Chloramphenicol ccdb resistance gateway cassette

Escherichia coli DH5a, Rosetta (DE3), and Agrobacterium tumefaciens GV3101, GV3103 or C58C1 cells were grown in Luria-Bertani (LB) medium at 37 and 28°C, respectively. For liquid culture, A. tumefaciens cells were grown in yeast extract beef (YEB) liquid medium at 28°C, overnight. Antibiotics were used at this following final concentrations (µg/mL): for E. coli : kanamycin (50), tetracycline (5) , gentamicin (10) , chloramphenicol (25) , carbenicillin (50) and spectinomycin (50) ; for A. tumefaciens : kanamycin (25), tetracycline (5) , gentamicin (20) and carbenicillin (25) . For Y2H experiments, two different yeast strains, L40 and Y187, were used for mating. The Y2H plasmids pP6 [39] and pB27 [40] plasmids provided by Hybrigenics (Paris) were modified into pNP377 and pNP378 Gateway Destination vectors by inserting the chloramphenicol/ccdB resistance Gateway cassette (Invitrogen). Recombined pDEST pNP377 and pNP378 plasmids were introduced in yeast cells using the LiAc/SS carrier DNA/PEG method described here (see Yeast Transformation). Yeast cells were grown at 30°C on rich medium YPGA or yeast minimal media SD-base (Takara Bio) supplemented with histidine 20 mg/L, tryptophan 20 mg/L or leucine 10 mg/L. Interaction test by mating was performed following standard procedures.