Transformation of Bacteria & Yeast

🔁 Why Introduce DNA into Cells?

Recombinant DNA must be inside a living host to:

• Be replicated for purification

• Be expressed into protein

• Be protected from degradation

➡ Plasmids & phages are the delivery vehicles
➡ Bacteria & yeast are the production factories

🦠 Transformation in Bacteria

📚 Key Terminology

🧪 Why Use Bacteria?

• Each bacterial colony originates from one cell + one plasmid

• Allows isolation of specific recombinant DNA

• Enables protein production (e.g. insulin in E. coli)

⚡ Methods of Bacterial Transformation

🧊 Chemical Competency (Heat Shock)

1. Treat bacteria with divalent cations (Ca²⁺, Mg²⁺) to neutralize charge

2. Incubate with DNA on ice

3. Heat shock at 42 °C → DNA enters

4. Add media to recover

5. Plate on selective antibiotic media

⚡ Electroporation

1. Wash cells in low-salt buffer

2. Apply electric field to form pores

3. DNA enters

4. Recovery & plating same as chemical method

✅ Higher efficiency than heat shock
✅ Useful for large plasmids or library construction

🧬 Selection Using Plasmids

🔬 pBR322

• Contains Amp^R and Tet^R

• Insert gene into resistance site → disrupts it

• Requires replica plating to identify insertion

🔵 pUC Plasmids – Blue-White Screening

Requires lacZ′⁻ bacterial strain.

🧬 Phage DNA Introduction

🧬 M13 & Phagemid Transformation

• M13 RF DNA behaves like plasmid → transformed normally

• Phagemids (e.g. pUC8 + M13) also follow plasmid method

• Need helper phage to produce phage particles

🧬 λ Phage Vectors

• Inefficient to transform as plasmid

• Instead:

  • Assemble phage in vitro

  • Infect bacteria with packaged phage

🧫 Visualising Infection – Plaques

🍞 Transformation of Yeast

🚫 Challenge: Yeast Have a Cell Wall

• DNA can’t enter unless wall is removed

• Solution: Create spheroplasts

🧪 Generating Spheroplasts

⚠ Only 1 copy of plasmid is taken up per cell
✅ Yeast can accept huge plasmids (up to 2 Mbp)

✅ Plasmid Maintenance & Selection in Yeast

• Yeast don’t respond to antibiotics

• Use auxotrophic selection

🧪 Selectable Marker Genes

➡ Grow on poor media lacking marker → only transformed yeast survive

Example:

• URA marker plasmid

• Grows on rich media (YEPD)

• Also grows on minimal media (YNBG) only if plasmid is present

🧪 Other Yeast Transformation Methods

• Electroporation

• Microinjection

• Gene gun (biolistics)

✅ Summary Table: Bacterial & Yeast Transformation