due 24 hours

Experiment II: Gene Mapping by Homologous Recombination

September 13, 2016

Introduction •  Rifampin resistant mutants form spontaneously

when a culture of Escherichia coli HR171is growing.

Mapping

•  To map the mutation we are going to measure the frequency of recombination between your rifR sample and the wild type rifS .

•  We will be using two methods of measuring recombination: – Conjugation – Transduction

Conjugation

•  Transfer genetic information using a conjugative plasmid

•  If an Hfr strain is used, the plasmid will integrate into the chromosome and the entire chromosome will be transferred

•  The further a gene is on the chromosome from the origin of transfer (oriT) the less likely it will be transferred

Transduction

•  Transfer genetic information using a bacteriophage

•  DNA from one bacterium is packaged into a phage head which is inserted into another bacterium upon infection

•  The closer two genes are to each other on a chromosome, the more likely they are to be packaged and transferred together

Today •  Preparing for Conjugation and Transduction

•  Transduction: –  Add 5 µl of 0.1 M CaCl2 in a 1.5 ml Eppendorf

tube –  100 µl of culture of E. coli KL227 –  Add 50 µl of P1 (we will add for you) –  Incubate at 30°C for 10 min –  Plate 50 µl on LB plate with chloramphenicol –  Incubate plate at 30°C, overnight

Today •  Conjugation: •  Need to have single colonies!

– Innoculate 2 tubes •  Inoculate 2ml of LB + rif (the tube with the red top) with one colony from your rif plate (recipient).

i.e. Your rpoB mutant from last week

•  Inoculate 2ml of LB with one colony from of Hfr KL16 (donor)

•  Incubate in 37°C shaker at 200 rpm

Please  Cleanup    

•  Place glass culture tubes in tray in the back of lab •  Empty biohazard cylinder into orange bin at end of

bench •  Wipe down bench with disinfectant •  Wash your hands

•  Thank you!

Suggested Reading: •  In the 3rd Edition of Snyder and Champness: •  167-184 (Gene mapping using Hfr crosses,

transduction) •  243-263 (Conjugation background) •  332-339 (Transduction/Phage background) •  429-435 (Homologous recombination

background) •  If you have a different edition, look in the

table of contents of your book for titles similar to the ones listed