-Rad).

A common practice students use to better understand transformation is by using GFP. Green Fluorescent Protein (GFP) is a tiny protein, 27 kDa in mass, that requires no cofactors or substrates to be expressed. When expressed it emits a green fluorescence at 509 nm allowing the organism expressing it to glow in the dark. It is extracted from the bioluminescent jellyfish, Aequorea victoria. It was used in an experiment in order to observe its effect on the structure and function of IS2 transposase by tagging for fluorescence-based random mutagenesis (Raj, M. Alpha, et al. 2015).

A resistance to antibiotics for humans can be extremely hazardous in the future but a resistance to antibiotics is a widely used tool in microbiology. It allows scientists to detect bacteria containing plasmids when the cells are in a controlled setting that provides pressure on the bacteria to keep the plasmid in place (Patrick 2014). The ampicillin resistance (AMPR) gene is a widely used tool because it is carried along with tetracycline resistance on the plasmid vector pBR322, a superior cloning vector for cloning in E. coli approved by the National Institutes of Health (Sutcliffe 1978).

This experiment was carried out to answer: “If GFP, a jellyfish gene, can successfully be cloned into and expressed in the E. Coli bacteria?” Of the four plates used in the experiment, two contained the pGLO plasmid and if the plasmid is successfully transferred it will be known by its resistance to the antibiotic ampicillin. As antibiotics are meant for, they break down cell walls of the bacteria thus destroying the bacteria, such as E. Coli. But if the bacteria wishes to remain alive its genetic code must incorporate a code that prevents and resists the ampicillin, this is where the pGLO plasmid comes into play to assist in the bacteria’s survival. The other variable in the experiment, arabinose, is a simple sugar molecule which is known to activate the gene that codes for GFP production in E. Coli (PGLO Transformation Protocol 2005). A growth in colonies on the plate containing +pGLO/LB/Amp/Ara is most probable due to it being the only plate implementing the arabinose sugar.

Materials and Methods

Two micro test tubes were used and labeled “+pGLO” and “-pGLO.” Into each test tube, 250 µl of the transformation solution (CaCl2) was transferred into each test tube using sterile transfer pipets. A single colony of the E. Coli bacteria was then inserted into each of the test tubes. Next, the pGLO plasmid DNA was only inserted into the “+pGLO” test tube. The tubes were incubated in an ice bath for 10 minutes. Afterwards, the tubes were

then heat shocked, that is the test tubes were then placed in a hot water bath raised to the temperature of 42°C, for exactly 50 seconds. Once the 50 seconds passed, they were then quickly transferred to the ice bath for an additional two minutes to complete the heat shock method. Next, 250 µl of the LB nutrient broth were added to each test tube and incubated at room temperature for 10 minutes. Then, 100 µl of the contents from the +pGLO test tube was transferred into two nutrient agar plates. Similarly, 100 µl of the contents from the -pGLO test tube was transferred into two other nutrient agar plates. To each of the agar plates, a sterile loop was used to evenly distribute the contents across the plates’ surface. Finally, the plates were then stored overnight in an incubator set to 37°C in an upside down position.

Experimental Data

LB: LB Nutrient Broth Amp: Ampicillin Antibiotic Ara: Arabinose Sugar

Data Analysis and Conclusion

From the gathered data, the four plates with different conditions resulted in four different outcomes. Starting with the plates that don’t contain the pGLO plasmid, the LB nutrient broth plate resulted in a lawn of E. Coli which would be expected from bacteria in a heated environment. The second plate with the LB nutrient broth and ampicillin resulted in no reaction due to ampicillin being an antibiotic that doesn’t allow E. Coli to survive. The first of the two plates that contain the pGLO plasmid, the LB nutrient and ampicillin plate resulted in white colonies throughout the surface of the plate. This happened because the E. Coli took up the pGLO plasmid which contained the AMPR gene and thus conferred resistance to the ampicillin. Lastly, the fourth plate containing the LB nutrient broth, ampicillin, and arabinose sugar resulted in the fluorescence and glow that is observed in the Aequorea victoria jellyfish. This occurred because the GFP gene is under the control of a conditional promoter. Meaning, in order for the promoter to be activated a condition must be met to allow gene expression. In this case, the condition is arabinose.

I originally hypothesized that the nutrient agar plate containing the arabinose sugar would be most likely to have a growth in colonies due to the arabinose sugar molecule being an activator for the gene that codes for the Green Fluorescent Protein production in

E. Coli. The results of this experiment supported the hypothesis. The data also mainly supports that the E. Coli genome was successfully transformed since the colonies that grew on the “+pGLO/LB/Amp/Ara” plate also fluoresced when put under the UV light.

Transformation is an important procedure that has a significant impact in society, a large portion in the medical field but is cleverly utilized in the bioremediation field, such as using bacteria with genetically transformed genes to enable them to digest oil spills (Bio-Rad). As an Environmental Science major, I strive to keep the planet sustainable for life to continue forward and utilizing bioremediation is an amazing way to help. If possible, a future experiment I would like to conduct using similar tactics from this lab is genetically transforming airborne bacteria with a gene that would allow for it to absorb the excess CO2 in the Earth’s atmosphere in order to combat the effects it has been causing to earth’s climate.

Literature Cited

Bacterial Transformation. 2013, pp. 1–8, Bacterial Transformation.

“Bio-Rad.” PGLO Bacterial Transformation Kit, pp. 1–69., www.bio- rad.com/webroot/web/pdf/lse/literature/Bulletin_1660033EDU.pdf.

Patrick, Marcy. “Plasmids 101: Antibiotic Resistance Genes.” Plasmids 101: Antibiotic Resistance Genes, Addgene, 30 Jan. 2014, blog.addgene.org/plasmids-101- everything-you-need-to-know-about-antibiotic-resistance-genes.

“PGLO Transformation Protocol.” Department.monm.edu, 2005, department.monm.edu/ chemistry/chemistry330/fall2005/hhawkins/pGLO_transform_protocol.htm.

Raj, M. Alpha, et al. “First Report on Rapid Screening of Nanomaterial-Based Antimicrobial Agents against β-Lactamase Resistance Using PGLO Plasmid Transformed Escherichia Coli HB 101 K-12.” SpringerLink, Springer Berlin Heidelberg, 25 Oct. 2015, link.springer.com/article/10.1007/ s13204-015-0506-7#citeas.

Sutcliffe, J. Gregor. Nucleotide Sequence of the Ampicillin Resistance Gene of Escherichia Coli Plasmid pBR322. 8th ed., vol. 75, Proc. Natl. Acad. Sci. USA, 1978, pp. 3737– 3741, Nucleotide Sequence of the Ampicillin Resistance Gene of Escherichia Coli Plasmid pBR322.

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