Yeastmaker yeast transformation system 2 user manual

Manufactured by Takara Bio

Sourced in United States

The Yeastmaker Yeast Transformation System 2 User Manual provides instructions for the use of the Yeastmaker system, which is designed to facilitate the transformation of yeast cells. The manual outlines the core functions and procedures for operating the system.

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Lab products found in correlation

Matchmaker gold yeast two hybrid system, by Takara Bio (2 mentions) Anti myc polyclonal antibody, by Abcam (1 mentions) Yeast protein extraction reagent, by Takara Bio (1 mentions) Yeast two hybrid system user manual, by Takara Bio (1 mentions) Pgadt7 vector, by Takara Bio (1 mentions) Yeast strain y2h gold, by Takara Bio (1 mentions) Matchmaker gold yeast two hybrid system user manual, by Takara Bio (1 mentions) Y2hgold yeast strain, by Takara Bio (1 mentions) Pgbkt7, by BD (1 mentions)

13 protocols using yeastmaker yeast transformation system 2 user manual

Yeast Two-Hybrid Assay for OsIRO2-OsFIT Interaction

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The yeast two-hybrid assays were carried out according to the manufacturer's protocol. The OsIRO2 N-terminal fragment (aa 1-150) was subcloned to the pGBKT7 plasmid as bait. Yeast transformation was performed according to the Yeastmaker Yeast Transformation System 2 User Manual (Clontech). More than 1 x 10 6 yeast clones were screened in synthetic defined (SD) medium minus Trp, Leu, His, and Ade. The plasmids of the positive clones were extracted and then retransformed into yeast for the double check on selective SD plates. Positive clones were selected for sequencing.
In yeast two-hybrid assays, the full-length OsFIT was subcloned to pGADT7 and then co-transformed with pGBKT7-OsIRO2-N. Yeast transformation was performed according to the Yeastmaker Yeast Transformation System 2 User Manual (Clontech). The primers used are listed in Supplemental Table S2.

Liang G., Zhang H., Li Y., Pu M., Yang Y., Li C., Lu C., Xu P., & Yu D. (2020). FER-LIKE FE DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (OsFIT) interacts with OsIRO2 to regulate iron homeostasis.

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Yeast Two-Hybrid Assay with Membrane Protein Interactions

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Yeast two-hybrid (Y2H) assays were performed according to the Yeastmaker Yeast Transformation System 2 User Manual (Clontech). Each pair of indicated genes were cloned into pGBKT7-DEST for fusing with GAL4 DNA binding domain (BD) or pGADT7-DEST for fusing with GAL4 activation domain (AD). Yeast competent cells (Y2H Gold) was co-transformed with bait and prey constructs, followed by 10-fold serial dilution and plating onto synthetic defined (SD) yeast leucine and tryptophan dropout medium (SD/-Leu/-Trp) or leucine, tryptophan, histidine and adenine dropout medium (SD/-Leu/-Trp/-His/-Ade). The transformants were allowed by 4- to 6-day growth on the dropout mediums at 28°C. For immunoblot detection of protein expression in yeast, the co-transformed yeast cells were propagated in liquid SD/-Leu/-Trp medium, and the cultures were harvested at OD₆₀₀ of 0.5. The total proteins were extracted by using Yeast Protein Extraction Reagent (Takara), followed by immunoblot detection with anti-HA monoclonal or anti-Myc polyclonal antibody (Abcam). Membrane yeast two hybrid (MYTH) assays were exactly performed according to the user manual of DUALmembrane starter kits (Dualsystems Biotech). Yeast competent cells (NMY51) were co-transformed with each pair of the indicated constructs, and plated onto SD/-Leu/-Trp and SD/-Leu/-Trp/-His/-Ade mediums.

Qin L., Liu H., Liu P., Jiang L., Cheng X., Li F., Shen W., Qiu W., Dai Z, & Cui H. (2024). Rubisco small subunit (RbCS) is co-opted by potyvirids as the scaffold protein in assembling a complex for viral intercellular movement. PLOS Pathogens, 20(3), e1012064.

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Yeast Two-Hybrid Screening of Rice Proteins

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Y2H assay was performed according to the YEASTMAKER Yeast Transformation System 2 User Manual (Clontech). The cDNA of OsPID was cloned into pGADT7, and the cDNAs of LAX1, OsMADS16, LOC_Os02g27030, LOC_Os04g31910 and LOC_Os06g12580 were cloned into pGBKT7 with Gibson Assembly, respectively. The primers used here were listed in Table S10. The paired bait and prey plasmids were co‐transformed into yeast stain AH109. Protein interactions were tested under the growth conditions on synthetic dropout medium without Trp, Leu, Ade and His at 28°C.

Wu H., Xie D., Tang Z., Shi D, & Yang W. (2020). PINOID regulates floral organ development by modulating auxin transport and interacts with MADS16 in rice. Plant Biotechnology Journal, 18(8), 1778-1795.

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Yeast Two-Hybrid Assay for Protein-Protein Interactions

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Yeast two-hybrid interaction assays were performed according to the Yeast Maker Yeast Transformation System 2 User Manual (Clontech). The coding sequences of AtSCC3 and AtBMI1A were subcloned into pGBKT7 or pGADT7, respectively. The constructs were then co-transformed into yeast (AH109). The yeast cells containing the bait and the prey constructs were grown on selective plates (SD-Leu-Trp-His-Ala and SD-Leu-Trp) for analysis. The results were tested after 3–7 days of growth at 30˚C. The primers used are listed in Supplementary Data 8. Values came from three biological replicates.

Zhang Y., Ma M., Liu M., Sun A., Zheng X., Liu K., Yin C., Li C., Jiang C., Tu X, & Fang Y. (2023). Histone H2A monoubiquitination marks are targeted to specific sites by cohesin subunits in Arabidopsis. Nature Communications, 14, 1209.

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Autoactivation and Toxicity Evaluation of TgROP16 Bait Protein in Yeast

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To test autoactivation and toxicity of bait protein TgROP16 in yeasts, bait plasmids containing the above mentioned three fragments of ROP16 were individually transformed into Saccharomyces cerevisiae strain Y2H Gold according to the Yeastmaker Yeast Transformation System 2 User Manual (Clontech Laboratories, Inc., Mountain View, CA, United States). Transformants were then plated onto agar plates containing appropriate selection media, including SD/-Trp, SD/-Trp/X (40 μg/ml X-α-Gal) or SD/-Trp/X/A (40 μg/ml X-α-Gal and 125 ng/ml Aureobasidin A). The plates were incubated at 30°C for 3 days and the growth (color and size) of transformants were recorded. The empty vector pGBKT7 was included as a control.

Pan M., Zhou Y., Wang Y., Li L., Song Y., Hou L, & Zhao J. (2017). Screening and Identification of the Host Proteins Interacting with Toxoplasma gondii Rhoptry Protein ROP16. Frontiers in Microbiology, 8, 2408.

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Yeast Transformation and Growth

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Yeast transformation was performed according to the Yeastmaker Yeast Transformation System 2 User Manual (Clontech). Growth was determined as described in the yeast two-hybrid system user manual (Clontech).

Pu M.N, & Liang G. (2023). The transcription factor POPEYE negatively regulates the expression of bHLH Ib genes to maintain iron homeostasis. Journal of Experimental Botany, 74(8), 2754-2767.

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Yeast Two-Hybrid Library Screening of Arabidopsis

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Total RNAs were extracted from 5-day-old Arabidopsis seedlings pretreated with 50 μM ABA (QIAGEN). The cDNA library was constructed into pGADT7 vector (Clontech) according to Matchmaker Gold Yeast Two-Hybrid System (Clontech, Cat. no. 630489). The cDNA library in pGADT7 and pGBKT7-SPY were co-transformed into Yeast strain Y2H Gold (Clontech) to ensure no autoactivation before the screening, and library screening was performed according to “Yeastmaker Yeast Transformation System 2 User Manual” (Clontech, PT1172-1).

Liang L., Wang Q., Song Z., Wu Y., Liang Q., Wang Q., Yang J., Bi Y., Zhou W, & Fan L.M. (2021). O-fucosylation of CPN20 by SPINDLY Derepresses Abscisic Acid Signaling During Seed Germination and Seedling Development. Frontiers in Plant Science, 12, 724144.

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Yeast Two-Hybrid System Assay

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The cDNA encoding MaNCP1 was amplified and ligated into pGADT7 vector to construct the recombinant plasmid pGADT7-MaNCP1. The promoter sequence of MaNmrA was amplified and ligated into the pHIS2 vector to generate pHIS2-MaNmrA, followed by transferring into the Y187 strain to construct Y187 (pHIS2-MaNmrA) and spreading on the selective media lacking tryptophan and histidine (SD/-Trp/-His) with different concentration of 3-Amino-1,2,4-triazole (3-AT), a competitive inhibitor of HIS3 expression products, to suppress the leakage expression of HIS3 products at the background level and eliminate false positives. Then, pGADT7-MaNCP1 and pHIS2-MaNmrA were co-transformed into the Y187 and spotted on the selective medium lacking leucine, tryptophan and histidine (SD/-Leu/-Trp/-His) with or without 3-AT to observe the growth of the yeast. The preparation and transformation of yeast competent cells were conducted according to the Yeastmaker Yeast Transformation System2 User Manual (Clontech). Y187 cells transformed with pGADT7-53 and pHIS2-53 vectors was used as the positive control. Y187 cells transformed with empty pHIS2 and pGADT7-MaNCP1 or empty pGADT7 and pHIS2-MaNmrA vectors were used as the negative control.

Li C., Xu D., Hu M., Zhang Q., Xia Y, & Jin K. (2022). MaNCP1, a C2H2 Zinc Finger Protein, Governs the Conidiation Pattern Shift through Regulating the Reductive Pathway for Nitric Oxide Synthesis in the Filamentous Fungus Metarhizium acridum. Microbiology Spectrum, 10(3), e00538-22.

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Yeast Two-Hybrid Screening Protocol

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Small-scale matings were performed between each of the respective bait plasmids in Y2HGold and each respective prey plasmid in Y187 according to instructions in the Matchmaker^® Gold Yeast Two-Hybrid System User Manual (PT4084-1, Takara Bio USA, 2010, p12). Screening of the interaction of proteins that were toxic in Y187 was performed using co-transformation of Y2HGold with these plasmids and each of the respective GLRaV-3 pGBKT7 constructs, according to the Yeastmaker™ Yeast Transformation System 2 User Manual (PT1172-1, Clontech Laboratories USA, 2010, p7). Y187[pGADT7-T] was mated with Y2HGold[pGBKT-p53] and Y2HGold[pGBKT7-Lam] to serve as positive and negative controls, respectively. Mated and co-transformed yeast was plated on DDO selective media, and the presence of both plasmids was confirmed by a colony PCR using Y2H screening primers (Table S1), as described above. One colony from each respective mating or co-transformation was streaked onto DDO, DDOXA, and QDOXA plates and grown at 30 °C for 3–5 days to screen for interacting protein pairs.

Mostert I., Bester R., Burger J.T, & Maree H.J. (2023). Identification of Interactions between Proteins Encoded by Grapevine Leafroll-Associated Virus 3. Viruses, 15(1), 208.

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Yeast Two-Hybrid Assay for VqAL4

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For transcriptional activation assays, the full‐length CDS of VqAL4 was inserted into the vector pGBKT7 (BD) to construct the pGBKT7‐VqAL4 (BD‐VqAL4) fusion vector. The plasmid DNA of BD‐VqAL4 was transferred into the Y2HGold yeast strain (Clontech) according to the instructions of the Yeastmaker Yeast Transformation System 2 User Manual (Clontech). pGADT7‐T co‐transformed with pGBKT7‐53 was used as a positive control, and pGADT7‐T co‐transformed with pGBKT7‐Lam was used as a negative control. The transformants were cultured on SD/−Trp, SD/−Trp + X‐α‐gal (40 μg/ml), and SD/−Trp + X‐α‐gal (40 μg/ml) + AbA (200 ng/ml) media at 30°C for 3 days (Wang et al., 2019 (link)). The primers used in this study are listed in Table S3.

Yan C., Yang N., Li R., Wang X., Xu Y., Zhang C., Wang X, & Wang Y. (2022). Alfin‐like transcription factor VqAL4 regulates a stilbene synthase to enhance powdery mildew resistance in grapevine. Molecular Plant Pathology, 24(2), 123-141.

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