the additional plasmid has to be introduced into the cells containing
already the bait and prey plasmids, and the new transformants
selected on SC + 2% glucose medium lacking tryptophan, leucine
and uracil. As control, the same transformation should be conducted
using an empty plasmid (seeNote 4).2.2 Yeast
and Bacterial Strains
- When the bait plasmid contains a LexA module, a yeast strain
carryinglexAoperators regulating the expression of reporter
genes has to be used in the two-hybrid assay. We use theSaccha-
romyces cerevisiaeTHY-AP4 strain (MATa, ura3, leu2, lexA::
lacZ::trp1, lexA::HIS3, lexA::ADE2)[20]. This strain requires
the complementation of the culture medium with uracil, leu-
cine, tryptophan, histidine and adenine, for growth (seeNote
5 ). An advantage of this strain is that it carries the yeast biosyn-
thetic geneHIS3under the control of thelexAoperator, so that
the interaction between AMPK subunits and putative partners
can be carried out both by nutritional selection for histidine
prototrophy and by an assay forβ-galactosidase activity (which
results from the expression of thelacZgene under the control of
lexAoperator). To maintain the strain, cells are grown in com-
plete YPD medium (seebelow). - Bacterial Strain:Escherichia coli KC8 (pyrF::Tn5, leuB600,
trpC-9830, hisB463).
2.3 Culture Media 1. Complete YPD medium plates [21]: 2% (w/v) glucose, 2%
(w/v) peptone, 1% (w/v) yeast extract, 2% (w/v) agar, adjusted
to pH 6.0.
- Synthetic complete (SC) + 2% glucose medium plates [21]: To
prepare 400 ml of medium, autoclave 340 ml of water contain-
ing 8 g agar. Cool down the medium to 55C, and add 20 ml
of 40% (w/v) glucose solution and 40 ml of a 10solution of
YNB + amino acid mix [6.7 g of yeast nitrogen base without
amino acids plus 0.95 g of an amino acid mix (1.25 g arginine,
1.25 g methionine, 1.88 g tyrosine, 1.88 g isoleucine, 1.88 g
lysine, 3.13 g phenylalanine, 6.25 g glutamic acid, 6.25 g
aspartic acid, 9.38 g valine, 25 g serine and 25 g threonine)
in 100 ml]. When necessary, 4 ml of each of the following stock
solutions should be added to 400 ml of SC medium: 10 mg/ml
leucine, 10 mg/ml tryptophan, 10 mg/ml histidine, 2.5 mg/
ml uracil, 2.5 mg/ml adenine. - M9 minimal medium plates forE. coli KC8cells: To prepare
400 ml of medium, autoclave 315 ml of water containing 8 g
agar. Cool down the medium to 55C, and add 40 ml of a 10
solution of M9 salts (sterile filtered 128 g/l Na 2 HPO 4 .12
H 2 O, 30 g/l KH 2 PO 4 , 5 g/l NaCl, 10 g/l NH 4 Cl), 5.5 ml
of 1 M NaOH, 4 ml of 40% (w/v) glucose, 40 ml of 10amino
Y2H Interaction Analyses 145