5 The role of quorum-sensing in the virulence of Pseudomonas ...

of 30µl, 10µl of digested pWSK29 and 10µl digested E106-serW, 3µl 10x ligase
buffer, 1µl T4 ligase (Promega, UK) and 6µl nanopure water was added and left at
4ºC overnight. All the ligation mixtures were heat-inactivated at 65ºC for 20 minutes,
vacuum-dried, washed in 500µl 70% ethanol, followed by centrifugation at 10,000g
for 5 minutes, vacuum-dried and re-suspended in 10µl nanopure water.
A 50µl of aliquot of chemical competent cells were defrosted on ice. 2µl of the
ligation mixture was added to the cells and was incubated on ice for 30 minutes. The
mixture was incubated at 42ºC, 15 seconds and then immediately incubated on ice for
2 minutes. 200µl LB was added to the mixture and left to incubate at 37ºC for 1 hour.
100µl of the mixture plus 40µl X-Gal (20 mg/ml) was plated on LA plates containing
ampicillin (100µg/ml) and incubated overnight at 37ºC.
Forty µl X-Gal (20 mg/ml) plus 160µl nanopure water were spread onto a fresh plate
of LA plates containing ampicillin (100µg/ml). Twenty white colonies were patched
per enzyme onto this plate and the plate was incubated overnight at 37ºC. Following
incubation 10 white colonies were selected and overnight LB cultures were prepared.
A mini plasmid preparation was performed on all 10 colonies and the appropriate
restriction digest was performed (either KpnI or PstI) to check size of insert. A single
clone for each insert size was selected and prepared for sequencing.
2.4 Preparation of Pseudomonas aeruginosa for in vivo work
2.4.1 Preparation of bacterial stocks
A single colony of P. aeruginosa was grown overnight in 5ml LB, 200rpm, 37ºC.
600µl was removed and centrifuged at 13,000rpm for 1 minute. This pellet was added
to 20ml of TSB (Sigma, UK) supplemented with 20% fetal calf serum (FCS - Sigma,
UK). The culture was then incubated at 37ºC, 200rpm for 3 hours. The culture was
aliquoted into 1.5ml microcentrifuge tubes and stored at -70ºC.
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