due 24 hours

Experiment II, day 5

Preparing phage lysates and analyzing transconjugants

September 27, 2016

Transduction

•  First you need to dilute your Lysogen (E. coli KL227 with P1Cm c1-100), in order to get the cultures you inoculated on Thursday growing again).

Transduction: •  Dilute your P1 lysogen 1:5 in a separate tube, (for

example, 1 ml of culture in 4 mls sterile LB). We can also provide you with a tube of lysogen, should you need it.

•  Check the OD625 nm , (blank first). •  Incubate at 32°C in shaker until OD625 nm reaches

0.6-0.7. •  Transfer lysogen culture to 42°C water bath shaker

for 30 min, check OD625 nm ~ every 15 min. •  Transfer lysogen culture to 38°C waterbath and

shake for 1.5-2 hours, check OD625 nm ~ every 30 min.

•  Check culture for lysis- dead cells will fall to bottom of tube, clear supernatant will contain phage.

Lifecycle of a bacteriophage:

Conjugation (just a reminder…) •  The donor strain (KL16) was sensitive to

streptomycin, and carried genes for the amino acids (KL16: his+trp+arg+strSrifS).

•  The recipient strain (HR171) was resistant to streptomycin, and did not have genes for the amino acids (HR171: his-trp-arg-strRrifR).

•  The only way for the recipient to grow on plates lacking an amino acid was if the donor had transferred the plasmid, along with some of the chromosome (amino acid genes), to the recipient.

Conjugation, continued: •  Colonies that grew on Trp, Arg plate are His+

transconjugants. •  All the colonies picked on Tuesday were His+.

– The percent of His+ transconjugants is 100% •  Count the number of colonies on Arg plate (these

are Trp+). – Divide number of Trp+ transconjugants by His+.

Selected marker hisG

% of recombinants

trpA argH rifS

100 40 6 7

Conjugation:

•  Observe patched plates, – count the number of colonies on each plate, – calculate the percent of recombinants, – add group data to spreadsheet on computer

at the front of the lab.

Conjugation, continued:

•  Gradient of transfer: – Graph of recombinant

percentages versus Distance in min. along the chromosome.

– Log scale.

•  End of conjugation part of Experiment II.

his  

trp  

arg  

1  

10  

100  

0   10   20   30   40   50   60   70   80  

%    o f   re co m b in an

ts  

Distance  in  min  

Gradient  of  Marker  Transfer  

rif