Genetic Transformation Overview

Genetic Transformation Overview

• Genetic Transformation:

  • The process where an organism’s genetic material is altered through the introduction of foreign DNA, crucial in biotechnology.

  • Natural occurrence seen in sexual reproduction and bacteria acquiring DNA from other bacteria/environments.

• Significance in Biotechnology:

  • Agriculture: Genetically modified crops for frost/pest resistance (e.g., antifreeze proteins in strawberries).

  • Bioremediation: Bacteria engineered to break down pollutants.

  • Medicine: Gene therapy to treat genetic disorders (e.g., cystic fibrosis).

Plasmids

• Definition: Small, circular DNA molecules present in bacteria, separate from chromosomal DNA.

• Function: Act as vectors to transfer genes; can facilitate horizontal gene transfer through bacterial conjugation.

• Example: pGLO plasmid, used in experiments; contains genes for Green Fluorescent Protein (GFP) and beta-lactamase (bla).

Antibiotics and Selection

• Antibiotics: Compounds that inhibit bacterial growth or kill bacteria (e.g., ampicillin inhibits cell wall synthesis).

• Role in Transformation: Resistance genes in plasmids (like bla) serve as selectable markers; only transformed bacteria survive on antibiotic media.

Transcription Factors

• Definition: Proteins that regulate gene expression by binding to DNA sequences (promotors) and influencing RNA polymerase activity.

• Example: AraC protein in pGLO, activating GFP transcription in the presence of arabinose.

Experimental Procedure: Transformation of E. coli

• Heat-shock transformation: Method to introduce plasmid (pGLO) into E. coli following specific temperature changes.

• Key Steps:

  1. Prepare transformation tubes (GLO and -pGLO) with calcium chloride solution.

  2. Add E. coli colonies to the tubes.

  3. Introduce the pGLO plasmid to the +pGLO tube only.

  4. Subject tubes to heat shock (42°C for 50 seconds) followed by rapid cooling.

  5. Allow recovery with nutrient broth.

  6. Plate on selective media (LB agar with/without ampicillin and arabinose)

Regulation of GFP Expression

• Arabinose Presence: Activates transcription factor AraC, inducing GFP production; no arabinose results in no GFP expression, thus no fluorescence.

• Expected Results:

  • Transformed cells containing pGLO will fluoresce when grown on media with arabinose; survival only occurs in the presence of ampicillin due to the bla gene.

Review Questions

1. LB Agar: Nutrient medium for bacterial growth, supporting transformation and selection.

2. Ampicillin: Antibiotic for selecting transformed bacteria expressing beta-lactamase from the pGLO plasmid.

3. pGLO: Plasmid containing genes for GFP and antibiotic resistance.

4. Arabinose: Sugar inducing transcription of the GFP gene.

5. Predictions about bacterial growth and transformation efficiency on different plates based on the experimental design.

Key Terms to Understand

• Gene: DNA segment coding for proteins.

• Plasmid: Circular bacterial DNA that can replicate independently.

• Horizontal Gene Transfer: Genetic material movement outside of standard parent-offspring transmission.

• Selectable Marker: Gene assisting in identifying transformed cells.

• RNA Polymerase: Enzyme that transcribes DNA into RNA during gene expression.

Experimental Material List

• E. coli starter plate

• LB agar plates (different variations)

• Transformation solution (TS)

• LB nutrient broth

• Inoculation loops, disposable pipets, micro test tubes, and other lab items.