Two‐Hybrid Dual Bait System

互联网2013-12-31

1997

Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

The yeast two?hybrid system, or interaction trap, is one of the most versatile methods available with which to identify and establish protein?protein interactions. The dual bait system is one adaptation of the classic approach. This system facilitates the simultaneous comparison of two distinct baits with one prey. One protein of interest is expressed as a fusion to the DNA?binding protein LexA (bait 1), while a second protein of interest is expressed as a fusion to the DNA?binding protein cI (bait 2). Strains of yeast engineered for screening of these dual baits possess four separate reporter genes. A plasmid expressing an activation domain?fused protein (prey), which can be either a defined protein interactor or a cDNA library, is also expressed to allow dual hybrid?mediated transcriptional activation. Curr. Protoc. Mol. Biol. 86:20.7.1?20.7.32. © 2009 by John Wiley & Sons, Inc.

Keywords: interactions; yeast two?hybrid; interaction trap; interaction mating; increased specificity

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Introduction
Basic Protocol 1: Preparation of Baits and Library: Characterizing Bait Proteins
Basic Protocol 2: Preparation of Baits and Library: Transforming and Characterizing the Library
Basic Protocol 3: Selecting an Interactor
Basic Protocol 4: Analysis of Primary Interactors
Alternate Protocol 1: Screening for Interaction Trap Positives by Yeast Plasmid Recovery
Support Protocol 1: Colorimetric Assay of β‐Galactosidase and β‐Glucuronidase Activity by Agarose Overlay
Support Protocol 2: Detection of Bait Protein Expression
Support Protocol 3: Preparation of High‐Quality Digested Library Plasmid
Commentary
Figures
Tables

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Materials

Basic Protocol 1: Preparation of Baits and Library: Characterizing Bait Proteins

Materials

DNA encoding the proteins of interest
LexA‐fusion plasmids and controls (Table 20.7.1 ): pMW103 (Figs. and ), pEG202‐Krev1 (control), and pEG202‐Krit (control)
cI‐fusion plasmids and controls (Table 20.7.2 ): pGLS23 (Figs. and ), pGLS22‐EE₁₂₃₄₅ L (control), and pGLS22‐Ras (control)
Yeast strains (Table 20.7.3 ): PRT50
YPD plates and liquid media (unit 13.1 )
pLacGus (Fig. )
90‐ or 100‐mm complete minimal (CM) medium dropout plates (unit 13.1 ) with 2% (w/v) glucose (Glu) or 2% (w/v) galactose (Gal) and 1% raffinose (Raff):
- Glu/CM −Lys
- Glu/CM −Ura −His
- Glu/CM −Trp (also in 24 × 24–cm plates)
- Gal‐Raff/CM −Lys
- Gal‐Raff/CM −Ura −His
- Gal‐Raff/CM −Ura −His −Leu
- Gal‐Raff/CM −Ura −His −Lys
H₂ O, sterile

96‐well microtiter plate
Insert grid from a rack of 200‐µl micropipet tips
Tape
200‐µl micropipet tips, sterile
Metal frogger (e.g., Dankar Scientific) or plastic replicator (Bel‐Blotter; Bel‐Art Products or Fisher)

Additional reagents and equipment for subcloning DNA fragments (unit 3.16 ) or alternative cloning strategies (e.g., in vivo recombination; Ma et al., ), lithium acetate transformation of yeast (unit 13.7 ), Xgal overlay assay (see protocol 6 ), and immunoblotting and immunodetection (see protocol 7 )

Table 0.7.1 MaterialsMost Commonly Used LexA‐Fusion Plasmids and ControlsRecommended cI‐Fusion Plasmids and ControlsLEU2/LYS2 Selection Strains

Plasmid name	Yeast selection	E. coli selection	Comment/description	Contact information
LexA Fusion plasmids
pMW103	HIS3	Km^R	Basic plasmids to clone bait as fusion with LexA . Expression is driven by the ADH1 promoter.	RB a
pEG202	HIS3	Ap^R	Basic plasmids to clone bait as fusion with LexA . Expression is driven by the ADH1 promoter.	Origene b , MoBiTec^c
pJK202	HIS3	Ap^R	pEG202 derivative, incorporating nuclear localization sequences between LexA and polylinker (enhanced ability to translocate bait to nucleus)	Origene b
pNLexA	HIS3	Ap^R	Polylinker is upstream of LexA , which allows fusion of LexA to C terminus of bait, leaving amino‐terminal residues of bait unblocked	Origene b
pGilda	HIS3	Ap^R	GAL1 promoter and CEN‐ARS backbone facilitate a tightly controlled, galactose‐inducible bait expression; should be used if continuous presence of the bait is toxic to yeast	Origene b
pDD	HIS3	Km^R	GAL1 promoter and CEN‐ARS backbone facilitate a tightly controlled, galactose‐inducible bait expression; should be used if continuous presence of the bait is toxic to yeast	R. Hopkins a
Control LexA‐fused baits
pEG202‐Krev1	HIS3	Ap^R	A negative control for activation and positive control for interaction with Krit1 and RalGDS	I. Serebriiskii a
pEG202‐Krit1	HIS3	Ap^R	A moderate positive control for activation	I. Serebriiskii a
pRFHM1 (control)	HIS3	Ap^R	The homeodomain of bicoid cloned into pEG202 backbone (see above); the resulting non‐activating fusion is recommended as a negative control for activation and interaction assays, and as a positive control for repression assay.	Origene b
pSH17‐4 (control)	HIS3	Ap^R	GAL4 activation domain cloned into pEG202 backbone (see above) is recommended as a positive control for transcriptional activation.	Origene b
Plasmid name (reading frames)	Yeast selection	E. coli selection	Comment/description c
cI‐Fusion plasmid
pGLS23 d (A, B)	HIS5	Cm^R	ADH1 promoter expresses cI followed by polylinker
Control cI‐fused baits
pGKS3‐Krev	HIS5	Cm^R	Expresses cI‐Krev fusion protein. Use as negative control for activation assay and positive control for interaction with Krit1.
pGLS22‐Ras	HIS5	Cm^R	Expresses cI‐Ras fusion protein. Use as negative control for activation assay and positive control for interaction with Raf d .
pGLS22‐EE₁₂₃₄₅ L	HIS5	Cm^R	Expresses cI‐EE₁₂₃₄₅ L fusion protein. Use as positive control for activation assay.
Name	Relevant genotype	Number of operators	Comment/description	Contact information
PRT50	MATα, trp1 , his3 , his 4, ura3 , lexAop‐LEU2 , cIop‐Lys2	6 lexA , 3 cI	Provides the most sensitive LexA‐responsive LEU2 reporter	I. Serebriiskii e
PRT475	MATa, his3 , HIS5, leu2 , trp1 , ura3 , lexAop‐LEU2 , cIop‐LYS2	4 lexA , 3 cI	Mating partner for PRT50 strain; can be also used as a reporter strain itself.	I. Serebriiskii e

^a Contact R. Brent at ; R. Hopkins at ; and Ilya Serebriiskii at .

^b See http://www.origene.com or http://www.mobitec‐germany.com.

Table 0.7.2 MaterialsMost Commonly Used LexA‐Fusion Plasmids and ControlsRecommended cI‐Fusion Plasmids and ControlsLEU2/LYS2 Selection Strains

Plasmid name	Yeast selection	E. coli selection	Comment/description	Contact information
LexA Fusion plasmids
pMW103	HIS3	Km^R	Basic plasmids to clone bait as fusion with LexA . Expression is driven by the ADH1 promoter.	RB a
pEG202	HIS3	Ap^R	Basic plasmids to clone bait as fusion with LexA . Expression is driven by the ADH1 promoter.	Origene b , MoBiTec^c
pJK202	HIS3	Ap^R	pEG202 derivative, incorporating nuclear localization sequences between LexA and polylinker (enhanced ability to translocate bait to nucleus)	Origene b
pNLexA	HIS3	Ap^R	Polylinker is upstream of LexA , which allows fusion of LexA to C terminus of bait, leaving amino‐terminal residues of bait unblocked	Origene b
pGilda	HIS3	Ap^R	GAL1 promoter and CEN‐ARS backbone facilitate a tightly controlled, galactose‐inducible bait expression; should be used if continuous presence of the bait is toxic to yeast	Origene b
pDD	HIS3	Km^R	GAL1 promoter and CEN‐ARS backbone facilitate a tightly controlled, galactose‐inducible bait expression; should be used if continuous presence of the bait is toxic to yeast	R. Hopkins a
Control LexA‐fused baits
pEG202‐Krev1	HIS3	Ap^R	A negative control for activation and positive control for interaction with Krit1 and RalGDS	I. Serebriiskii a
pEG202‐Krit1	HIS3	Ap^R	A moderate positive control for activation	I. Serebriiskii a
pRFHM1 (control)	HIS3	Ap^R	The homeodomain of bicoid cloned into pEG202 backbone (see above); the resulting non‐activating fusion is recommended as a negative control for activation and interaction assays, and as a positive control for repression assay.	Origene b
pSH17‐4 (control)	HIS3	Ap^R	GAL4 activation domain cloned into pEG202 backbone (see above) is recommended as a positive control for transcriptional activation.	Origene b
Plasmid name (reading frames)	Yeast selection	E. coli selection	Comment/description c
cI‐Fusion plasmid
pGLS23 d (A, B)	HIS5	Cm^R	ADH1 promoter expresses cI followed by polylinker
Control cI‐fused baits
pGKS3‐Krev	HIS5	Cm^R	Expresses cI‐Krev fusion protein. Use as negative control for activation assay and positive control for interaction with Krit1.
pGLS22‐Ras	HIS5	Cm^R	Expresses cI‐Ras fusion protein. Use as negative control for activation assay and positive control for interaction with Raf d .
pGLS22‐EE₁₂₃₄₅ L	HIS5	Cm^R	Expresses cI‐EE₁₂₃₄₅ L fusion protein. Use as positive control for activation assay.
Name	Relevant genotype	Number of operators	Comment/description	Contact information
PRT50	MATα, trp1 , his3 , his 4, ura3 , lexAop‐LEU2 , cIop‐Lys2	6 lexA , 3 cI	Provides the most sensitive LexA‐responsive LEU2 reporter	I. Serebriiskii e
PRT475	MATa, his3 , HIS5, leu2 , trp1 , ura3 , lexAop‐LEU2 , cIop‐LYS2	4 lexA , 3 cI	Mating partner for PRT50 strain; can be also used as a reporter strain itself.	I. Serebriiskii e

^c Contact Ilya Serebriiskii at

^d Control plasmids are cloned in a pGLS22 background; pGLS22 is identical to pGLS23 except for an extra Eco RI site outside the polylinker.

Table 0.7.3 MaterialsMost Commonly Used LexA‐Fusion Plasmids and ControlsRecommended cI‐Fusion Plasmids and ControlsLEU2/LYS2 Selection Strains

Plasmid name	Yeast selection	E. coli selection	Comment/description	Contact information
LexA Fusion plasmids
pMW103	HIS3	Km^R	Basic plasmids to clone bait as fusion with LexA . Expression is driven by the ADH1 promoter.	RB a
pEG202	HIS3	Ap^R	Basic plasmids to clone bait as fusion with LexA . Expression is driven by the ADH1 promoter.	Origene b , MoBiTec^c
pJK202	HIS3	Ap^R	pEG202 derivative, incorporating nuclear localization sequences between LexA and polylinker (enhanced ability to translocate bait to nucleus)	Origene b
pNLexA	HIS3	Ap^R	Polylinker is upstream of LexA , which allows fusion of LexA to C terminus of bait, leaving amino‐terminal residues of bait unblocked	Origene b
pGilda	HIS3	Ap^R	GAL1 promoter and CEN‐ARS backbone facilitate a tightly controlled, galactose‐inducible bait expression; should be used if continuous presence of the bait is toxic to yeast	Origene b
pDD	HIS3	Km^R	GAL1 promoter and CEN‐ARS backbone facilitate a tightly controlled, galactose‐inducible bait expression; should be used if continuous presence of the bait is toxic to yeast	R. Hopkins a
Control LexA‐fused baits
pEG202‐Krev1	HIS3	Ap^R	A negative control for activation and positive control for interaction with Krit1 and RalGDS	I. Serebriiskii a
pEG202‐Krit1	HIS3	Ap^R	A moderate positive control for activation	I. Serebriiskii a
pRFHM1 (control)	HIS3	Ap^R	The homeodomain of bicoid cloned into pEG202 backbone (see above); the resulting non‐activating fusion is recommended as a negative control for activation and interaction assays, and as a positive control for repression assay.	Origene b
pSH17‐4 (control)	HIS3	Ap^R	GAL4 activation domain cloned into pEG202 backbone (see above) is recommended as a positive control for transcriptional activation.	Origene b
Plasmid name (reading frames)	Yeast selection	E. coli selection	Comment/description c
cI‐Fusion plasmid
pGLS23 d (A, B)	HIS5	Cm^R	ADH1 promoter expresses cI followed by polylinker
Control cI‐fused baits
pGKS3‐Krev	HIS5	Cm^R	Expresses cI‐Krev fusion protein. Use as negative control for activation assay and positive control for interaction with Krit1.
pGLS22‐Ras	HIS5	Cm^R	Expresses cI‐Ras fusion protein. Use as negative control for activation assay and positive control for interaction with Raf d .
pGLS22‐EE₁₂₃₄₅ L	HIS5	Cm^R	Expresses cI‐EE₁₂₃₄₅ L fusion protein. Use as positive control for activation assay.
Name	Relevant genotype	Number of operators	Comment/description	Contact information
PRT50	MATα, trp1 , his3 , his 4, ura3 , lexAop‐LEU2 , cIop‐Lys2	6 lexA , 3 cI	Provides the most sensitive LexA‐responsive LEU2 reporter	I. Serebriiskii e
PRT475	MATa, his3 , HIS5, leu2 , trp1 , ura3 , lexAop‐LEU2 , cIop‐LYS2	4 lexA , 3 cI	Mating partner for PRT50 strain; can be also used as a reporter strain itself.	I. Serebriiskii e

^e Contact at .

Basic Protocol 2: Preparation of Baits and Library: Transforming and Characterizing the Library

Materials

Yeast strains (Table 20.7.3 ), fresh: PRT475
YPD liquid media (unit 13.1 ) without G418 ( appendix 1K )
H₂ O, sterile
TE buffer ( appendix 22 )/0.1 M lithium acetate
Library DNA in pJG4‐5 (Fig. ) or pYesTrp (Table 20.7.4 )
Carrier DNA, freshly denatured
Negative control plasmids (Table 20.7.4 ; optional): pJG4‐5 or pYesTrp2, pJG4‐5‐Raf1, pJG4‐5‐Krit, and pYesTrp2‐RalGDS
40% (w/v) PEG 4000/0.1 M lithium acetate/TE buffer, pH 7.5
Dimethyl sulfoxide (DMSO)
90‐mm and 24 × 24–cm Glu/CM −Trp plates (unit 13.1 )
TE buffer ( appendix 22 ), sterile (optional)
Glycerol solution: 65% (w/v) sterile glycerol/0.1 M MgSO₄ /25 mM Tris·Cl, pH 8.0 ( appendix 22 )

Orbital shaker, 30°C
50‐ml conical tubes, sterile
42°C heat block
3‐ to 4‐mm glass balls, sterile (Thomas Scientific or Fisher)

Additional reagents and equipment for lithium acetate transformation of yeast (unit 13.7 )

Table 0.7.4 MaterialsMost Commonly Used Activation Domain Fusion Plasmids and Controls

Plasmid	Yeast selection	E. coli selection	Comment/description	Contact information
pJG4‐5	TRP1	Ap^R	Library construction plasmid; GAL1 promoter provides efficient expression of a gene fused to a cassette consisting of nuclear localization sequence, transcriptional activation domain, and HA epitope tag	Origene, MoBiTec f
pYesTrp	TRP1	Ap^R	GAL1 promoter expresses nuclear localization domain, transcriptional activation domain, V5 epitope tag, multiple cloning sites; contains f1 ori and T7 promoter/flanking site. Used to express cDNA libraries	Invitrogen f
Control activation domain–fusions
pJG4‐5‐Raf	TRP1	Ap^R	A positive control for interaction with Ras	I. Serebriiskii g
pYesTrp‐RalGDS	TRP1	Ap^R	A positive control for interaction with Ras and Krev	I. Serebriiskii g
pJG4‐5‐Krit	TRP1	Ap^R	A positive control for interaction with Krev	I. Serebriiskii g

^f See http://www.origene.com, http://www.mobitec‐germany.com, or http://www.invitrogen.com.

^g Contact at .

Basic Protocol 3: Selecting an Interactor

Materials

Complete minimal (CM) medium dropout plates (or medium; unit 13.1 ) with 2% (w/v) glucose (Glu) or 2% (w/v) galactose (Gal) and 1% raffinose (Raff):
- Glu/CM −Ura −His
- Glu/CM −Ura −His −Trp
- Glu/CM −Ura −His −Trp −Leu
- Glu/CM −Ura −His −Trp −Lys
- Glu/CM −Leu (optional)
- Glu /CM −Lys (optional)
- Gal‐Raff/CM −Ura −His −Trp
- Gal‐Raff/CM −Ura −His −Trp −Leu
- Gal‐Raff/CM −Ura −His −Trp −Lys
- Gal‐Raff/CM −Lys
- Gal‐Raff/CM −Leu (optional)
- Gal‐Raff/CM −Ura −His −Trp −Lys −Leu (optional)
Bait strains containing appropriate combinations of plasmids (i.e., pMW103‐Bait1 + pLacGus + pGLS23‐Bait2) on Glu/CM −Ura −His (see protocol 1 )
Pretransformed library strain (see protocol 2 )
H₂ O, sterile
90‐ or 100‐mm‐diameter YPD plates (unit 3.1 )
Glycerol solution: 65% (w/v) sterile glycerol/0.1 M MgSO₄ /25 mM Tris·Cl, pH 8.0
30°C incubator with and without shaker
1.5‐ml microcentrifuge tube, sterile
3‐ to 4‐mm sterile glass balls (Thomas Scientific or Fisher)
Markers or wax pencils of different colors
Insert grid from a rack of 200‐µl pipet tips
Metal frogger (e.g., Dankar Scientific) or plastic replicator (i.e., Bel‐Blotter; Bel‐Art Products or Fisher)
96‐well microtiter plate

Additional reagents and equipment for lithium acetate transformation of yeast (unit 13.7 ) and protocol 6

Basic Protocol 4: Analysis of Primary Interactors

Materials

Glu/CM −Ura −His −Trp master plate (see protocol 3 )
β‐Glucuronidase solution: dilute crude β‐glucuronidase type HP‐2 from H. pomatia (Sigma) 1:50 in 50 mM Tris·Cl, pH 7.5 ( appendix 22 )/10 mM EDTA; prepare fresh
Library plasmid specific primers—e.g., for JG4‐5:
Forward primer (FP1): 5′‐CTG AGT GGA GAT GCC TCC
Reverse primer (FP2): 5′ CTG GCA AGG TAG ACA AGC CG
Hae III (unit 3.1 )
PRT50 containing pMW103‐Bait1 + pLacGus + pGLS23‐Bait2 (see protocol 1 )
PRT50 containing pEG202‐Krev1 + pLacGus + pGLS22‐Ras (see protocol 1 )
Raf1 and Krit1 PCR fragment (see protocol 8 ; optional)
Glu/CM −Ura −His −Trp dropout plates (unit 13.1 )

Metal frogger (e.g., Dankar Scientific) or plastic replicator (i.e., Bel‐Blotter; Bel‐Art Products or Fisher)
96‐well microtiter plate
Horizontal shaker, 37°C
Tape
150‐ to 212‐µm glass beads (e.g., Sigma G‐1145)
Vortex with flat‐top surface

Additional reagents and equipment for PCR (unit 15.1 ), restriction endonuclease digestion (unit 3.1 ), agarose gel electrophoresis (unit 2.5 ), purification of DNA fragments (unit 2.6 ), lithium acetate transformation of yeast (unit 3.17 ), sequencing (Chapter 7), rapid miniprep isolation of yeast DNA (unit 3.6 ), and miniprep isolation of bacterial DNA (unit 1.6 ).

Alternate Protocol 1: Screening for Interaction Trap Positives by Yeast Plasmid Recovery

Materials

Plates containing spotted yeast colonies to be assayed
Chloroform
Xgal agarose: prepare 1% low‐melting agarose in 100 mM KHPO₄ , pH 7.0, and heat to boil; cool to ∼60°C and add Xgal to 0.25 mg/ml; prepare fresh
Xgluc agarose: prepare as described for Xgal agarose, except replace Xgal with Xgluc

Support Protocol 1: Colorimetric Assay of β‐Galactosidase and β‐Glucuronidase Activity by Agarose Overlay

Materials

Master plates containing primary transformants of each bait (see protocol 1 ) and positive control
Selective medium (unit 13.1 )
2× Laemmli sample buffer (unit 20.1 )
Antibodies to LexA and cI (Invitrogen)

Additional reagents and equipment for minigel electrophoresis (unit 2.5 )

Support Protocol 2: Detection of Bait Protein Expression

Empty library plasmid: pJG4‐5
Restriction enzymes (e.g., Eco RI and Xho I; unit 3.1 )
pJG4‐5‐Raf1 (Table 20.7.4 )
pJG4‐5‐Krit1 (Table 20.7.4 )
PRT475 (Table 20.7.3 )
Glu/CM −Trp dropout plates (unit 13.1 )

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Figures

Figure 20.7.1 Outline of the dual bait system and the key control points. An activation domain (AD)‐fused prey interacts with Bait1 (LexA‐fused) to drive transcription of lexAop ‐responsive reporters ( LEU2 and LacZ ), but does not interact with Bait2 (cI‐fused), and thus does not turn on transcription of cIop ‐responsive reporters ( LYS2 and GusA ). As shown in this figure, the cI‐bait, represents a negative control for prey binding; the system can also be configured so prey interacts with only cI‐bait or both baits. To maximize the utility of the system, one can adjust experimental parameters affecting expression of baits and reporter readouts. Points of flexibility in the dual bait‐hybrid system are indicated in the figure by circled numbers and include: (1) varying expression level of baits, (2) varying cellular localization of baits, (3) varying sensitivity of reporters, (4) diversifying plasmid markers to broaden compatibility with different yeast two‐hybrid systems and to facilitate isolation of library plasmid in E. coli , (5) enriching polylinkers and choice of reading frames to facilitate cloning of baits, and (6) development of robust yeast strains suitable both for bait testing and interaction mating.

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Figure 20.7.2 Flow chart of the two‐hybrid screen done by interaction mating. The third stage allows some flexibility, reflected in the availability of different protocols (see and ).

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Figure 20.7.3 Plasmid map of the 10101‐nt basic dual bait vector pMW103. This plasmid, a derivative of pEG202, uses the strong constitutive ADH1 promoter to express bait proteins as fusions to the DNA‐binding protein LexA. The plasmid contains the HIS3 selectable marker and the 2 µ origin of replication to allow selection and propagation in yeast, the kanamycin resistance gene and the pBR origin of replication to allow selection and propagation in E. coli . A detailed map of the LexA‐fusion polylinker is given in Figure .

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Figure 20.7.4 Polylinkers of the two‐hybrid basic vectors. (A ) The LexA‐fusion vector polylinker for (top) pMW103 (also see Fig. ) and (bottom) pNLexA. (B ) The cI‐fusion vector polylinker (e.g., pGLS23; Fig. ). Note both the A (top) and B (bottom) reading frames are shown. These are the AAT and GAA reading frames, respectively. (C ) The AD‐fusion (library) vectors polylinker for (top) pJG4‐5 (also see Fig. ), which is also known as pB42AD and displayTarget, and pYesTrp2. Only restriction sites that are available for insertion of coding sequences are shown; those shown in bold type are unique. The asterisk ~undefined) in panel A denotes that Nco I is unique in pEG202 but not pMW103.

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Figure 20.7.5 Plasmid map of the 6187‐nt basic dual bait vector pGLS23. This plasmid uses the strong constitutive TEF promoter to express bait proteins as fusions to the DNA‐binding protein cI. The plasmid contains the 2µ origin of replication ( ori ) and HIS5 marker to allow propagation in yeast, and the ColE1 origin of replication and chloramphenicol resistance marker to allow selection in E. coli . Note that control plasmids described below are cloned in a pGLS22 background. pGLS22 is identical to pGLS23 except that it has an extra Eco RI site outside of the polylinker.

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Figure 20.7.6 Plasmid map of the 11099‐nt basic dual bait vector pLacGus, which is also known as pDR8. pLacGus is a dual reporter plasmid. Eight LexA operators upstream of the lacZ reporter make it the most sensitive of the lexA‐responsive lacZ reporters. The cI‐responsive gusA reporter (three cI operators) has a comparable sensitivity range. These two cassettes are separated by cytC terminator. The plasmid contains the URA3 selectable marker and the 2 µ origin of replication to allow selection and propagation in yeast, and the kanamycin resistance gene and the pUC origin of replication to allow selection and propagation in E. coli .

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Figure 20.7.7 Detailed library screening flow chart. See Basic Protocols 2 and 3 for details.

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Figure 20.7.8 Plasmid map of the basic dual bait vector pJG4‐5. This plasmid expresses cDNAs or other coding sequences inserted into the unique Eco RI and Xho I sites as translational fusion to a cassette consisting of the SV40 nuclear localization sequence, the acid blob B42, and the hemagglutinin (HA) epitope tag. Expression of sequences is under the control of the GAL1 galactose‐inducible promoter. The plasmid contains the TRP1 selectable marker and the 2 µ origin of replication to allow selection and propagation in yeast, and the ampicillin resistance gene and pUC origin of replication to allow selection and propagation in E. coli . The fusion cassette is shown at the bottom. A detailed map of the AD‐fusion polylinker is given in Figure .

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Figure 20.7.9 Detailed flow chart for characterization and second confirmation of primary positives. See and for details.

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Literature Cited

Literature Cited
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Internet Resource
	http://www.fccc.edu/research/labs/golemis/InteractionTrapInWork.html
	Analysis of the two‐hybrid usage; database of false positives detected in two‐hybrid experiments; database of available libraries, strains, and specialized vectors (maps, sequences) for Interaction Trap/Dual Bait system; protocols related to the two‐hybrid screening and β‐galactosidase assays in yeast