2.2 Culture Media 1. 2Tryptone-Yeast (TY) medium: 16 g/L tryptone, 10 g/L
yeast extract, 5 g/L NaCl. Add 2% (wt/vol) glucose and Ap
100 μg/mL after autoclaving.
- Tryptone-Yeast extract (TYE) agar: 10 g/L tryptone, 5 g/L
yeast extract, 8 g/L NaCl, 20 g/L agar. Add 2% (wt/vol)
glucose and 100μg/mL Ap after autoclaving. - Luria-Bertani (LB) broth: 10 g/L NaCl, 10 g/L tryptone,
5 g/L yeast extract. Sterilize by autoclaving. - Luria-Bertani (LB) agar: LB plus 15 g/L agar. Sterilize by
autoclaving. - Terrific Broth (TB) medium: dissolve 12 g tryptone, 24 g yeast
extract, and 4 mL glycerol in 900 mL of H 2 O. After autoclav-
ing, add aseptically 100 mL of potassium phosphate stock
solution for TB medium. - Nematode Growth Medium (NGM) agar: dissolve 2.4 g of
NaCl, 2 g of peptone, and 13.6 g of agar in 777 mL of H 2 O.
After autoclaving cool to 50C and add pre-sterilized 0.8 mL
cholesterol stock solution, 0.8 mL 1 M CaCl 2 , 0.8 mL 1 M
MgSO 4 , and 20 mL 1 M KH 2 PO 4 pH 6.0. - Nematode Growth Medium (NGM) extra-peptone (EP) agar:
dissolve 4 g of NaCl, 6 g of peptone, 1.2 g of yeast extract, and
16 g of agar in 777 mL of H 2 O and autoclave. After autoclav-
ing cool to 50C and add pre-sterilized 0.8 mL cholesterol
stock solution, 0.8 mL 1 M CaCl 2 , 0.8 mL 1 M MgSO 4 , and
20 mL 1 M KH 2 PO 4 pH 6.0.
2.3 Bacteria, Phages,
and Nematodes
- For construction of an immunized phage display library, elec-
trocompetent suppressorEscherichia coliTG1 cells {SupE thi-1
Δ(lac-proAB)Δ(mcrB-hsdSM)5(rkmk)[F^0 traD36 proAB
laclqZΔM15]} are used. - For the expression of recombinant proteins, recombinant-
deficient strainE. coliXL-1 Blue cells {endA1 supE44 thi-1
hsdR17 recA1 gyrA96 relA1 lac[F^0 proAB lacIq ZΔM15 Tn10
(Tetr)]} are used.
3.P. aeruginosastrains PAO1 and PA14, and the clinical isolate
strain 04.232058R (referred as PA058 hereafter) used forCae-
norhabditis elegansslow killing assay, were kindly donated by
Prof. Tim Mitchell, University of Glasgow, UK. - The helper phage M13KO7 carries a modified gene II from
M13mp1 (G changed to T at position 6125, giving a Met to Ile
change at codon 40 of the gene II protein) and a plasmid origin
of replication (ori) derived from p15A. The mutated gene II
products interact less efficiently with its own phageorithan
Monoclonal Antibodies Inactivating AHL Signal Molecules 329