Pglo Transformation
Bacterial transformation is a technique widely practiced by scientists for research purposes. This experiment explored the transformation of E. coli cultures with pGLO plasmids to allow the bacterial cells to express a foreign protein and emit a fluorescent glow under UV light. The transformation was completed through the heat shock method. Both transformed and untransformed E. coli cultures were grown in four mediums. The four mediums were made of different combinations of the LB nutrient broth, ampicillin and arabinose C sugar. When exposed to UV light, only the transformed bacteria growing on the LB/ampicillin/arabinose plate had a fluorescent glow.
The pGLO plasmid was genetically engineered to contain the green fluorescent protein (GFP),
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A sufficient amount of this solution was poured into two petri dishes labelled “LB -pGLO” and “LB”. Into two dishes labelled “LB/amp -pGLO” and “LB/amp +pGLO”, the agar broth with ampicillin was poured in. Arabinose C sugar was then added to the broth and was poured into one dish labelled “LB/amp/ara +pGLO”(Fig. 1). They were left overnight to harden. Meanwhile, a streak plate was made (Fig. …show more content…
coli were seen. The bacterial cells’ ability to survive the ampicillin in the medium was a result of their transformation. As mentioned previously, the pGLO plasmid contained the beta-lactamase enzyme which is needed for antibiotic resistance. Since the E. coli on this plate underwent the transformation, they were able to uptake the beta-lactamase enzyme thereby making the bacterial cells ampicillin resistant.
As well as being able to successfully grow and reproduce, the E. coli in the LB/amp/ara +pGLO plate also emitted a fluorescent glow when exposed to UV light. This can be explained by the examining the medium in which they were grown in. The bacteria were transformed with the pGLO plasmid which contained the GFP and resulted in glowing bacteria, however, in order for this to occur the arabinose C sugar must be present in the medium. This sugar is responsible for the activation of the GFP6.
Recall that in the E. coli cells in LB/amp +pGLO plate were also transformed but did not express the fluorescent glow. Even though they had the GFP gene in them, they were unable to express this protein without the arabinose C sugar to activate transcription of the
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