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Introduction to Yeast Hybrid-related vectors, strains, and their functions?
(1) pGBKT7 Vector: Used for constructing Nuclear Yeast Two-Hybrid Bait-pGBKT7 plasmids or as an empty control.
(2) pGADT7 Vector: Used for constructing Nuclear Yeast Two-Hybrid and One-Hybrid Prey-pGADT7 plasmids or as an empty control.
(3) pABAi Vector: Used for constructing One-Hybrid Bait-pABAi plasmids.
(4) pGBKT7-53 Plasmid & pGADT7-T Plasmid: Used as a positive control pair for Nuclear Yeast Two-Hybrid verification and screening.
(5) pGBKT7-lam Plasmid & pGADT7-T Plasmid: Used as a negative control pair for Nuclear Yeast Two-Hybrid verification and screening.
(6) pOST1-NubI Plasmid & pBT3-N-bait Plasmid: Used as a functional validation control pair for Membrane Yeast Two-Hybrid verification and screening.
(7) pBT3-N Vector: Used for constructing bait proteins with the N-terminus in the cytosol and C-terminus in the organelle lumen (or extracellular space).
(8) pBT3-SUC Vector: Used for constructing bait proteins with the N-terminus in the organelle lumen (or extracellular space) and containing an N-terminal signal peptide.
(9) pBT3-STE Vector: Used for constructing bait proteins with the C-terminus in the cytosol, N-terminus in the organelle lumen (or extracellular space), and without an N-terminal signal peptide.
(10) pBT3-N and pBT3-STE: Both can be used for bait proteins with both N- and C-termini located in the cytosol.
(11) pPR3-N Vector: Used for constructing prey proteins with the C-terminus in the cytosol and N-terminus extracellular or in the organelle lumen.
(12) pPR3-C Vector: Used for membrane library construction and prey proteins without transmembrane domains.
(13) Ptsu2-APP Plasmid & pNubG-Fe65 Plasmid: Used as a positive control pair for Membrane Yeast Two-Hybrid verification and screening.
(14) Ptsu2-APP Plasmid & pPR3-N Plasmid: Used as a negative control pair for Membrane Yeast Two-Hybrid verification and screening.
(15) Y187 Strain: A GAL4 system yeast strain developed by Clontech for One-Hybrid and Two-Hybrid experiments. MATα type. Can be directly transformed with plasmids or used for library screening via mating with MATa strains (Y2HGold, AH109, etc.). Genotype: trp1, leu2. Reporter genes: lacZ, MEL1.
(16) Y1HGold Strain: A GAL4-AbA yeast One-Hybrid system strain developed by Clontech. MATα type. Can be directly transformed with plasmids for library screening. Genotype: ura3, leu2. Reporter gene: AbAr.
(17) Y2HGold Strain: A GAL4 system yeast Two-Hybrid strain developed by Clontech. MATa type. Can be directly transformed with plasmids or used for protein interaction verification/library screening via mating with MATα strains like Y187. Transformation markers: trp1, leu2. Reporter genes: AbAr, HIS3, ADE2, MEL1.
(18) NMY51 Strain: A yeast Two-Hybrid strain developed by DUALsystems BioTech for detecting membrane protein interactions. Can be directly transformed with plasmids for protein interaction verification or library screening. Genotype: *trp1, leu2-3*. Reporter genes: HIS3, ADE2, lacZ.
Explanation of reporter genes and plate types used in Yeast Hybrid assays?
(1) HIS3. Y2HGold cannot synthesize histidine and therefore cannot grow on media lacking this essential amino acid. When bait and prey proteins interact, the expression of the Gal4-responsive His3 gene allows cells to biosynthesize histidine and grow on minimal medium. 3-AT can be added to suppress background.
(2) ADE2. Y2HGold also cannot grow on minimal medium lacking adenine. However, when two proteins interact, Ade2 expression is activated, enabling these cells to grow on Ade- minimal medium.
(3) MEL1. MEL1 codes for α-galactosidase, an enzyme naturally present in many yeasts. Due to the two-hybrid interaction, α-galactosidase (MEL1) is expressed and secreted by the yeast cells. Yeast colonies expressing Mel1 turn blue in the presence of the chromogenic substrate X-α-Gal.
(4) AUR1-C. a dominant mutant version of the AUR1 gene, encodes inositol phosphorylceramide synthase. *AUR1-C* is expressed in the Y2HGold yeast strain due to protein-protein interaction bringing the GAL4 transcription activation and DNA-binding domains into proximity. AbA can be added for background suppression.The pABAi vector carries the Ura3 gene. The pGADT7 vector carries the Leu gene. The pGBKT7 vector carries the Trp gene.
Plates used for screening:
Single Dropout: SD/-Ura, SD/-Leu /+AbA
Double Dropout: DDO [SD/-Leu/-Trp], DDO/X [SD/-Leu/-Trp/X-α-Gal]
Triple Dropout: TDO [SD/-Leu/-Trp/-His]
Quadruple Dropout: QDO [SD/-Leu/-Trp/-His/-Ade]