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Streak
L. pneumophila recipient strain of interest (see
Note 1) from a frozen stock on a CYE plate and incubate it
for 72 hours at 37°C.

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With
a sterile loop scrape off enough bacteria from the plate to
inoculate 15 mL of AYE medium at an OD600 ~ 0.1.

In
parallel, grow E. coli MFDpir donor strain in 7.5 mL
LB broth containing 100 µg/mL ampicillin and 1% DAP.

Incubate
both cultures overnight at 37°C with agitation to stationary phase
(OD600 = 4-5 for L. pneumophila)

Centrifuge
the cultures at 5,000 g for 10 minutes. Discard supernatant.
Resuspend the L. pneumophila cell pellet in 1.5 mL
sterile water and the E. coli cell pellet in 750 µL sterile
PBS.

Mix
both concentrated pellets by pipetting.

Spot
100 µL of this mixture on non-selective CYED plates (i.e, CYE
plates without iron and cysteine but with DAP) without spreading. Up
to 20 spots can be arranged on one plate. When the liquid is
completely absorbed, incubate plates 5 to 6 hours at 37°C. During
this incubation time, both the conjugation mediated by the RP4
conjugative plasmid and the following mutagenesis by transposition
will take place.

Resuspend
each spots in 1 mL sterile water and plate 500 µL of each
suspension on CYE plates containing 10 µg/mL gentamicin to obtain
the transconjugants (i.e, the mutants). Make a serial
dilution using 100 µL of the remaining suspension to approximate
the number of mutants obtained. Incubate plates 72 hours at 37°C.

Count
the total number of mutants obtained on all plates. This number
varies greatly depending on the tested strain (See
Note 1). Permissive strains may yield from 250,000 to 500,000
mutants. A library containing
fewer than 200,000
mutants will
not be suitable for a Tn-seq study.

Scrape
off all the mutants from all the plates and resuspend them in 25 mL
AYE medium. Add 25 mL glycerol 30% and mix gently. Aliquot 500 µL
of the suspension in cryotubes and store at -80°C.

The
following day, thaw a frozen aliquot and plate serial dilutions to
determine the titer of the library in CFU/mL. Typically, 250,000 to
500,000 mutants resuspended in 50 mL of AYE+glycerol yields a titer
of 1010 CFU/mL.

Your
mutants library is now ready to be used in any Tn-seq screen.

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