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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. 37 0 C. Bacteria. To grow bacteria, need:. - PowerPoint PPT Presentation
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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
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