Biology Reference
In-Depth Information
Table 1
Common bait and prey vectors used for Y2H
Plasmid
Fusion
Epitope
Yeast selection
Bacterial selection
Reference and/or map
pGBKT7 (1)
BD/bait c-Myc
TRP1
Kanamycin
Clontech Cat. No. 630443
pGBT9 (3)
BD/bait None
TRP1
Ampicillin
[
7
]
pAS2-1 (1)
BD/bait None
TRP1
Ampicillin
[
8
]
pGADT7 (1) AD/prey HA
LEU2
Ampicillin
Clontech Cat. No. 630442
pACT2 (2)
AD/prey HA
LEU2
Ampicillin
[
9
]
BD or AD fusions are expressed from full length (1) or truncated (2 and 3) ADH promoters to produce high
(1), medium (2), or low constitutive expression level
Table 2
Genotypic and phenotypic characteristics of PJ69-4A and AH109
Transformation
markers
Strain
Genotype
Reporters
Reference
PJ69-4A
MATa leu2-3,112 ura3-52 trp1-901 his3-200
gal4
HIS3, ADE2,
LacZ
trp1, leu2
[
10
]
GAL2-ADE2 LYS2::GAL1-
HIS3 MET2::GAL7- LacZ
Δ
gal80
Δ
AH109
MATa, leu2-3, 112, ura3-52, trp1-901,
his3-200, gal4
HIS3, ADE2,
MEL1
trp1, leu2
[
10
]
Δ
Δ
, GAL2-ADE2,
LYS2 : : GAL1-HIS3, URA3 : : MEL1-lacZ
, gal80
The
GAL1, GAL2,
and
GAL7
upstream activating sequences (UASs) are recognized and bound by the Gal4 BD.
The
trp1, his3, gal4,
and
gal80
mutations are all deletions;
leu2-3, 112
is a double mutation. PJ69-4A requires
addition of leucine, uracil, tryptophan, histidine, adenine, lysine, and methionine in minimal media to grow
(detailed in Subheading
2.3
) [
10
]
.
In addition using either
lacZ or
MEL1,
which encode
-galactosidase respec-
tively, allows for quantitative studies of the protein-protein
interactions.
For additional information on the growth and maintenance of
yeast, see the Guide to Yeast Genetics and Molecular Biology [
11
].
The complete phenotype and genotype of the PJ69-4A and AH109
strains are shown in Table
2
. The protocols presented in this chap-
ter have been optimized for PJ69-4A.
β
-galactosidase and
α
Prepare all media using deionized water. For solutions use ultra-
pure water and analytical grade reagents. All reagents must be pre-
pared and stored at room temperature (unless otherwise
indicated).
1.
Minimal medium (MM).
It is used for general yeast growth. It
contains Yeast Nitrogen Base (YNB, 1.7 g/l), ammonium
2.3 Media and
Supplements
