2 Array-Based Yeast Two-Hybrid Screens: A Practical Guide 23

a Recombination in yeast b Univector system c Gateway system

SfiI-A site

ORF ORF ORF

st
SfiI-B site
1 round PCR PCR
PCR

ORF
B1 ORF B2
ORF PCR product
SfiI-A digestion SfiI-B + BP reaction
2nd round PCR
polylinker
P1 ccdB P2

ORF empty vector gentr

pDONR vector
transformation of PCR Kanr
by-product
product into yeast ligation
R1 ccdB R2
ORF
ORF L1 ORF L2
homologous entry vector
recombination OriR6K pENTR vector gentr
AD or
in yeast Kanr
DBD tag
+
linearized Y2H + LR reaction
vector AD or R1 ccdB R2
Y2H destination DBD tag
AD or
DBD tag vector (bait or prey) Y2H destination e.g. ampr or kanr
Ampr vector (bait or prey)
vector fusion by by-product
AD or Cre recombination P1 ccdB P2
DBD tag ORF ORF Kanr AD or
DBD tag
B1 ORF B2
Y2H expression AD or Y2H expression vector
DBD tag OriR6K Y2H expression
vector (bait or prey) (bait or prey) vector (bait or prey)
Ampr e.g. ampr or kanr

Fig. 1. ORFeome cloning systems. (a) Homologous recombination in yeast: ORFs are amplified (first PCR) with gene-specific
primers that generate a product with common 5 and 3 20-nucleotide tails. A second PCR generates a product with common
5 and 3 70-nucleotide tails. The common 70-nucleotide ends allow cloning into linearized two-hybrid expression vectors
by cotransfection into yeast. The endogenous yeast homologous recombination machinery performs the recombination
reaction and results in a circular plasmid. (b) Univector plasmid-fusion system: ORFs are amplified with gene-specific
primers that generate a product with common 5 and 3 rare-cutting restriction sites. The PCR product is cloned into a pUNI
entry vector by DNA ligation. CreloxP-mediated site-specific recombination fuses the pUNI entry clone and yeast two-hybrid
expression plasmids (bait/prey) at the loxP site. As a result, the gene of interest is placed under the control of the yeast
two-hybrid expression vector promoter. (c) Gateway cloning: The ORFs are amplified with gene-specific primers that
generate a product with common 5 and 3 recombination sites (attB1 and attB2 ). The entry clones are made by recombining
the ORFs of interest with the flanking attB sites into the attP sites of a suitable Gateway entry vector (e.g., pDONR201 or
pDONR207) mediated by the Gateway BP Clonase II Enzyme Mix (Invitrogen). Subsequently, the fragment in the entry clone
can be transferred to any yeast two-hybrid destination vector that contains the attR sites by mixing both plasmids and
using the Gateway LR Clonase II Enzyme Mix.

second-round PCR, ~50-nucleotide tails (homologous to the

destination vector-cloning site) are attached to the first-round PCR
product (Fig. 1). The PCR product is then transfected into the
yeast cells together with the linearized vector and the recombina-
tion event between them takes place inside the yeast cell. The
advantage of this strategy is its much reduced cost. The disadvantage
is that plasmids have to be recovered from yeast which can be time
consuming and inefficient.

1.2.2. Univector Plasmid- Similar to the Gateway system, the Univector Plasmid-Fusion
Fusion System System (UPS) requires an entry vector containing the ORF. The
UPS uses CreloxP-based site-specific recombination to catalyze
plasmid fusion between the entry univector and destination vectors