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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 AntibioticsBacteria are often used
in Biotech to study genes. Why use bacteria?
Simple to grow
Grow rapidly
24 hours
370C
Bacteria and AntibioticsTo 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 AntibioticsAntibiotics – 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 – beta-galactasidase 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 –
GreenFluorescentProtein
Also ampr
What DNA will we transform into cells?
pUWL500
luciferase andampr
What DNA will we transform into cells?
pGEM3z-
Just 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