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