Experiment 15- Bacterial Transformation BIOL221 Lab

  • REVIEW: In Experiment 14, students learned about vertical and horizontal gene transfer.

    • Horizontal Gene Transfer: Transfer of genetic information between organisms of the same generation, without reproduction.

      • Example: Conjugation= involves direct cell-to-cell contact, where a donor bacterium transfers genetic material to a recipient cell through a pilus.

  • Transduction: Transfer of genetic material from one bacterium to another via a bacteriophage.

    • Transformation: Bacteria take up free DNA from the environment.

Natural Competence

  • Definition: The ability of certain bacteria to take up free DNA from their environment.

    • Not all bacteria possess natural competence; Only NATURALLY COMPETENT bacteria have this ability

  • Characteristics of Naturally Competent Bacteria:

    • can benefit/harm the bacteria (depending on the type of free DNA taken)

  • Benefits and Risks of DNA Uptake:

    • Benefits:

    • Can utilize free DNA as nutrient sources.

    • Can aid in chromosome repair.

    • Can lead to acquisition of new genotypes.

    • Risks:

    • Depends on the type of free DNA taken.

Membrane-Bound Proteins (Natural Competence) CONT…

  • Naturally competent bacteria utilize double-stranded free DNA and integrate it into their genome.

  • Mechanism:

    • DNA uptake through membrane-bound pore-forming proteins.

    • Once DNA is inside, one strand is degraded while the other is incorporated into the genome via recombinases.

  • Process Overview:

    • Pore-like proteins (secretins/pores) may be initially blocked by specialized bacterial proteins until free DNA is detected.

    • When free DNA is present, DNA-pulling proteins transport the strand further into the cell.

Chemical Competence

  • Scientists can replicate natural competence in laboratory settings through bacterial transformation. This process involves introducing foreign DNA into bacteria, allowing them to take up and express new genetic material.

  • Applications:

    • Cloning DNA and producing significant quantities of proteins for research or drug development.

    • Example: Inducing bacteria to produce human insulin for diabetes treatment.

  • Types of Transformation Techniques:

    • Electroporation

    • Chemical Transformation

Electroporation

  • Method: Utilizes an electric pulse via an electroporator machine to facilitate uptake of foreign DNA.

  • Mechanism:

    • Electric shock temporarily disturbs the bacterial cell membrane, allowing free DNA entry.

    • Visual materials may elaborate on the function and use of an electroporator.

Chemical Transformation

  • Method: Uses negatively charged salts (e.g., Calcium Chloride [CaCl2]) to assist free DNA (e.g., pGlo plasmid) adhere to the positively charged bacterial cell membrane.

  • Process:

    • Necessitates heat shock (e.g., 42°C for a brief period) to loosen the membrane, allowing the adhered free DNA to permeate into the cell.

    • Post-heat shock, cooling with ice helps restore membrane rigidity and seal openings.

  • Equation/Process Example:

    • CaCl2+extplasmidCaCl_2 + ext{plasmid}

    • Result: Membrane loosens, plasmid can enter (Temperature: 42°C for 45 seconds).

Overview of pGlo Plasmid

  • The pGlo plasmid contains several critical components:

    • Origin of Replication (ori): Facilitates self-replication of the plasmid in host bacteria.

    • Ampicillin Resistance Gene (aka..AmpR, bla): Provides resistance to ampicillin.

    • Arabinose Operon Components:

    1. araC: Gene encoding for regulatory protein araC.

    2. PBAD Promoter Site: Also known as araBAD; RNA polymerase binds to this site in the presence of arabinose for transcription to RNA.

    3. Green Fluorescent Protein (GFP): Encodes a protein that emits green fluorescence under UV light.

  • Transformation in Lab Context: Involves chemical transformation using pGlo plasmid.

The Arabinose Operon

  • Regulatory Mechanism: The operon is an example of both positive and negative gene regulation.

  • araC Structure:

    • Without Arabinose: araC remains UNCHANGED, resulting in repression of transcription by a repressor protein due to binding to the operon.

    • With Arabinose: The binding of arabinose CHANGES araC's conformation, leading to activation of transcription and production of PBAD enzymes.

Positive Gene Regulation Process

  • Activation:

    • Transcription of genes by an activator protein occurs when arabinose is bound to araC.

    • Enzymes Produced: araB, araA, and araD (aka..pBAD) enzymes for processing arabinose are generated.

Negative Gene Regulation Process

  • Inhibition:

    • Transcription of genes is suppressed by a repressor protein when araC is unbound by arabinose.

    • Enzyme Production: No enzymes are produced in the absence of arabinose.

Special Media

LB Agar + Amp + ARA

  • Nutrient Agar Specifics:

    • Contains ampicillin to inhibit growth of non-transformed cells.

    • Negative Control: Bacteria without pGlo or those that did not transfect will not grow due to lack of ampicillin resistance.

    • Positive Control: Bacteria transformed with pGlo are expected to grow, but without arabinose, they won't fluoresce.

    • LB Agar + Amp + ARA: Includes both ampicillin and arabinose; transformed bacteria grow and fluoresce under UV light.

Expected Results

Media Type: