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

Bacteria Bacteria are often used in Biotech to study genes. Simple to grow Grow rapidly 24 hours 37 0 C Why use bacteria?

Bacteria To grow bacteria, need: 1.Correct nutrients – LB agar (solid) or LB broth (liquid). 2. Correct temperature – 37 0 C. 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 amp r = ampicillin-resistance gene 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 amp r

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 CaCl 2 In solution CaCl 2  Ca Cl - Positively charged Ca 2+ coats negatively charged pores. DNA can move into cell. Heat Shock Animation