1. Bacterial Transformation – bacteria take up and express foreign DNA, usually a plasmid.
Plasmid – circular piece of DNA

2. Steps of Bacterial Transformation
Choose a bacterial host.
E.coli is a model organism.
Well studied
No nuclear membranes
Has enzymes necessary for replication
DNA/
Chrom.

3. Steps of Bacterial Transformation
2. Choose a plasmid to transform.
Characteristics of a useful plasmid.
Single recognition site
Plasmid only cuts in one place, so this ensures that the plasmid is reformed in the correct order.
Origin of replication
Allows plasmid to replicate and make copies for new cells.
Marker genes
Identifies cells that have been transformed.
 gene for antibiotic resistance – bacteria is plated on media
with an antibiotic, and only bacteria that have taken up a
plasmid will grow
 gene that expresses color – bacteria that have taken up a
recombinant plasmid are a different color than bacteria
that have taken up a NONrecombinat vector.

4. Steps of Bacterial Transformation
3. Prepare bacterial cells for transformation of plasmid.
Treat with calcium chloride, which allows plasmid to pass through bacterial cell walls. This is the most common method.
Electroporation - brief electric pulse
Directly inject plasmid into bacterial cell.

5. Steps of Bacterial Transformation
4. Plate transformation solution on appropriate media.
Contains nutrients for bacteria.
Contains antibiotic to distinguish transformed bacteria from NONtransformed bacteria.
5. Incubate plates overnight.
E.coli grows in the human body,
and is therefore incubated at
body temperature (37°C)
6. Analyze plates.