Pioneering E. coli Transformation: Unveiling Gene Regulation and Bioremediation Potential

The aim of this laboratory experiment was to investigate the impact of the pGLO plasmid on various colonies of E. coli bacteria. The pGLO plasmid is a genetically modified structure containing three key genes, including an ampicillin resistance gene for antibiotic survival, a GFP gene that produces green fluorescent protein under specific conditions, and an araC gene acting as a trigger for GFP gene activation in the presence of a particular sugar. Students conducted a transformation, transferring the modified plasmid to E.

coli using a solution with CaCl. Heat shock was applied to facilitate plasmid insertion into the bacteria. Four distinct growth plates were employed to simulate different growth conditions.

The first plate (-pGLO / LB) lacked ampicillin and contained bacteria without the plasmid, expected to show substantial growth as the bacteria could thrive on the LB broth. However, the colonies should not fluoresce due to a repressor protein binding to the GFP gene promoter, hindering its transcription until ARA is present, showcasing GFP gene regulation.

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The second plate (-pGLO / LB / AMP) was not anticipated to exhibit growth since the bacteria, lacking the pGLO plasmid, were not resistant to ampicillin. The third plate (+pGLO/ LB / AMP) should demonstrate moderate growth as the bacteria possess the plasmid, enabling them to grow on ampicillin and LB. However, the colonies' color should remain white due to the repressor binding to the operator in the absence of sugar, preventing GFP gene transcription. The fourth plate (+pGLO / LB / AMP / ARA) was expected to display glowing bacteria as the presence of ARA would deactivate the repressor, allowing GFP transcription.

Through this experiment, students gained insights into plasmids and gene regulation.

Observations of E.Coli Plates With and Without pGLO

Upon completing the experiment, the group confirmed the success of the transformation as evidenced by the glowing E. coli in the +pGLO / LB / AMP / ARA plate, indicating the acquisition of the plasmid with the GFP and AMP genes. The presence of arabinose sugar allowed an ARA inducer molecule to deactivate the repressor, enabling the transcription of the inserted GFP gene. The growth of bacteria in the +pGLO / LB / AMP plate further supported the successful transformation, demonstrating the functionality of the antibiotic resistance gene. Conversely, the absence of growth in the -pGLO / LB / AMP plate highlighted the importance of this antibiotic resistance gene for bacterial survival in an ampicillin-containing environment.

To enhance future experiments, the group recognized the need to improve the incubation time of the test tubes containing LB nutrient broth, as insufficient incubation may have impacted the results. Moving forward, the group aims to optimize the timing for better experimental outcomes.

In this experiment, the group achieved the production of genetically modified bacteria with desired phenotypes. The ability of each E. coli cell to regulate the expression of the GFP gene using an inducer molecule showcased the advantages of controlling gene expression. This regulatory capability allows cells to conserve energy by activating or deactivating specific genes based on the presence or absence of particular environmental factors. The experiment emphasized the potential applications of bacterial transformation, particularly in bioremediation, where genetically modified bacteria could be employed to eliminate pollutants in environments such as oil spills. Through genetic engineering, plasmids containing genes for pollutant digestion could be introduced into bacteria, enabling them to break down toxins and contribute to environmental cleanup, such as degrading hydrocarbons in oil.