5 - Biol 241 2010 Lecture 9&10: Bacterial Genetics: 1....

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Biol 241 - 2010 1. Advantages of using E. coli as a genetic model system: - It is haploid. Phenotypes reflect genotypes. - It grows fast, with a doubling time of 20 minutes in rich media at 37°C. - The ease of manipulating auxotrophic mutations. 2. Three routes of transferring genetic materials into bacteria: a. Conjugation – bacterial mating. b. Transformation – uptake of extracellular DNA. c. Transduction – mediated by phage. Conjugation: Initial experiment (Lederberg and Tatum, 1946): Two auxotrophic E. coli strains, A ( met - , bio - , thr + , leu + , thi + ) and B ( met + , bio + , thr - , leu - , thi - ) were mixed. Prototrophs were generated at the frequency of 10 -7 . As each strain carried more than one auxotrophic mutation, revertants (changed back to wild type by mutations) were unlikely. Furthermore, plating of either A or B alone on minimal media plates did not generate prototrophs. The conclusion was that there was a transfer of genetic materials between the two strains. Mechanism of conjugation - Different bacteria strains were involved, and the transfer of genetic material is unidirectional. - Donor strain is designated as F + (F for fertility) and the recipient strain as F - . - Cell contact was required for conjugation, demonstrated by the U-tube experiment (Bernard Davis). - F + cells have F factor, which can be lost under certain conditions and regained when grown with F + cells. Therefore, F factor is a mobile element. F factor is a circular dsDNA and acts as an autonomous genetic unit (it is basically a plasmid). In F + x F - mating, the recipient becomes F + . It thought that a single-stranded nick is made in the F factor in the donor. The DNA then moves into the recipient via the pilus. The F factors in both donor and recipient are then replicated and sealed by ligase. As a result, both cells are now F + . Hfr (High frequency recombinant) In 1950, a strain isolated from E. coli K12 treated with mutagen underwent recombination at the frequency of 10 -4 (10 3 more efficient than what was observed by Lederberg and Tatum). -
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This note was uploaded on 02/09/2010 for the course BIOL 241 taught by Professor Henrychang during the Spring '10 term at Purdue University-West Lafayette.

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5 - Biol 241 2010 Lecture 9&10: Bacterial Genetics: 1....

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