with the phagemidoriof replication. It also carries a gene for
Km resistance to aid selection of infected bacteria.- Wild-typeCaenorhabditis elegansstrain Bristol N2.
2.4 Antibodies 1. Anti-M13 monoclonal HRP conjugate antibody (GE Health-
care, 27-9421-01).
- Donkey anti-sheep IgG (whole molecule) antibody HRP
(Sigma, A3415). - Donkey anti-sheep IgG (whole molecule) antibody (Sigma,
S2763). - Goat anti-human Cκ(bound and free) antibody (Sigma, K-
3502). - Goat anti-human Cκlight chain (bound and free) peroxidase
antibody (Sigma, A7164). - Human whole IgG standard (кisotype) (Sigma, I2511).
- Sheep IgG standard (Sigma, I5131).
2.5 Antigen
Conjugates and Free
Antigens
1.N-Dodecanoyl-homoserine lactone (C 12 -HSL),N-3-oxodo-
decanoyl-homoserine lactone (3OC 12 -HSL), andN-3-hydro-
xydodecanoyl-homoserine lactone (3OHC 12 -HSL) are
commercially available or can be custom synthesized by differ-
ent companies.- Synthetic analogues of C 12 -HSL, 3OC 12 -HSL, and 3OHC 12 -
HSL with a carboxylic group at the end of the acyl side-chain
were custom synthesized (seeNote 1) and coupled to bovine
serum albumin (BSA) or bovine thyroglobulin (TG) via the
carboxylic group [7].
2.6 Plasmids 1. Phagemid vector pHEN2a can be used for phage display library
construction. This is a pUC119-based phagemid vector
derived from pHEN1 with origins of replication from ColEI
and M13 to facilitate the growth inE. coliand packaging in
filamentous phage, respectively [8]. Restriction sites SfiI/NcoI
and NotI permit cloning of antibody fragments fused in
between thepelBleader sequence and the phagemid gene III
protein sequence. A hexa-histidine tag and c-myc tag are pres-
ent after the NotI cloning site, followed by an amber stop
codon introduced by site-directed mutagenesis. When
expressed in a non-suppressor strain of E. coli, the amber
codon is read as a stop and the expressed single chain Fv can
be easily purified and detected using the hexa-histidine tag or
the c-myc tag.
- The expression vector pIMS147 allows improving the solubil-
ity of single chain antibody fragments by co-expressing theE.
coliSkp chaperone [9]. This dicistronic vector is inducible with
330 Soumya Palliyil