Biomedical Engineering Reference
In-Depth Information
principle of homologous recombination. By introducing common 5
and 3
specific recombi-
nation sequences downstream of the BD or AD domains in both bait and prey vectors, it is
possible to generate a single PCR product which will directionally recombine with a series
of compatible vectors to generate in-frame bait or prey constructs (Figure 8.2). Conven-
tionally, bait and prey vectors are assembled in PJ69-4A MATa [11] or PJ69-4
α
MAT
α
[5]
host strains respectively. This facilitates mating of bait and prey clones during subsequent
interaction studies.
Procedures described in this chapter involve the use of bait and prey vectors containing the
att
B1 and
att
B2 recombination sites, which are also used in GATEWAY
TM
cloning systems.
Therefore, PCR products generated for standard GATEWAY
TM
BP reactions (Invitrogen)
can also be used to generate bait or prey clones by
in vivo
gap repair when co-transfected
with either pGBAD-B/pACTBD-B or pGBAE-B/pACTBE-B vectors [11]. This series of
vectors can also facilitate the rapid detection of bait or prey clones containing in-frame
inserts (Figure 8.2).
Unlike conventional Y2H vectors, which encode either DB or AD domains, the
pGBAD-B/pACTBD-B and pGBAE-B/pACTBE-B series of vectors encode both DB and
AD domains separated by an in-frame linker containing the A1 and A2 recombination
sequences (Figure 8.2). In the intact empty vector the DB and AD domains are expressed
as one in-frame fusion protein. As a result, Y2H reporter genes are constitutively turned
on, allowing yeast to grow efficiently, producing white colonies on low adenine selection
media. In contrast, vectors containing an insert with an in-frame stop codon will only
express the 5
Gal4 domain. Fusion proteins expressed from these vectors will not
independently drive transcription of biosynthetic reporter genes. Consequently, yeast
containing inserts with in-frame stop codons produce colonies with a characteristic pink/red
color on media containing low amounts of adenine [11].
8.2.2 Generating recombination-compatible inserts
for gap repair cloning
PCR products used for gap repair cloning are composed of three regions: the 5
upstream
recombination sequence; the protein coding region; and the 3
downstream recombination
sequence (Figure 8.2). In this procedure the 5
recombination sequence termed the 'A1
tag' includes the
att
B1 sequence, which is in-frame with upstream BD or AD domains
(see Protocol 8.1). Reverse primers are designed to include the 'A2 tag' (including the
att
B2 sequence) downstream of an in-frame stop codon. Inserts or domains lacking a stop
codon must be used in combination with the pGBAE-B (bait) or pACTBE-B (prey) vectors,
which use a frame-shift strategy to prevent read-through transcription of downstream AD or
BD domains. Significantly, the pGBAE-B and pACTBE-B vectors still allow clones with
in-frame inserts to be selected by pink/white color selection [11].
PROTOCOL 8.1 Primer Design and PCR Amplification of Inserts
for Use in Gap Repair Reactions
Equipment and reagents
PCR tubes
•
PCR machine
•
