Bacterial Transformation – bacteria take up and express
foreign DNA, usually a plasmid.
•Plasmid – circular piece of DNA
Steps of Bacterial Transformation
1. Choose a bacterial host.
a. E.coli is a model organism.
i. Well studied
ii. No nuclear membranes
iii.Has enzymes necessary for replication
DNA/
Chrom.
Steps of Bacterial Transformation
2. Choose a plasmid to transform.
a.
Characteristics of a useful plasmid.
i. Single recognition site
• Plasmid only cuts in one place, so this ensures that the
plasmid is reformed in the correct order.
ii. Origin of replication
• Allows plasmid to replicate and make copies for new cells.
iii. 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.
Steps of Bacterial Transformation
3. Prepare bacterial cells for transformation of plasmid.
a. Treat with calcium chloride, which allows plasmid to pass
through bacterial cell walls. This is the most common
method.
b. Electroporation - brief electric pulse
c. Directly inject plasmid into bacterial cell.
Steps of Bacterial Transformation
4. Plate transformation solution on appropriate media.
a. Contains nutrients for bacteria.
b. Contains antibiotic to distinguish transformed bacteria
from NONtransformed bacteria.
5. Incubate plates overnight.
a. E.coli grows in the human body,
and is therefore incubated at
body temperature (37°C)
6. Analyze plates.