Genetic pGLO Transformation Laboratory Report

Abstract

The laboratory experiment focused on genetic transformation, a process where cells take up and express new genetic material, specifically DNA. In this experiment, we used the pGLO transformation kit to introduce a gene that codes for Green Fluorescent Protein (GFP) into E. coli bacteria. Genetic transformation allows organisms to acquire new traits, which can be visually identified after the transformation. This experiment not only demonstrates the concept of genetic transformation but also highlights the use of green fluorescent protein for quick determination of protein expression.

Introduction

Genetic transformation is a fundamental process in molecular biology, wherein cells acquire and express new genetic material, typically DNA, resulting in altered traits. This experiment aimed to demonstrate genetic transformation using E. coli bacteria and the pGLO transformation kit, which introduces the Green Fluorescent Protein (GFP) gene into the bacterial cells.

After the transformation, the altered E. coli cells should exhibit observable changes, such as the ability to fluoresce green when exposed to specific conditions.

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This experiment not only serves as an educational tool to illustrate genetic transformation but also provides students with hands-on experience in genetic engineering techniques.

Materials and Methods

The genetic transformation experiment was conducted using the following materials:

• E. coli bacterial culture
• pGLO plasmid DNA
• Luria-Bertani (LB) nutrient agar plates
• Ampicillin
• AraC (Arabinose)
• UV light source

The procedure was as follows:

1. Starter plates containing E. coli colonies were examined for observable traits, including color, number, and distribution of colonies.
2. A sample of the original pGLO plasmid DNA was exposed to UV light to check for fluorescence.
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3. E. coli colonies were grown on LB plates, some containing ampicillin and arabinose, and some without ampicillin.
4. The bacterial growth on each plate was observed, noting the number of colonies and their appearance.
5. The color of the bacterial colonies was examined under normal room lighting and long-wave UV light.

Data Analysis

The data collected from the experiment included the number and appearance of bacterial colonies on different plates, as well as the presence or absence of fluorescence in the pGLO plasmid DNA sample.

Results

The results of the experiment are as follows:

• The pGLO plasmid DNA sample did not fluoresce under UV light.
• On the LB plates, E. coli colonies were observed on both plates with and without ampicillin.
• LB/amp and LB/amp/ara plates had numerous colonies, while the LB (-) pGLO plate displayed a lawn of bacteria, making individual colony counting impossible.
• The color of bacterial colonies was whitish on the (+) pGLO/amp plate and the (-) pGLO plates. However, the colonies on the (+) pGLO/LB/amp/ara plate appeared white under normal room lighting but fluoresced green when exposed to long-wave UV light.

Approximately 75 colonies were observed on each of the two (+) pGLO plates, while the LB plate exhibited a uniform dispersion of bacteria without distinguishable individual colonies.

Discussion

The experiment aimed to investigate the effect of ampicillin on the growth of E. coli. The presence of bacterial colonies on the LB/amp plate suggests that some bacteria were resistant to ampicillin, indicating that ampicillin did not completely inhibit their growth. This observation contradicts the expectation that ampicillin should restrict bacterial growth or eliminate it.

The LB (-) pGLO plate displayed bacterial colonies that resembled non-transformed E. coli. These bacteria were not treated with the pGLO plasmid and, therefore, did not express any new genetic traits. They were similar to the non-transformed E. coli present on the starter plates.

Transformed cells, which have taken up the pGLO plasmid and express the ampicillin resistance gene, are expected to grow on the LB/amp/ara plates. The presence of bacterial colonies on these plates confirms the successful genetic transformation, as these colonies possess the ability to grow in the presence of ampicillin.

Comparing the LB/amp (-) pGLO and the LB/amp (+) plates allows us to determine if genetic transformation occurred. Cells not expressing the ampicillin resistance gene (LB/amp (-) pGLO) did not grow on the LB/amp plates. In contrast, cells treated with the pGLO plasmid (LB/amp (+)) exhibited growth on the LB/amp plates. This demonstrates that the pGLO plasmid contains a gene for ampicillin resistance.

Conclusion

The genetic transformation experiment successfully illustrated the process of introducing new genetic material into E. coli cells. The use of the pGLO transformation kit allowed for the visualization of genetic transformation through the expression of the GFP gene. This visual indicator, along with the growth of bacteria on selective plates, confirmed the successful genetic transformation of E. coli.

Recommendations

For future experiments involving genetic transformation, it is essential to further investigate the unexpected growth of E. coli on ampicillin-containing plates. Understanding the mechanisms behind ampicillin resistance in certain bacterial colonies can provide valuable insights into the interaction between antibiotics and bacteria. Additionally, students should be encouraged to explore the practical applications of genetic transformation in biotechnology and genetic engineering.

References

1. Komives, C., Rech, S., & Mcneil, M. (2004). Laboratory Experiment on GENE SUBCLONING For Chemical Engineering Students. Retrieved 28 December 2007, from http://www.engr.sjsu.edu/ckomives/Courses/Biochemical%20Engineering%20Laboratory/additional%20materials/published%20article.pdf
2. Reynolds, J. (2004). Bacterial Transformation. Retrieved 28 December 2007, from http://www.rlc.dcccd.edu/mathsci/reynolds/micro/lab_manual/transformation.html