Bacteria Bacteria – single-celled organisms that are prokaryotic (no true nucleus)

Bacteria

Bacteria – single-celled organisms that are prokaryotic (no true nucleus)

BacteriaBacteria are often used

in Biotech to study genes.

Simple to grow

Grow rapidly

24 hours

370C

Why use bacteria?

BacteriaTo grow bacteria, need:

1.Correct nutrients – LB agar (solid) or LB broth (liquid).

2. Correct temperature – 370C.

3. Can divide every 20 minutes.

Transformation

Changing an organism’s traits by adding a new gene.

Why transform bacteria?

Study genes of complex organisms in much simpler ones.

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

- Replace “disease” genes with “healthy” genes.

- 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% (plate hold about 1 million colonies)

How do you know which cells have been transformed?

Selectable marker – gene which allows only transformed cells to survive.

Ex. Antibiotic resistance genes

What DNA will we transform into cells?

Plasmid – small, circular DNA

small “easy” to get into cells

familiar genes will be used to make proteins

vector can carry other genes into bacteria

What DNA will we transform into cells?

pVIB

luciferase – gene encoding Bioluminescence video

ampr = ampicillin-resistance gene

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 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