Lab Report: Bacterial Transformation

Categories: Chemistry

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Abstract

In this lab, the objective was to induce the transformation of E. coli bacteria to exhibit resistance to ampicillin and to express green fluorescent protein (GFP) under the influence of arabinose. Four plates were prepared, including a control plate (-pGLO LB), a plate with +pGLO LB/Amp, a plate with +pGLO LB/Amp/Ara, and a plate with -pGLO LB/Amp. The results showed that the presence of the pGLO plasmid conferred resistance to ampicillin, and the combination of +pGLO with LB/Amp/Ara led to the expression of GFP, resulting in glowing bacteria under UV light.

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This experiment demonstrated successful genetic transformations and provided insights into gene regulation in bacteria.

Introduction

In this lab, the goal was to transform the bacteria E. coli to glow in the dark under a black light. This transformation involved manipulating the genetic makeup of the bacteria using plasmids. Four plates were set up with agar for the bacteria to feed on and grow.

These plates included:

A control plate (-pGLO LB): Containing only LB agar to serve as a baseline.
A plate with +pGLO LB/Amp: Containing LB agar and ampicillin to assess ampicillin resistance.
A plate with +pGLO LB/Amp/Ara: Containing LB agar, ampicillin, and arabinose to induce GFP expression and fluorescence.
A plate with -pGLO LB/Amp: Containing LB agar and ampicillin but lacking the pGLO plasmid.

The key experiment aimed to determine whether the presence of the pGLO plasmid would confer ampicillin resistance and whether the addition of arabinose would activate GFP production, causing the bacteria to glow in the dark.

The gene regulation process in bacteria plays a critical role in this experiment. Gene expression is controlled during transcription, where genes can be turned on or off. Repressors bind to operators in the cell, halting transcription. Gene activation occurs when a metabolite binds to the repressor, preventing it from binding to the operator, thus allowing transcription to proceed. The addition of specific substances to the bacteria in this lab aimed to manipulate this gene regulation process, turning the GFP gene on or off.

Materials and Methods

The complete list of materials and procedures can be found in the science manual for the Bacterial Transformation Lab.

Results

Plate	Contents	Colonies	Observations
+pGLO LB/Amp	pGLO, LB, Ampicillin	49	Colonies present; beige color
+pGLO LB/Amp/Ara	pGLO, LB, Ampicillin, Arabinose	53	Colonies present; glowing greenish color under UV light
-pGLO LB/Amp	LB, Ampicillin	None	No bacterial growth observed
-pGLO LB	LB	TNTC	Plate filled with beige bacteria

Analysis

Based on the results obtained, several conclusions can be drawn:

The plate with +pGLO LB/Amp exhibited resistance to ampicillin, as indicated by the presence of colonies. This resistance was due to the presence of the pGLO plasmid, which transformed the bacteria.
The plate with +pGLO LB/Amp/Ara displayed glowing bacteria under UV light, demonstrating the successful activation of the GFP gene. Arabinose played a crucial role in this process.
The plate with -pGLO LB/Amp showed no bacterial growth because the plasmid necessary for ampicillin resistance was absent.
The plate with -pGLO LB served as the control, with abundant bacterial growth, as no changes were made to the bacteria or the plate.

Additionally, this lab raises questions about genetic transformation in different organisms. It suggests that single-celled organisms are better suited for total genetic transformation due to their ability to efficiently take up new genes. Among the options presented (bacterium, earthworm, fish, or mouse), bacteria are the ideal choice for genetic transformation, given their single-celled nature and rapid reproduction rate.

Mathematical calculations were also involved in the analysis. For example, in the +pGLO LB/Amp/Ara plate, there were 53 colonies. To determine the total amount of pGLO DNA (μg), the concentration of DNA (μg/μl) was multiplied by the volume of DNA (μl). In this case, 0.08 μg/μl x 510 μl = 40.8 μg. The fraction of DNA used was calculated by dividing the volume spread on the LB/Amp plate (μl) by the total volume in the test tube (μl), resulting in 100/510 or 10/51, which equals 0.2. To find the pGLO DNA spread (μg), the total amount of DNA used in μg was multiplied by the fraction of DNA used, resulting in 40.8 μg x 10/51 = 8.16 μg. Finally, the transformation efficiency was calculated by dividing the total number of cells growing on the agar plate by the amount of DNA spread on the agar plate, resulting in 53/8.16, which equals approximately 6.5 as the transformation efficiency.

Conclusion

In conclusion, the Bacterial Transformation Lab was successful in achieving its objectives. The results confirmed that the presence of the pGLO plasmid conferred resistance to ampicillin and that the addition of arabinose induced the expression of GFP, resulting in glowing bacteria. The control plate exhibited typical bacterial growth with no genetic modifications.

The experiment was error-free, and the results were consistent with the expected outcomes. If repeated, the lab could be conducted on a larger scale to further investigate the effects of genetic transformations. This would provide valuable insights into the manipulation of genes in bacteria and the potential for various other gene expressions. Overall, the lab was both educational and enjoyable, demonstrating the transformation of E. coli into antibiotic-resistant and fluorescent bacteria.