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What is the experimental workflow for Nuclear Yeast library construction and screening?
1、Library Construction:
Total RNA extraction → mRNA isolation & purification → Double-stranded cDNA synthesis & purification → In vitro ligation of ds cDNA into pGADT7 vector → E. coli library culture → Library plasmids → Transformation into Y187 yeast host → Y187 yeast library culture.
2、Two-Hybrid Screening:
(1) Mating method:
Bait recombinant pGBKT7 plasmid construction → Autoactivation test: Co-transformation of bait recombinant plasmid & empty pGADT7 into Y2HGold yeast host → Mixing Y2HGold yeast culture containing bait with Y187 yeast library culture → Plating on selective plates based on autoactivation results → Picking positive colonies → Culturing positive yeast & plasmid extraction → PCR identification of positives → Sending for sequencing → Sequencing result analysis.
(2) Co-transformation method:
Bait recombinant pGBKT7 plasmid construction → Autoactivation test: Co-transformation of bait recombinant plasmid & empty pGADT7 into Y2HGold yeast host → Transformation of library plasmids into Y2HGold competent cells containing bait → Plating on selective plates based on autoactivation results → Picking positive colonies → Culturing positive yeast & plasmid extraction → PCR identification of positives → Sending for sequencing → Sequencing result analysis.
3、One-Hybrid Screening:
Bait recombinant pABAi plasmid construction → Autoactivation test: Transformation of linearized bait recombinant plasmid & empty pGADT7 into Y1HGold yeast host → Transformation of library plasmids into Y1HGold competent cells containing bait → Plating on selective plates based on autoactivation results → Picking positive colonies → Culturing positive yeast & plasmid extraction → PCR identification of positives → Sending for sequencing → Sequencing result analysis.
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.