Genetic Transformation of E. coli by the P. glo Plasmid

Overview

• Discusses the process of genetic transformation in E. coli using the P. glo plasmid.
• Experiment focuses on altering the DNA of E. coli to make it resistant to ampicillin and cause it to fluoresce.

Key Concepts

Transformation

• Definition: Changing the genetic makeup of an organism.
• Applied to E. coli, a genus and species, Escherichia coli.
  • Characteristics:
    • Reproduces rapidly (up to 1000 times/day).
    • Small, inexpensive, and readily available.
    • Certain strains can cause illness, but not the one used here.
    • E. coli are prokaryotes (no nucleus, have a nucleoid).

Plasmids

• Small, circular pieces of DNA, separate from the bacterial chromosome.
• Can be transferred between bacteria.
• Self-replicating: Can multiply independently within a bacterial cell.

Chemicals Involved

• LB (Luria Broth): Nutrient medium for growing E. coli.
• AMP (Ampicillin): Antibiotic that kills bacteria, used to test resistance.
• ARA (Arabinose): Sugar that serves as a food source, used for gene induction.

The P. glo Plasmid

• Trademark plasmid with two main genes:
  1. BLA gene:
     • Codes for β-lactamase enzyme.
     • Confers resistance to ampicillin by breaking it down.
  2. GFP gene:
     • Codes for Green Fluorescent Protein.
     • Causes cells to fluoresce under black light.

Methods for Introducing Plasmids

• Competence: Making E. coli able to take up plasmids, achieved by:
  • Chemical means: Using calcium chloride (transformation solution).
  • Physical means: Heat shock to facilitate plasmid uptake.

Gene Induction

• Inducers: Chemicals that activate specific genes to produce proteins.
  • AMP: Induces β-lactamase gene to confer antibiotic resistance.
  • ARA: Induces GFP gene to produce fluorescent protein.

Mechanism

• Promoters at the start of genes are key to inducing protein production.
• Arabinose typically induces the production of arabinase.
• In this experiment, the arabinose promoter is repurposed to produce GFP.

Experimental Task

• Observe the transformation and effects of the P. glo plasmid on E. coli.

Note: This is a summary for Experiment 9, highlighting key processes and expected observations.