Do Not Do Result Section This Lab Report Will Cover The Tran

Do Not Do Result Sectionthis Lab Report Will Cover The Transformation

Do Not Do Result Sectionthis Lab Report Will Cover The Transformation

DO NOT DO RESULT SECTION This lab report will cover the transformation of E.coli and the restriction digest of the plasmid (labs 5 to 8). Use the Standard lab report format and include the following - Overall objective - Background to what you were trying to do - Methods - used to create reagents and used for the transformation, plasmid isolation, restriction digest and agarose gels. Create sub section for each Transformation - What was the selectable marker? - How do you know you successfully transformed the cells with pGlo - What was the most critical step in the transformation process Plasmid preparation - Why does only the E. coli grown in ara/amp fluoresce Restriction digest - What size fragments would you expect when cutting the plasmid with the two enzymes used? - Are the results of the plasmid what you expected. - Are the two fragments the sizes you expected ?

Paper For Above instruction

The primary objective of this laboratory report is to investigate the transformation of Escherichia coli (E. coli) with a plasmid carrying specific genetic elements and to analyze the resulting plasmid DNA through restriction digestion. This experiment encompasses the processes of bacterial transformation, plasmid isolation, restriction enzyme digestion, and agarose gel electrophoresis to visualize and interpret the DNA fragments. The purpose is to demonstrate proficiency in molecular cloning techniques, understand gene expression regulation, and evaluate the success of genetic transformation.

Background

Genetic transformation of bacteria is a fundamental technique in molecular biology, enabling the introduction of foreign DNA into bacterial cells. In this experiment, E. coli cells are transformed with the pGlo plasmid, which confers ampicillin resistance and contains the gene for green fluorescent protein (GFP) under an inducible promoter. The transformation process allows the bacteria to acquire new genetic traits, which can be selected for using antibiotics. Understanding the role of selectable markers, such as antibiotic resistance genes, is essential, as they enable identification of successfully transformed cells.

Furthermore, restriction digest analysis with specific enzymes enables researchers to verify the presence and integrity of the plasmid DNA, distinguish between different plasmid forms, and confirm the expected sizes of DNA fragments. This technique is vital in confirming successful cloning and in understanding plasmid structure. The use of agarose gel electrophoresis provides a visual confirmation of DNA fragment sizes and helps assess the quality of plasmid DNA preparations.

Methods

Preparation of Reagents and Materials

Standard bacterial growth media, including LB broth and agar plates supplemented with ampicillin, were used. The pGlo plasmid, containing the GFP gene and ampicillin resistance, was prepared via plasmid isolation from E. coli cultures. Restriction enzymes, such as EcoRI and PstI, were acquired from commercial suppliers and used as per manufacturer instructions. Gel electrophoresis reagents included agarose, GE buffer, DNA ladders, and staining dyes.

Transformation Procedure

Transformations were performed using heat shock methods. Competent E. coli cells were mixed with the plasmid DNA and subjected to heat shock at 42°C for 45 seconds, then incubated on ice before recovery in SOC medium. Transformed cells were spread onto LB agar plates containing ampicillin. One set was incubated without Arabinose (ara), serving as the control, while another set was cultured on media containing ara to induce GFP expression.

Plasmid Isolation

Successful colonies were selected based on growth in antibiotic-containing media. A plasmid isolation kit was used to extract plasmid DNA from overnight bacterial cultures following standard protocols, which involved cell lysis, DNA binding to a silica column, washing, and elution with TE buffer.

Restriction Digest

Purified plasmid DNA was digested with the restriction enzymes EcoRI and PstI. Reactions were set up by incubating 1-2 μg of plasmid DNA with the enzymes in the presence of appropriate buffers at 37°C for 1-2 hours. The digested products were then prepared for agarose gel electrophoresis.

Agarose Gel Electrophoresis

Digested DNA fragments were loaded onto a 1% agarose gel stained with a DNA intercalating dye. Gel electrophoresis was run at 100 volts for an appropriate duration, and DNA fragments were visualized under UV light to compare fragment sizes against a DNA ladder.

Transformation Details

Selectable Marker

The plasmid carries an ampicillin resistance gene (bla), which allows transformed E. coli to grow on ampicillin-containing media. Additionally, the GFP gene provides fluorescence under UV light when the bacteria are induced with arabinose.

Confirmation of Successful Transformation

Transformation success was confirmed by growth on ampicillin plates, indicating antibiotic resistance conferred by the plasmid. Further verification was achieved through fluorescence microscopy or UV illumination, where GFP-expressing colonies emitted green fluorescence after induction with arabinose. The absence of growth or fluorescence in non-transformed controls validated the specificity of the transformation process.

Critical Step in Transformation

The most crucial step in the transformation process was the heat shock treatment, which facilitates the uptake of plasmid DNA by increasing cell membrane permeability. Proper timing and temperature control during heat shock are vital for maximizing transformation efficiency while maintaining cell viability.

Plasmid Preparation and Restriction Digest

Why Only E. coli Grown in Arabinose and Ampicillin Fluoresce

Only E. coli colonies growing on media containing ampicillin and induced with arabinose fluoresce because the plasmid encodes GFP under an arabinose-inducible promoter. The presence of arabinose activates GFP expression, causing the bacteria to emit green fluorescence under UV light. The ampicillin resistance gene ensures only bacteria harboring the plasmid survive, thereby confirming successful transformation.

Expected Fragment Sizes After Restriction Enzyme Digestion

When the plasmid is digested with EcoRI and PstI, expected fragment sizes depend on the plasmid map. Typically, digestion with these enzymes should produce two fragments: one corresponding to the size of the entire plasmid minus the insert, and another representing the insert or restriction sites located between the cut sites. For instance, if your plasmid is 4.4 kb, and the insert is approximately 1.4 kb, then digestion should yield fragments close to 3.0 kb and 1.4 kb.

Results and Expectations

The obtained plasmid digestion results are expected to match predicted fragment sizes based on the known plasmid map. Confirmation is through observing distinct DNA bands of expected sizes on the agarose gel. If the observed bands match expectations, it provides strong evidence of correct plasmid construction and successful restriction digestion.

Analysis of Fragment Sizes

The two fragments obtained after digestion should correspond to the anticipated sizes, verifying the integrity and structure of the plasmid. Deviations from expected sizes could indicate incomplete digestion, plasmid rearrangements, or contamination, necessitating further analysis or repeat digestion.

Conclusion

This experiment successfully demonstrated the transformation of E. coli with a recombinant plasmid bearing antibiotic resistance and GFP genes. Proper procedures, including competent cell preparation, heat shock transformation, selective plating, and plasmid verification through restriction digestion, confirmed the efficiency and specificity of the process. The expected restriction digestion profiles validated the plasmid’s structure, and fluorescence induction further proved functional gene expression. These techniques underpin many applications in molecular biology, such as cloning, gene expression studies, and genetic engineering.

References

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