Basic Protocol 1: Producing and Characterizing a Bait Strain Materials - DNA encoding the protein of interest
- Plasmids (see Table 4.4.1 ): e.g., pEG202 (Fig. ), pSH18‐34 (Fig. ), pSH17‐4, pRFHM1
- Yeast strain: e.g., EGY48 (ura3 trp1 his3 3LexA ‐operator‐LEU2 ; see Table 4.4.2 )
- 100‐mm complete minimal (CM) medium dropout plates (Treco and Lundblad, ) with 2% (w/v) glucose (Glu) or 2% (w/v) galactose (Gal):
- Glu/CM –Ura –His
- Gal/CM –Ura –His
- Gal/CM –Ura –His –Leu
- Glu/CM Xgal and Gal/CM Xgal plates (Treco and Lundblad, )
- CM dropout liquid medium (Treco and Lundblad, ) with 2% (w/v/) glucose: Glu/CM −Ura −His
- 2× Laemmli sample buffer (see recipe )
- Antibody to LexA or fusion domain
- H 2 O, sterile
- 30°C incubator
- 100°C water bath
- Additional reagents and equipment for subcloning (Struhl, ), lithium acetate transformation of yeast (Becker and Lundblad, ), filter lift or liquid assay for β‐galactosidase (Reynolds et al., ), SDS‐PAGE (Gallagher, ), and immunoblotting (Gallagher et al., ) Table 4.4.1 Materials Interaction Trap Plasmids a a , b b Interaction Trap Plasmids Interaction Trap Yeast Selection Strains f Interaction Trap Yeast Selection Strains
| | | Selection | | | Plasmid name/source | In yeast | In E. coli | Comment/description | | LexA fusion plasmids | | pEG202 c , d | HIS3 | Apr | Contains an ADH promoter that expresses LexA followed by polylinker | | pJK202 | HIS3 | Apr | Like pEG202, but incorporates nuclear localization sequences between LexA and polylinker; used to enhance translocation of bait to nucleus | | pNLexA d | HIS3 | Apr | Contains an ADH promoter that expresses polylinker followed by LexA for use with baits where their amino‐terminal residues must remain unblocked | | pGilda d | HIS3 | Apr | Contains a GAL1 promoter that expresses same LexA and polylinker cassette as pEG202 for use with baits where their continuous presence is toxic to yeast | | pEE202I | HIS3 | Apr | An integrating form of pEG202 that can be targeted into HIS3 following digestion with Kpn I; for use where physiological screen requires lower levels of bait to be expressed | | pRFHM1 d (control) | HIS3 | Apr | Contains an ADH promoter that expresses LexA fused to the homeodomain of bicoid to produce nonactivating fusion used; as positive control for repression assay, negative control for activation and interaction assays | | pSH17‐4 d (control) | HIS3 | Apr | ADH promoter expresses LexA fused to GAL4 activation domain; used as a positive control for transcriptional activation | | pMW101 e | HIS3 | Cmr | Same as pEG202, but with altered antibiotic resistance markers; basic plasmid used for cloning bait | | pMW103 e | HIS3 | Kmr | Same as pEG202, but with altered antibiotic resistance markers; basic plasmid used for cloning bait | | pHybLex/Zeo | Zeor | Zeor | Bait cloning vector compatible with interaction trap and all other two‐hybrid systems; minimal ADH promotor expresses LexA followed by extended polylinker | | Activation domain fusion plasmids | | pJG4‐5 c , d | TRP1 | Apr | Contains a GAL1 promoter that expresses nuclear localization domain, transcriptional activation domain, HA epitope tag, cloning sites; used to express cDNA libraries | | pJG4‐5I | TRP1 | Apr | An integrating form of pJG4‐5 that can be targeted into TRP1 by digestion with Bsu 36I (New England Biolabs); to be used with pEE202I to study interactions that occur physiologically at low protein concentrations | | pYESTrp | TRP1 | Apr | Contains a GAL1 promoter that 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) | | pMW102 e | TRP1 | Kmr | Same as pJG4‐5, but with altered antibiotic resistance markers; no libraries yet available | | pMW104 e | TRP1 | Cmr | Same as pJG4‐5, but with altered antibiotic resistance markers; no libraries yet available | | LacZ reporter plasmids | | pSH18‐34 d | URA3 | Apr | Contains eight LexA operators that direct transcription of the lacZ gene; one of the most sensitive indicator plasmids for transcriptional activation | | pJK103 d | URA3 | Apr | Contains two LexA operators that direct transcription of the lacZ gene; an intermediate reporter plasmid for transcriptional activation | | pRB1840 d | URA3 | Apr | Contains one LexA operator that directs transcription of the lacZ gene; one of the most stringent reporters for transcriptional activation | | pMW112 e | URA3 | Kmr | Same as pSH18‐34, but with altered antibiotic resistance marker | | pMW109 e | URA3 | Kmr | Same as pJK103, but with altered antibiotic resistance marker | | pMW111 e | URA3 | Kmr | Same as pRB1840, but with altered antibiotic resistance marker | | pMW107 e | URA3 | Cmr | Same as pSH18‐34, but with altered antibiotic resistance marker | | pMW108 e | URA3 | Cmr | Same as pJK103, but with altered antibiotic resistance marker | | pMW110 e | URA3 | Cmr | Same as pRB1840, but with altered antibiotic resistance marker | | pJK101 d (control) | URA3 | Apr | Contains a GAL1 upstream activating sequence followed by two LexA operators followed by lacZ gene; used in repression assay to assess bait binding to operator sequences | | Strain | Relevant genotype | Number of operators | Comments/description | | EGY48 g | MAT α trp1 , his3 , ura3 , lexAops‐LEU2 | 6 | lexA operators direct transcription from the LEU2 gene; EGY48 is a basic strain used to select for interacting clones from a cDNA library | | EGY191 | MATα trp1 , his3 , ura3 , lexAops‐LEU2 | 2 | EGY191 provides a more stringent selection than EGY48, producing lower background with baits with instrinsic ability to activate transcription | | L40b | MATα trpl , leu 2, ade 2, GAL4, lexAops‐HIS34 , lexAops‐lacZ8 | | Expression driven from GAL1 promoter is constitutive in L40 (inducible in EGY strains); selection is for HIS prototrophy. Integrated lacZ reporter is considerably less sensitive than pSH18‐34 maintained in EGY strains. | |
| | a All plasmids contain a 2µm origin for maintenance in yeast, as well as a bacterial origin of replication, except where noted (pEE202I, pJG4‐5I). b Interaction Trap reagents represent the work of many contributors: the original basic reagents were developed in the Brent laboratory (Gyuris et al., ). Plasmids with altered antibiotic resistance markers (all pMW plasmids) were constructed at Glaxo in Research Triangle Park, N.C. (Watson et al., ). Plasmids and strains for specialized applications have been developed by the following individuals: E. Golemis, Fox Chase Cancer Center, Philadelphia, Pa. (pEG202); cumulative efforts of I. York, Dana‐Farber Cancer Center, Boston, Mass. and M. Sainz and S. Nottwehr, U. Oregon (pNLexA); D.A. Shaywitz, MIT Center for Cancer Research, Cambridge, Mass. (pGilda); R. Buckholz, Glaxo, Research Triangle Park, N.C. (pEE202I, pJG4‐5I); J. Gyuris, Mitotix, Cambridge, Mass. (pJG4‐5); R.L. Finl | |
热门搜索词：
移动版：