November 12, 2008 handout

1. Transformation: DNA donor is dead, and may not be a close relative of recipient
a. Artificial: uses of artificial plasmids
i. Artificial plasmids: plasmid (circles of DNA w/ genes & oriC site) that’s had new genes inserted into it by humans
ii. Expose culture of bacteria (E. coli) to plasmids, heat/electricity will shock bacteria and some will die and others will take in plasmids and express the plasmid’s characteristics
iii. Examples: insulin for diabetics; insulin is processed from E. coli containing plasmid (circle of DNA with genes that have a promoter sequence & oriC site) with human insulin gene & gene for ampicillin-R (antibiotic resistance)
iv. How it works:
1. Plasmid from bacteria is cut with restriction enzymes (cuts DNA into sections at specific sequences) and treated with DNA swatches w/ genes that we want ( insulin gene & ampicillin resistance gene) & then the plasmids are treated with DNA ligase (patches pieces together) to get circle of DNA with insulin gene and ampicillin resistance gene
2. Get plasmid into bacteria by electric or heat shock then plate onto nutrient agar with ampicillin and so the bacteria that grew on the plate must have ampicillin resistance and insulin genes
b. Natural: takes up naked DNA (linear swatch of double stranded DNA)
i. only allows genetic exchange between closely related bacteria
ii. competent (able to take up DNA from the environment)
1. bacteria take up DNA from environment because in environmentally stressful situations (change in environment) bacteria are more likely to take up DNA
2. genetic exchange (especially transformation) is more likely to occur in more genetically variable populations
iii. How it works:
1. Bacteria in environment with a lot of naked DNA; DNA binds to sites on bacterial cell wall
2. One strand of dsDNA swatch gets destroyed, while the other is brought into the bacterial cell
3. Single strand of DNA that was brought into the cell is covered in single strand binding protein so it is not destroyed
4. If any of the swatch is homologous (same/similar DNA sequence) with bacterial chromosome; the homogolous parts of base will pair with bacterial chromosome
5. Next time it divides one cell will get copy of new swatch and another one gets original DNA
2. Conjugation-DNA donor & recipient are both alive, and occur in
a. Pilus-made out of pilin protein (expressed) that makes pilus
i. fairly closely related
b. In gram-negative bacteria
i. F+ bacteria=have pilin gene
ii. F- bacteria=recipient
iii. F+ bacteria uses pilus to attach to and pull F-bacteria close, F- bacteria then copies plasmid & sends copy to F- through merged cell envelopes
iv. Now both donor and recipient are F+
c. In gram-positive bacteria
i. At high population density, pheromone is secreted
1. Pheromone will attract bacteria to each other and then will clump the bacteria

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