Bacterial Transformation:

Background:

The Discovery of Transformation:

In the early 1900s, pneumonia was causing many deaths

What is a plasmid vector?

• A lab-made plasmid used to insert new genes into bacteria

• A typical recombinant plasmid has:

  • ORI: where replication starts

  • Antibiotic resistance gene: to help us identify which bacteria took in the plasmid

  • Multiple cloning site: a spot where we can cut and insert DNA

  • Promoter: starts transcription of the inserted gene

  • Selection Marker: helps us detect successful transformation (like glowing proteins or antibiotic resistance)

How does transformation work?

1. Bacteria are treated to make their membranes leaky (like with calcium chloride and heat shock)

2. The plasmid enters the bacterial cell

3. Only bacteria with the plasmid survive on antibiotic plates

Griffith’s Experiment (Early 1900s)”

He used two different strains to perform an experiment

• S-strain = smooth = has a protective capsule = virulent (harmful)

• R-strain = rough = no capsule = non-virulent

Steps:

• First step: Griffith took some of the S-strain bacteria and heated it, creating a Heat-killed S-strain

• Second step: He gathered some mice and divided them into four groups

  1. Inject S strain into mice —> Mice died

  2. Inject Non-virulent R strain —> Mice lived

  3. Inject heat-killed S strain —> Mice lived

  4. Inject R-strain + heat-killed S-strain —> Mice died

• Conclusion: Something from the dead S-strain turned the harmless R- R-strain deadly

• This “transforming principle” passed the ability to make the capsule

What Was the Transforming Principle?:

• Later, Avery, Maclead, and McCarty showed it was desoxyribonucleic acid (DNA)

  So what happened in his experiment?:

• DNA from the S-strain got into the R-strain, transforming it!

Modern Transformation Using E.coli:

• What We Use:

  • E. coli bacteria (easy to work with)

  • Plasmids = Small circles of DNA that can carry genes (like antibiotic resistance)

• How It Works:

• E.coli are made competent meaning they can take up DNA from the environment

  • This means under certain environmental conditions like a chemical, electrical, or heat shock, the cell wall can become temporarily permeable and allow the uptake of DNA from the environment

• They take in the plasmid from their surroundings, which can either be in the cytoplasm or a new host cell

• If the bacteria loses the plasmid at any point, the cell will also lose its antibiotic resistance

• If the plasmid has an antibiotic resistance gene, the bacteria will survive in antibiotic-containing media

In Today’s Lab:

• We will learn how to transform E.coli cells with a plasmid containing an antibiotic resistance gene while practicing microbiological techniques