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This Week

This Week. Score Conjugation Plates, Start High Frequency of Recombination (HFR) experiment, Continue Nasonia experiment. H igh F requency of R ecombination (Hfr). ...bacteria exhibiting a high frequency of recombination ,

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This Week

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  1. This Week • Score Conjugation Plates, • Start High Frequency of Recombination (HFR) experiment, • Continue Nasonia experiment.

  2. High Frequency of Recombination(Hfr) ...bacteria exhibiting a high frequency of recombination, • an alteration DNA sequence such that the genotype of subsequent individuals differs from the parent, …specifically, strains with a chromosome integrated F factor that is able to mobilize and transfer part of the chromosome to the F- cell.

  3. ...F factor integration site, ...host (bacteria chromosome) integration site. Hfr Cells F factor Bacterial Chromosome Inserted F plasmid

  4. F Pilus Attaches to F- Cell

  5. Hfr DNA is Cut

  6. F factor and Chromosomal DNA are Transferred

  7. Double Crossover Recombination Requires Crossing over

  8. DNA not Incorporated into Chromosome are Digested

  9. F factor inserts in different regions of the bacterial chromosome, Also inserts in different orientations.

  10. Origin of Replication Hfr Order of transfer strain H thr azi ton lac pur gal his gly thi 1 thr thi gly his gal pur lac ton azi 2 lac pur gal his gly thi thr azi ton 3 gal pur lac tonazi thr thi gly his

  11. Indicates direction of transfer. A a A F factor A a Hfr F- Hfr DNA that is not incorporated in the F- strand, and DNA that has crossed out of the F- strand is digested.

  12. A A Hfr F- F factor A transfers first. A A Hfr F- A transfers last. Leading Gene: the first gene transferred is determined empirically.

  13. Hfr Order of transfer strain H thr azi ton lac pur gal his gly thi 1 thr thi gly his gal pur lac ton azi 2 lac pur gal his gly thi thr azi ton 3 gal pur lac tonazi thr thi gly his

  14. E. coli Map • 0 minutes is at the threonine, • 100 minutes is required to transfer complete genome,

  15. pp. 3 assignment due 5/28/2008 car::tn10 (0.6) tetR rpoB+ (90) rifS lac (7) metB- (89) car (0.6) tetS rpoB- (90) rifR metB+ (89) cysG- (72) cysG+ (72) gyrA+ (48) nalR gyrA- (48) nalS 1st Selection: tetR, nalR

  16. car::tn10 (0.6) tetR rpoB+ (90) rifS lac (7) metB- (89) car (0.6) tetS rpoB- (90) rifR metB+ (89) cysG- (72) cysG+ (72) gyrA+ (48) nalR gyrA- (48) nalS 1st Selection: tetR, nalR

  17. Selection Scheme Supplements Marker Selected met, cys cys met no selection

  18. car::tn10 (0.6) tetR rpoB+ (90) rifS lac (7) metB- (89) car (0.6) tetS rpoB- (90) rifR metB+ (89) cysG- (72) cysG+ (72) gyrA+ (48) nalR gyrA- (48) nalS Met +, Cys + Met +, Cys - Met -, Cys - Met -, Cys +

  19. Met+ Cys+ Met- Cys+ Met+ Cys- Met- Cys - Plates: A __ B __ C __ D __ Plates: A __ B __ C __ D __ Plates: A __ B __ C __ D __ Plates: A __ B __ C __ D __ car::tn10 (0.6) tetR rpoB+ (90) rifS lac (7) metB- (89) car (0.6) tetS rpoB- (90) rifR metB+ (89) cysG- (72) cysG+ (72) gyrA+ (48) nalR gyrA- (48) nalS

  20. car::tn10 (0.6) tetR rpoB+ (90) rifS lac (7) metB- (89) car (0.6) tetS rpoB- (90) rifR metB+ (89) cysG- (72) cysG+ (72) gyrA+ (48) nalR gyrA- (48) nalS Met+, Cys+ Plates: A __ B __ C __ D __

  21. car::tn10 (0.6) tetR rpoB+ (90) rifS lac (7) metB- (89) car (0.6) tetS rpoB- (90) rifR metB+ (89) cysG- (72) cysG+ (72) gyrA+ (48) nalR gyrA- (48) nalS Met_, Cys+ Plates: A __ B __ C __ D __

  22. car::tn10 (0.6) tetR rpoB+ (90) rifS lac (7) metB- (89) car (0.6) tetS rpoB- (90) rifR metB+ (89) cysG- (72) cysG+ (72) gyrA+ (48) nalR gyrA- (48) nalS Met_, Cys- Plates: A __ B __ C __ D __

  23. car::tn10 (0.6) tetR rpoB+ (90) rifS lac (7) metB- (89) car (0.6) tetS rpoB- (90) rifR metB+ (89) cysG- (72) cysG+ (72) gyrA+ (48) nalR gyrA- (48) nalS Met+, Cys- Plates: A __ B __ C __ D __

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