This Week Score Conjugation Plates,

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Bacteria replication, recombination, and transformation
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This Week Score Conjugation Plates, Start High Frequency of Recombination (HFR) experiment, Continue Nasonia experiment.

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.

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

F Pilus Attaches to F- Cell

Hfr DNA is Cut

F factor and Chromosomal DNA are Transferred

Recombination Requires Crossing over Double Crossover

DNA not Incorporated into Chromosome are Digested

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

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 ton azi thr thi gly his

Indicates direction of transfer. F factor A A a 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.

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

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 ton azi thr thi gly his

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

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

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

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

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

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

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

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

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

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

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