![Page 1: Zone of Inhibition Area around antibiotic disk with no bacterial growth. Bacteriocidal – no bacteria living in ZOI. Bacteriostatic – bacteria are not reproducing](https://reader035.vdocument.in/reader035/viewer/2022062722/56649f295503460f94c41ce8/html5/thumbnails/1.jpg)
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.
![Page 2: Zone of Inhibition Area around antibiotic disk with no bacterial growth. Bacteriocidal – no bacteria living in ZOI. Bacteriostatic – bacteria are not reproducing](https://reader035.vdocument.in/reader035/viewer/2022062722/56649f295503460f94c41ce8/html5/thumbnails/2.jpg)
Bacteria and Antibiotics
Bacteria – single-celled organisms that are prokaryotic (no true nucleus)
![Page 3: Zone of Inhibition Area around antibiotic disk with no bacterial growth. Bacteriocidal – no bacteria living in ZOI. Bacteriostatic – bacteria are not reproducing](https://reader035.vdocument.in/reader035/viewer/2022062722/56649f295503460f94c41ce8/html5/thumbnails/3.jpg)
Bacteria and AntibioticsBacteria are often used
in Biotech to study genes. Why use bacteria?
Simple to grow
Grow rapidly
24 hours
370C
![Page 4: Zone of Inhibition Area around antibiotic disk with no bacterial growth. Bacteriocidal – no bacteria living in ZOI. Bacteriostatic – bacteria are not reproducing](https://reader035.vdocument.in/reader035/viewer/2022062722/56649f295503460f94c41ce8/html5/thumbnails/4.jpg)
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.
![Page 5: Zone of Inhibition Area around antibiotic disk with no bacterial growth. Bacteriocidal – no bacteria living in ZOI. Bacteriostatic – bacteria are not reproducing](https://reader035.vdocument.in/reader035/viewer/2022062722/56649f295503460f94c41ce8/html5/thumbnails/5.jpg)
1.Takes time to adjust.
2. 2n
3. Run out of nutrients
![Page 6: Zone of Inhibition Area around antibiotic disk with no bacterial growth. Bacteriocidal – no bacteria living in ZOI. Bacteriostatic – bacteria are not reproducing](https://reader035.vdocument.in/reader035/viewer/2022062722/56649f295503460f94c41ce8/html5/thumbnails/6.jpg)
Bacteria and AntibioticsAntibiotics – any chemical which
prevent bacteria from growing.1.Bacteriocidal- kills bacteria.
2. Bacteriostatic- prevents bacteria from reproducing. Originals are not dead!
![Page 7: Zone of Inhibition Area around antibiotic disk with no bacterial growth. Bacteriocidal – no bacteria living in ZOI. Bacteriostatic – bacteria are not reproducing](https://reader035.vdocument.in/reader035/viewer/2022062722/56649f295503460f94c41ce8/html5/thumbnails/7.jpg)
Sterile Technique
Preventing contamination:
- of you by experiment.
- of experiment by you.
Ex. Gloves, keeping lids closed, alcohol, flame.
![Page 8: Zone of Inhibition Area around antibiotic disk with no bacterial growth. Bacteriocidal – no bacteria living in ZOI. Bacteriostatic – bacteria are not reproducing](https://reader035.vdocument.in/reader035/viewer/2022062722/56649f295503460f94c41ce8/html5/thumbnails/8.jpg)
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.
![Page 9: Zone of Inhibition Area around antibiotic disk with no bacterial growth. Bacteriocidal – no bacteria living in ZOI. Bacteriostatic – bacteria are not reproducing](https://reader035.vdocument.in/reader035/viewer/2022062722/56649f295503460f94c41ce8/html5/thumbnails/9.jpg)
Ways to get DNA into a cell:Electroporation
Using electricity to poke holes in cell membrane; DNA rushes into cell.
![Page 10: Zone of Inhibition Area around antibiotic disk with no bacterial growth. Bacteriocidal – no bacteria living in ZOI. Bacteriostatic – bacteria are not reproducing](https://reader035.vdocument.in/reader035/viewer/2022062722/56649f295503460f94c41ce8/html5/thumbnails/10.jpg)
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.
![Page 11: Zone of Inhibition Area around antibiotic disk with no bacterial growth. Bacteriocidal – no bacteria living in ZOI. Bacteriostatic – bacteria are not reproducing](https://reader035.vdocument.in/reader035/viewer/2022062722/56649f295503460f94c41ce8/html5/thumbnails/11.jpg)
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
![Page 12: Zone of Inhibition Area around antibiotic disk with no bacterial growth. Bacteriocidal – no bacteria living in ZOI. Bacteriostatic – bacteria are not reproducing](https://reader035.vdocument.in/reader035/viewer/2022062722/56649f295503460f94c41ce8/html5/thumbnails/12.jpg)
Ways to get DNA into a cell:Heat Shock
Using heat to open pores in cell membrane. (see sketch on board).
![Page 13: Zone of Inhibition Area around antibiotic disk with no bacterial growth. Bacteriocidal – no bacteria living in ZOI. Bacteriostatic – bacteria are not reproducing](https://reader035.vdocument.in/reader035/viewer/2022062722/56649f295503460f94c41ce8/html5/thumbnails/13.jpg)
Transformation Efficiency
# of bacterial cells on plate# bacterial cells possible on plate
For heat shock, this number is <0.01%
![Page 14: Zone of Inhibition Area around antibiotic disk with no bacterial growth. Bacteriocidal – no bacteria living in ZOI. Bacteriostatic – bacteria are not reproducing](https://reader035.vdocument.in/reader035/viewer/2022062722/56649f295503460f94c41ce8/html5/thumbnails/14.jpg)
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
![Page 15: Zone of Inhibition Area around antibiotic disk with no bacterial growth. Bacteriocidal – no bacteria living in ZOI. Bacteriostatic – bacteria are not reproducing](https://reader035.vdocument.in/reader035/viewer/2022062722/56649f295503460f94c41ce8/html5/thumbnails/15.jpg)
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)
![Page 16: Zone of Inhibition Area around antibiotic disk with no bacterial growth. Bacteriocidal – no bacteria living in ZOI. Bacteriostatic – bacteria are not reproducing](https://reader035.vdocument.in/reader035/viewer/2022062722/56649f295503460f94c41ce8/html5/thumbnails/16.jpg)
What DNA will we transform into cells?
pGREEN
GFP –
GreenFluorescentProtein
Also ampr
![Page 17: Zone of Inhibition Area around antibiotic disk with no bacterial growth. Bacteriocidal – no bacteria living in ZOI. Bacteriostatic – bacteria are not reproducing](https://reader035.vdocument.in/reader035/viewer/2022062722/56649f295503460f94c41ce8/html5/thumbnails/17.jpg)
What DNA will we transform into cells?
pUWL500
luciferase andampr
![Page 18: Zone of Inhibition Area around antibiotic disk with no bacterial growth. Bacteriocidal – no bacteria living in ZOI. Bacteriostatic – bacteria are not reproducing](https://reader035.vdocument.in/reader035/viewer/2022062722/56649f295503460f94c41ce8/html5/thumbnails/18.jpg)
What DNA will we transform into cells?
pGEM3z-
Just ampr
![Page 19: Zone of Inhibition Area around antibiotic disk with no bacterial growth. Bacteriocidal – no bacteria living in ZOI. Bacteriostatic – bacteria are not reproducing](https://reader035.vdocument.in/reader035/viewer/2022062722/56649f295503460f94c41ce8/html5/thumbnails/19.jpg)
What type of bacterial cells are we going to transform?
E. Coli that are “competent”.
Competent – ready to be transformed.
![Page 20: Zone of Inhibition Area around antibiotic disk with no bacterial growth. Bacteriocidal – no bacteria living in ZOI. Bacteriostatic – bacteria are not reproducing](https://reader035.vdocument.in/reader035/viewer/2022062722/56649f295503460f94c41ce8/html5/thumbnails/20.jpg)
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