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

Bacterial Chromosome

Inserted F plasmid

...F factor integration site,

...host (bacteria chromosome) integration site.

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 transferstrain

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

F factor

Hfr F-

A

a

Indicates direction of transfer.

A

A

a

Hfr DNA that is not incorporated in the F- strand, and DNA that has crossed out of the F- strand is

digested.

F factor

Hfr F-

A

Leading Gene: the first gene transferred is determined empirically.

A

Hfr

A

F-A

A transfers first.

A transfers last.

Hfr Order of transferstrain

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,

lac (7)

car::tn10 (0.6)tetR

car (0.6)tetS

rpoB- (90)rifR

rpoB+ (90)rifS

metB- (89)

metB+ (89)

cysG- (72) cysG+ (72)

gyrA- (48)

nalS

gyrA+ (48)

nalR

1st Selection: tetR, nalR

pp. 3 assignmentdue 5/28/2008

lac (7)

car::tn10 (0.6)tetR

car (0.6)tetS

rpoB- (90)rifR

rpoB+ (90)rifS

metB- (89)

metB+ (89)

cysG- (72) cysG+ (72)

gyrA- (48)

nalS

gyrA+ (48)

nalR

1st Selection: tetR, nalR

Plate A proline, arginine, uracil

Plate B proline, arginine, uracil,methionine

Plate C proline, arginine, uracil,cysteine

Plate D proline, arginine, uracil,methionine, cysteine

Supplements Marker Selectedmet, cys

cys

met

no selection

Selection Scheme

lac (7)

car::tn10 (0.6)tetR

car (0.6)tetS

rpoB- (90)rifR

rpoB+ (90)rifS

metB- (89)

metB+ (89)

cysG- (72) cysG+ (72)

gyrA- (48)

nalS

gyrA+ (48)

nalR

Met +, Cys + Met +, Cys - Met -, Cys +Met -, Cys -

Met+ Cys+ Plates: A __ B __ C __ D __

lac (7)

car::tn10 (0.6)tetR

car (0.6)tetS

rpoB- (90)rifR

rpoB+ (90)rifS

metB- (89)

metB+ (89)

cysG- (72) cysG+ (72)

gyrA- (48)

nalS

gyrA+ (48)

nalR

Met+ Cys- Plates: A __ B __ C __ D __Met- Cys+ Plates: A __ B __ C __ D __Met- Cys - Plates: A __ B __ C __ D __

lac (7)

car::tn10 (0.6)tetR

car (0.6)tetS

rpoB- (90)rifR

rpoB+ (90)rifS

metB- (89)

metB+ (89)

cysG- (72) cysG+ (72)

gyrA- (48)

nalS

gyrA+ (48)

nalR

Met+, Cys+ Plates: A __ B __ C __ D __

lac (7)

car::tn10 (0.6)tetR

car (0.6)tetS

rpoB- (90)rifR

rpoB+ (90)rifS

metB- (89)

metB+ (89)

cysG- (72) cysG+ (72)

gyrA- (48)

nalS

gyrA+ (48)

nalR

Met_, Cys+ Plates: A __ B __ C __ D __

lac (7)

car::tn10 (0.6)tetR

car (0.6)tetS

rpoB- (90)rifR

rpoB+ (90)rifS

metB- (89)

metB+ (89)

cysG- (72) cysG+ (72)

gyrA- (48)

nalS

gyrA+ (48)

nalR

Met_, Cys- Plates: A __ B __ C __ D __

lac (7)

car::tn10 (0.6)tetR

car (0.6)tetS

rpoB- (90)rifR

rpoB+ (90)rifS

metB- (89)

metB+ (89)

cysG- (72) cysG+ (72)

gyrA- (48)

nalS

gyrA+ (48)

nalR

Met+, Cys- Plates: A __ B __ C __ D __