A dose–response experiment should also be conducted for the
E. coliAHL monitor strain.- AHL monitor strain: The unstable GFP derivative GFP(ASV),
containing alanine (A), serine (S), and valine (V) at the C-
terminus, is used as a reporter [15]. The GFP-based monitor
plasmid pJBA132, which carries the Vibrio fischeri region
encodingluxRand a fusion betweenPluxIandgfp(PluxI-gfp
(ASV)), has been cloned into the stable broad-host-range vec-
tor pME6031 (pVS1 replicon, accession no. AF118811). The
E. colimonitor strain MT102 is a restriction-minus derivative
of MC1000 (araD139(Δara-leu)7697Δlac,thi,hsdR) (for
further detailssee[12, 14]). The JB525-gfp(ASV) isE. coli
MT102 containing the plasmid pJBA132. - Luria Bertani (LB) liquid medium: 1% (wt/vol) bacto-
tryptone, 0.5% (wt/vol) yeast extract, 1% (wt/vol) sodium
chloride in distilled water. Autoclave. - LB agar medium: LB liquid medium plus 1.5% (wt/vol) agar.
Autoclave. - Blue agar plates.
- 250 ml conical flasks.
- Shaking incubator at 37C.
- 15 and 50 ml plastic tubes.
- Centrifuge capable of 5000g.
2.2 Alginate Beads 1. Protanal: Seaweed alginate with approximately 60% guluronic
acid content (10/60) is dissolved in 0.9% NaCl to a concentra-
tion of 11 mg/ml and autoclaved. Store at 4C until use. Can
be stored for several months.
- TRIS-buffer: 0.1 M Tris Base (C 4 H 11 NO 3 ), adjust the pH to
7.0 by adding 25% HCl and autoclave. Store at 4C until use.
Can be stored for several months. - TRIS-buffer with 0.1 M CaCl 2 : mix 100 ml TRIS-buffer with
1.47 g (0.1 M) CaCl 2 on a magnetic stirrer. - Bead making: the viscous suspension of bacteria and protanal is
placed in a cylindrical reservoir and forced through an 18-G
cannular by compressed air (0.4 bar), while another jet of air is
forced coaxially along the needle to blow off the alginate
droplets (1.0 bar) [19] (for a figureseein [ 20]). Clean the
equipment in 70% (vol/vol) ethanol.
2.3 Mice 1. Female BALB/c mice at 9–11 weeks of age. BALB/c mice have
been shown to have a Th 2 immune response as seen in chroni-
cally infected cystic fibrosis patients and are susceptible to
infection withP. aeruginosa[21]. The mice should be main-
tained on standard mouse chow and water ad libitum for a
ImagingN-Acyl Homoserine Lactone Quorum Sensing In Vivo 205