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Zone of Inhibition
Area around
antibiotic disk
with no bacterial
growth.
Bacteriocidal – no bacteria living in ZOI.
Bacteriostatic – bacteria are not
reproducing in ZOI.
Note the lawn of bacteria.
Bacteria and Antibiotics
Bacteria – single-celled
organisms that are
prokaryotic (no true
nucleus)
Bacteria and Antibiotics
Bacteria are often used in
Biotech to study genes.
Why use bacteria?
Simple to grow
Grow rapidly
24 hours
370C
Bacteria and Antibiotics
To grow bacteria, need:
1. Correct nutrients – LB
agar (solid) or LB broth
(liquid).
2. Correct temperature –
370C.
3. Can divide every 20
minutes.
1. Takes time to adjust.
2. 2n
3. Run out of nutrients
Bacteria and Antibiotics
Antibiotics – any chemical which
prevent bacteria from growing.
1. Bacteriocidal - kills bacteria.
2. Bacteriostatic - prevents bacteria from
reproducing. Originals
are not dead!
Sterile Technique
Preventing
contamination:
- of you by
experiment.
- of experiment by
you.
Ex. Gloves, keeping lids closed, alcohol, flame.
Transformation
Changing an
organism’s traits
by adding a new
gene.
Why bacteria?
Simple organisms with few genes. One
change in genotype will probably lead to
change in phenotype.
Ways to get DNA into a cell:
Electroporation
Using electricity to
poke holes in cell
membrane; DNA
rushes into cell.
Ways to get DNA into a cell:
Virus Infection
- Remove disease
causing genes
from virus.
- Insert gene of
interest.
- Virus “infects”
cell with new
gene.
Ways to get DNA into a cell:
Gene Gun
- DNA attached to
gold pellet.
- Pellet fired into
cell.
- Typically used
with plants.
Why?
Gene Gun Animation
Ways to get DNA into a cell:
Heat Shock
Using heat to open
pores in cell
membrane. (see
sketch on board).
Transformation Efficiency
# of bacterial cells on plate
# bacterial cells possible on plate
For heat shock, this number is <0.01%
How do you know which cells
have been transformed?
Selectable marker – gene which gives an
obvious phenotype to transformed cell and
allows only transformed cells to survive.
Ex. Antibiotic resistance genes
What DNA will we transform into cells?
pVIB
luciferase – gene
encoding
bioluminescence
ampr = ampicillin-resistance gene – betagalactasidase which breaks down cell wall
Any bacteria with this gene can grow in presence
of ampicillin (antibiotic)
What DNA will we transform into cells?
pGREEN
GFP –
Green
Fluorescent
Protein
Also ampr
What DNA will we transform into cells?
pUWL500
luciferase and
ampr
What DNA will we transform into cells?
pGEM3zJust ampr
What type of bacterial cells are we
going to transform?
E. Coli that are
“competent”.
Competent –
ready to be
transformed.
How are we going to make competent
cells?
Treat cells with CaCl2
In solution CaCl2  Ca2+
+
2 Cl-
Positively charged Ca2+ coats negatively
charged pores. DNA can move into cell.
Heat Shock Animation