Transcript Slide 1
Bacteria Bacteria – single-celled organisms that are prokaryotic (no true nucleus) Bacteria Bacteria are often used in Biotech to study genes. Why use bacteria? Simple to grow Grow rapidly 24 hours 370C Bacteria To 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 – Green Fluorescent Protein 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