Dual bait Two Hybrid System

plasmids and strains

They are supposed to work in combination with lexA-based bait plasmids. Be sure to pick up the combination which is compatible by yeast markers...

cI Fusion plasmids

Plasmid name / reading
frames
Selection
in yeast/
 in E.coli
  

Comment/description
pGKS3  AB HIS3 ApR ADH promoter expresses cI followed by polylinker
pGKS4  AB KmR
pGKS-8 AB
ZeoR
 Dual purpose vector:ADH promoter expresses cI followed by polylinker, while cI-responsive gusA reporter cassette is integrated into the same backbone.
pGKS6 ABC ADH promoter expresses cI followed by polylinker
pGKS7 AB Modified ADH promoter ensures higher level of expression of cI
pGBS9 AB G418R KmR ADH promoter expresses cI followed by polylinker
pGBS10 AB Modified ADH promoter ensures higher level of expression of cI
pGMS11 A
ZeoR
 GAL promoter expresses cI followed by polylinker, for use with Baits whose continuous presence is toxic to yeast
pGMS12 B G418R KmR

cI-fused bait controls


Plasmid name / reading
frames
Selection
in yeast/
 in E.coli
  

Comment/description
pGKS3-Krev HIS3
ApR
 Expresses cI-Krev fusion protein. Use as negative control for activation assay, and positive control for interaction with Krit1
pGBS9-Krev G418R KmR
pGBS10-Krev
pGKS6-Krev
ZeoR
pGKS6-Krit Expresses cI-Krit fusion protein. Use as positive control for activation asssay
pGKS7-Krit
pGKS3-Krit HIS3
ApR
pGBS9-Krit G418R
KmR
pGBS10-Krit
pGMS12-Krit


cI-responsive GusA Reporter Plasmids

Plasmid name
/source
Selection
in yeast/
in E.coli
Number of operators
  

Comment/description
PRG64, 64a,64b
URA3
KmR
4 cI
 cI operators direct transcription of the gusA gene: the most sensitive indicator plasmids for transcriptional activation have 4 operators, intermediate reporters 2, and the most stringent reporters 1 operator
PRG62
2 cI
PRG61
1 cI
pDR8

(Invitrogen)
8 lexA 

3 cI

 lexA-responsive lacZ reporter the same as that in pMW112, while cI-responsive gusA reporter has lower background level that in pGKS8.

 

LEU2/LYS2 Selection Strains

 Name
Genotype
Number of operators
Comment/description
SKY48  MATatrp1,
his3, ura3, lexAop-LEU2, cIop-Lys2
6 lexA 3 cI
 Provides a more stringent selection for interaction partners of cI-fused Baits, and more sensitive lexA-responsive LEU2 reporter than the one in SKY191
SKY191
2 lexA 3 cI
 Provides a more stringent lexA-responsive LEU2 reporter, and more sensitive cI-responsive LYS2 reporter than the one in SKY48.
SKY473 MATahis3, leu2, trp1, ura3, lexAop-LEU2 cIop-LYS2
4 lexA

3 cI

 Mating partner for SKY strains; can be also used as a reporter strain itself. Sensitivity of LEU2 reporter is intermediate between sensitivity of LEU2 in SKY48 and SKY191. Sensitivity of LYS2 reporter is the same as sensitivity  of LYS2 in SKY191. 

cI antibodies are also available... Ø