Laboratory 6: Intro to Genetic Engineering: Bacterial Transformation

Laboratory 6: Intro to Genetic Engineering: Bacterial Transformation

Core Concept of Biology: Information Flow

Learning Goals:

• Determine what we mean by and explore our thoughts on genetic engineering.

• Describe how transforming the genetic makeup of an organism with cell machinery changes their traits in different environments, relating this to the central dogma of molecular biology.

• Use micropipettes and common molecular biology tools.

Important Terms

1. Bacteria

2. Plasmid

3. Genetic engineering

4. Transformation

5. Competency

6. pGLO

7. Negative control

8. Positive control

9. Aseptic Techniques

Safety Guidelines

• Use aseptic technique when handling E. coli culture!

  • The strain is non-pathogenic, meaning it does not cause disease, but lab safety procedures must still be followed.

• Close boxes of pipette tips unless you are using them within the next few seconds to avoid contamination from airborne microbes.

• Dispose of plastic materials such as microcentrifuge tubes, pipette tips, and E. coli culture plates in the BSL1 (dead/biohazard) bucket.

• Consult with your instructor on the disposal methods for LB broth tubes and transformation solution tubes (plastic goes into the BSL1 bucket, glass into the wire racks in the fume hood).

• Return pipette boxes and pipettes to the prep counter. Dispose of any KimWipes used. Rinse waste beakers and return them to the glassware cabinets.

• Wipe down lab benches with Ethanol (Et-OH/alcohol) solution to maintain a clean workspace.

Introduction to Genetic Engineering

• Definition:

  • Genetic engineering refers to the artificial and direct alteration of an organism's DNA, which can lead to traits that might not otherwise be present but are beneficial to humans.

• Distinction from Natural Processes:

  • Genetic engineering differs from natural genetic recombination processes like independent assortment, crossing over, transduction, or bacterial conjugation.

• Benefits vs Risks:

  • Genetic engineering is a tool that can have positive applications, such as the creation of vaccines and nutrient-rich crops (e.g., golden rice).

  • However, it also presents risks, such as genetically modified organisms (GMOs) that can disrupt ecosystems (e.g., fish that escape pens) or lead to pest resistance cycles in agriculture.

  • Longitudinal data on the long-term health impacts of genetic engineering are currently lacking.

• Clarification:

  • Practices like artificial selection and hormone application are not considered genetic engineering, as they do not involve direct modifications to DNA.

Characteristics of Prokaryotic Cells

• Genetic Simplicity:

  • Prokaryotic cells, such as bacteria, are easier to genetically engineer because they do not have membrane-bound organelles; their genetic material is not enclosed within a nucleus.

  • Most bacterial genetic material exists as a large, circular chromosome, which encodes essential genes for the bacterium's function and identity.

• Plasmids:

  • Bacteria can take up small circles of DNA known as plasmids. These plasmids can contain genes that confer particular traits.

  • Transformation refers to the process where bacterial cells take up plasmid DNA, allowing them to use their cellular machinery (like RNA polymerase and ribosomes) to express the genes contained within the plasmids.

• Competency:

  • Cells are termed naturally competent if they can take up plasmid DNA on their own. However, it is also possible to artificially induce competency in cells in laboratory settings.

Transformation Procedure Overview

Transformation Steps

1. Add bacterial cells to plasmid DNA and transformation solution that neutralizes the charges of the DNA and the bacterial cell walls.

2. Heat-shock the mixture to introduce the plasmid DNA into the cells.

3. Plate the transformed bacteria on different types of solid LB agar, which contains various combinations of antibiotics and sugars to determine which cells successfully took up the plasmid.

Bacterial Transformation Process

Organism Used

• Escherichia coli:

  • Commonly referred to as E. coli; the strain used in this experiment is non-pathogenic.

Plasmid Used

• pGLO Plasmid:

  • This plasmid is conventionally named with a lowercase “p” followed by a description of the traits encoded (e.g., glowing). It is small and serves as a reporter gene in research.

  • Focus genes on pGLO:

  • bla (beta-lactamase gene):

  • Confers antibiotic resistance, allowing survival of bacteria on media containing antibiotics like ampicillin.

  • araC:

  • Acts as a switch, controlling the expression of the gfp gene based on the presence of arabinose in the media.

  • gfp (Green Fluorescent Protein):

  • Codes for a protein that causes the bacteria to glow under ultraviolet (UV) light when arabinose is present in the LB medium.

Experimental Controls

• Negative Controls:

  • These are samples where no change is expected (e.g., plates that should not show bacterial growth).

• Positive Controls:

  • These are samples where an effect is expected to occur (e.g., plates where bacterial growth should be seen without additional variables).

Lab Activities

Preparation for Bacterial Structure Lab (Collecting Microorganisms)

• Microorganisms are present in the environment and can be enriched using nutrient agar for visibility as colonies.

• Factors for sampling:

  • Consider the environmental conditions of sampling locations (temperature, moisture, food sources).

Sampling Protocol

1. Obtain a nutrient agar plate and label it appropriately on the side with agar.

2. Identify two sampling sites to collect bacteria from. Do not sample sites that can be dangerous (e.g., nose, mouth).

3. Use sterile swabs for sampling and inoculating the agar plate.

4. Dispose of swabs in the biohazard bucket.

5. Return the inoculated plate to the proper section tray.

Bacterial Transformation Experiment

Materials Needed

• Starter plate with E. coli

• LB plates (basic media)

• LB plates with ampicillin

• LB plates with ampicillin and arabinose

• pGLO plasmid solution

• Sterile inoculating loops

• Transformation solution (blue tube TS)

• LB nutrient broth (red tube)

• Micropipettes (P1000 and tips)

• Microcentrifuge tubes

• Ice bath

• 42°C water bath and 37°C incubator

Transformation Protocol

1. Label microfuge tubes: "pGLO" and "no plasmid"; keep on ice.

2. Add 250µL of transformation solution to each tube using a P1000 pipette.

3. Use a sterile loop to transfer E. coli colony to both microfuge tubes, mixing gently.

4. Add approximately 10µL of pGLO plasmid to the "pGLO" tube; mix gently.

5. Heat shock in a 42°C water bath for 50 seconds and then return to ice for 2 minutes.

6. Add 250µL of LB to each tube and let sit at room temperature for 10 minutes.

7. Plate transformations from both tubes onto respective LB plates and label accordingly.

Disposal and Cleanup

• Properly dispose of all waste materials and wipe down work surfaces with ethanol.

Case Study: CRISPR/Cas9 - A Modern Breakthrough

• CRISPR/Cas9 Mechanism:

  • Discovered in 2007 by researchers including Dr. Jennifer Doudna, CRISPR is a series of DNA repeats that integrates viral DNA to protect against viruses.

  • Cas9 proteins can recognize and cut viral DNA, allowing for precise modifications to DNA sequences.

• Applications:

  • This technology acts as a gene-editing tool that offers potential treatment options for conditions like Huntington's Disease by allowing the repair of flawed genes.

  • The system's efficacy depends on the ability to cut and repair specific DNA sequences.

• Cellular Considerations:

  • Cells lacking the p53 transcription factor respond better to CRISPR editing. p53 is a crucial protein that regulates the cell cycle and prevents cancer.

  • Higher rates of editing can be associated with an increased risk of cancer due to the possibility of unintentional mutations during the repair process.