Yeast Two-Hybrid Service

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.

From the perspective of library construction:

(1) Flexible library construction methods: Depending on sample type, provided sample amount, etc., methods such as SMART, Gateway, In-Fusion in vitro recombination, and T4 DNA ligase in vitro ligation can be used for library construction.

(2) Controllable library insert size: The insert size can be controlled based on the type/size of protein the client wishes to study, to increase the possibility of screening for the target protein. For example, small molecular weight proteins like interleukins and growth factors; slightly larger molecular weight proteins like transcription factors and signaling pathway molecules.

(3) Libraries are universal for One- and Two-Hybrid screens: One type of library can be used for both One- and Two-Hybrid screening.

For library screening:

(1) Diverse screening methods: Library screening can be performed using mating or co-transformation.

(2) Controllable interaction strength: Screening pressure can be increased (or decreased) to screen for proteins with strong (or weak) interactions.

(3) Further validation of positive results: Positive results from library screening can undergo point-to-point verification to further exclude false positives.

The main service scope is as follows:

1、Nuclear Yeast One/Two-Hybrid library construction;

2、Membrane Yeast Two-Hybrid library construction;

3、Nuclear Yeast One/Two-Hybrid library screening;

4、Membrane Yeast Two-Hybrid library screening;

5、Nuclear Yeast One/Two-Hybrid point-to-point verification;

6、Membrane Yeast Two-Hybrid point-to-point verification

(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]