Pgbkt7 53

Manufactured by Takara Bio

Sourced in United States, Japan

PGBKT7-53 is a plasmid vector used for gene expression in yeast. It contains the TRP1 selectable marker and the GPD promoter for high-level expression of target genes.

Automatically generated - may contain errors

Lab products found in correlation

Spelling variants of this product

PGBKT7 (413 citations)

10 protocols using pgbkt7 53

Yeast Two-Hybrid Assay for MvfR-QslA Interaction

Check if the same lab product or an alternative is used in the 5 most similar protocols

Yeast two-hybrid experiment was performed using Matchmaker Gold Yeast System (Clontech) following the manufacturer’s instructions. Briefly, DNA fragments encoding QslA and different domains of MvfR were PCR amplified with primers listed in Supplementary Table S3. The resulting qslA gene was cloned in the prey vector pGADT7. The coding sequence of MvfR and its divided fragments were fused in-frame with the GAL4 DNA-binding domain of the bait vector pGBKT7. Each bait/prey pair was co-transformed into the yeast strain AH109.
AH109 cells carrying the transformed plasmid were grown for 3 days at 30°C on agar plates with all essential amino acids except for tryptophan, leucine, and histidine (SD-Leu-Trp-His), and adenine (SD-Leu-Trp-His-Ade). The transformants co-transformed with plasmids pGBKT7-53 and pGADT7-T (Clontech) were used as positive controls (Iwabuchi et al., 1993 (link); Li and Fields, 1993 (link)). The strength of the protein interactions was judged by the growth of the colony on selective media following the manufacturer’s instructions.

Fang Y.L., Chen B., Zhou L., Jin Z.J., Sun S, & He Y.W. (2018). The Anti-activator QslA Negatively Regulates Phenazine-1-Carboxylic Acid Biosynthesis by Interacting With the Quorum Sensing Regulator MvfR in the Rhizobacterium Pseudomonas aeruginosa Strain PA1201. Frontiers in Microbiology, 9, 1584.

+ Open protocol

+ Expand

Yeast Transformation Assay for T. brucei 4G Homologs

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Yeast strain PJ69-4A was cultivated overnight at 30°C in YPD medium (Ammerman et al. 2012 (link)). Each transformation used 1 mL cell suspension washed and resuspended in 100 µL TE/100 mM lithium acetate buffer and incubated at RT for 15 min. The cells were centrifuged and resuspended in 360 µL transformation buffer (1 × TE/1 mM LiOAc; 50% PEG 8000 and 2 mg/mL boiled salmon sperm DNA), simultaneously transformed with GBK (tryp+) and GAD (leu+) plasmids expressing TbE5 and individual T. brucei 4G homologs, and incubated for 30 min at RT. Subsequently, the cells were incubated at 42°C for 20 min, and then spun down. The pellet was resuspended in 2 mL dropout medium (minimal medium minus tryptophan and leucine) and incubated overnight at 30°C. After dropout incubation the OD₆₀₀ was checked and all cultures centrifuged, diluted to OD₆₀₀ 0.5 in dropout medium (minimal medium minus tryptophan, leucine, and histidine), applied on to 2% agar dropout medium plates containing 3-amino-1,2,4-triazole (3AT) in serial dilutions, and incubated at 30°C for 5 d. The positive control plates used plasmids pGADT7-T and pGBKT7-53 (CLONTECH Laboratories Inc.) for transformation.

Freire E.R., Vashisht A.A., Malvezzi A.M., Zuberek J., Langousis G., Saada E.A., Nascimento J.D., Stepinski J., Darzynkiewicz E., Hill K., De Melo Neto O.P., Wohlschlegel J.A., Sturm N.R, & Campbell D.A. (2014). eIF4F-like complexes formed by cap-binding homolog TbEIF4E5 with TbEIF4G1 or TbEIF4G2 are implicated in post-transcriptional regulation in Trypanosoma brucei. RNA, 20(8), 1272-1286.

+ Open protocol

+ Expand

Yeast-based Transcription Factor Screening

Check if the same lab product or an alternative is used in the 5 most similar protocols

The construction of yeast reporter strain and Y1H screening was carried out based on the method of Ouwerkerk and Meijer^{77 (link)} with modifications. Y1H bait constructs were prepared by cloning three repeats of the p53 binding site (p53BS) (5′-AGACATGCCT-3′) using the primer pair p53BS-F1/R1^{78 (link)}, four repeats of the putative SnToxA PnPf2 binding site (Pf2BS) (5′-AAGGACCGA-3′) using the primer pair Pf2BS-F1/R1 and four repeats of pf2bs (5′-AAGGAAATA-3′) using the primer pair pf2bs-F1/R1 into pINT1-HIS3NB (provided by Dr. P.B.F Ouwerkerk, Leiden University) (Supplementary Table S3). Repeats of binding sites were cloned into pINT1-HIS3NB. Each construct was linearised, transformed into the yeast strain Y187 (Clontech, CA, USA) and selected on YPAD supplemented with G418. Bait strains were grown on selective media (-His) containing 3-amino-1,2,4-triazole (Sigma-Aldrich, MO, USA). Mating of the yeast bait strains with the prey strains was conducted by mixing the two strains together and grown on YPAD medium. Confirmation of the specific interaction between the bait sequence and the target protein was performed by reintroduce the prey construct into the bait strain. The prey construct pGADT7-p53 was built by cloning partial p53 from pGBKT7-53 (Clontech, CA, USA) into pGADT7. Similarly, PnPf2 was amplified from cDNA using the primer pair Pf2-F2/R3 and ligated into pGADT7.

Jones D.A., John E., Rybak K., Phan H.T., Singh K.B., Lin S.Y., Solomon P.S., Oliver R.P, & Tan K.C. (2019). A specific fungal transcription factor controls effector gene expression and orchestrates the establishment of the necrotrophic pathogen lifestyle on wheat. Scientific Reports, 9, 15884.

+ Open protocol

+ Expand

Yeast-based TH1 Interaction Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

The full-length TH1 coding region from the wild type and the mutants was amplified by PCR using primer BD-TH1F/R (see Supplementary Table S3) and cloned into the pGBKT7 vector (Clontech). The resultant constructs were co-transformed with the empty pGADT7 vector (Clontech) into yeast strain AH109 containing the GAL4-UAS-β-galactosidase reporter gene. The transformants were grown on the solid medium lacking leucine and tryptophan. The β-Galactosidase activity was detected using the Colony-lift Filter Assay according to the manufacturer’s user manual. For dimer formation assay, the full-length TH1 coding region from the wild type and the mutants was amplified by PCR using primer AD-TH1F/R (see Supplementary Table S3) and cloned into the pGADT7 vector. The resultant constructs were co-transformed with the pGBKT7 vector containing different TH1 alleles into yeast strain AH109. The transformants were grown on selective solid medium SD-Leu-Trp (DDO) or SD-Leu-Trp-His-Ade (QDO). Yeast colony co-transformed with pGBKT7-53 and pGADT7-T (Clontech) was used as positive control, while yeast colony co-transformed with pGBKT7-Lam and pGADT7-T (Clontech) was used as negative control.

Peng P., Liu L., Fang J., Zhao J., Yuan S, & Li X. (2017). The rice TRIANGULAR HULL1 protein acts as a transcriptional repressor in regulating lateral development of spikelet. Scientific Reports, 7, 13712.

+ Open protocol

+ Expand

Yeast Two-Hybrid Assay for Protein-Protein Interactions

Check if the same lab product or an alternative is used in the 5 most similar protocols

Yeast vectors pGBKT7 (GAL4 DNA-binding domain, BD), pGADT7 (GAL4 activation domain, AD), and the yeast strain Y2HGold (Clontech, Mountain View, CA, USA) were used in the yeast two-hybrid assays. To test the interaction between AtNPR1 and the candidate target proteins (CsNPR3 and CsTGA5), cDNA sequences of AtNPR1 were cloned into the pGBKT7 vector in-frame with the GAL4 DNA-binding domain (BD). The cDNA sequences of target proteins were cloned into the pGADT7 vector in-frame with the GAL4 DNA-activating domain (AD). Co-transformation of BD and AD vectors in Y2HGold was performed to confirm the interaction. Empty BD and AD vectors were used as negative controls. Additionally, standard positive controls (pGBKT7-53 and pGADT7-T; Clontech) and standard negative controls (pGBKT7-Lam and pGADT7-T) were included. Yeast transformants were plated on double dropout medium (DDO) with -Trp and -Leu and were screened on quadruple dropout medium (QDO) with -Trp, -Leu, -Ade, and -His supplemented with X-α-Gal and aureobasidin A (QDO/X/A). QDO/X/A was supplemented with 100 µM SA (Sigma Aldrich, Saint Louis, MO, USA).

Qiu W., Soares J., Pang Z., Huang Y., Sun Z., Wang N., Grosser J, & Dutt M. (2020). Potential Mechanisms of AtNPR1 Mediated Resistance against Huanglongbing (HLB) in Citrus. International Journal of Molecular Sciences, 21(6), 2009.

+ Open protocol

+ Expand

Antibody Procurement and Plasmid Preparation

Check if the same lab product or an alternative is used in the 5 most similar protocols

C-Myc antibodies, GST-tag antibodies, and His-tag antibodies were purchased from Sangon Biotech, China. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG was purchased from Proteintech (USA). FITC-conjugated Goat anti-rat IgG (H + L) was purchased from BBI Life Sciences (USA), and Cy3-conjugated goat anti-rabbit IgG (H + L) secondary antibody was purchased from BOSTER, China. The beta tubulin rabbit polyclonal antibody (β-tubulin) and EIF4A2 rabbit polyclonal antibody were purchased from Proteintech (USA). The mAb to the TGEV M protein was kindly donated by Yu Bai from Wenzhou college of sciences and agriculture. Y2HGold and Y187 were purchased from Clontech, Japan. The Escherichia coli Rosetta strain was provided by Prof. Ji Xiang Li. The plasmids applied in the yeast two-hybrid, pFastBac™-M was stored in the laboratory at −80 °C, and both pGBKT7 and pGBKT7–53 were purchased from Clontech, Japan. pMD19-T simple was purchased from TaKaRa, Japan.

Song Z., Yang Y., Wang L., Wang K., Ran L., Xie Y., Huang L., Yang Z., Yuan P, & Yu Q. (2018). EIF4A2 interacts with the membrane protein of transmissible gastroenteritis coronavirus and plays a role in virus replication. Research in Veterinary Science, 123, 39-46.

+ Open protocol

+ Expand

Cell Culture and Plasmid Manipulation

Check if the same lab product or an alternative is used in the 5 most similar protocols

DF-1 cells and HEK 293 T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Plasmid pHSIE was kindly provided by Yi Zheng of the Shenzhen Key Lab of Gene and Antibody Therapy, Tsinghua University. Y2HGold yeast strain, Y187 yeast strain, pGADT7-Rec (cloning vector), pGBKT7 (cloning vector), pGADT7-T (control vector), pGBKT7-Lam (control vector), and pGBKT7-53 (control vector) were purchased from Clontech (Japan).

Gao X., Hu X., Tong L., Liu D., Chang X., Wang H., Dang R., Wang X., Xiao S., Du E, & Yang Z. (2016). Construction of a camelid VHH yeast two-hybrid library and the selection of VHH against haemagglutinin-neuraminidase protein of the Newcastle disease virus. BMC Veterinary Research, 12, 39.

+ Open protocol

+ Expand

Yeast Two-Hybrid Protein Interaction Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Strain AH109 of Saccharomyces cerevisiae and vectors pGBKT7, pGADT7, pGBKT7-53, pGADT7-T, and pGBKT7-Lam were commercially provided by Clontech Laboratories Inc. (CA, USA) for protein-protein interaction analysis. For the construction of bait and prey vectors, the oligonucleotide primers, as shown in Supplementary Table S1, were used for gene amplification of MiglnB, MiargB, MiaccB1, and MiaccB2 lacking their signal peptide sequences. The amplicon carrying the mature MiPII-coding sequence was ligated into the EcoRI- and PstI-digested pGBKT7 to generate the bait vector pGBKT7-glnB. Similarly, MiargB, MiaccB1, and MiaccB2 lacking their signal peptide sequences were ligated into EcoRI- and XhoI-treated pGADT7 to generate the prey vectors, pGADT7-argB, pGADT7-accB1, and pGADT7-accB2, respectively. The plasmid pair between pGBKT7-glnB and pGADT7-argB, pGADT7-accB1, or pGADT7-accB2 was co-transformed into AH109 yeast separately by electroporation (Bio-Rad, USA), which were inoculated on SD-Trp-Leu plates according to the Clontech Yeast Protocols Handbook (Clontech, CA, USA). Expression of the lacZ reporter gene was determined by measuring β-galactosidase activity using 2-nitrophenyl-β-D-galactopyranoside as a substrate. The plasmid pairs pGBKT7-53 and pGADT7, and pGBKT7-Lam and pGADT7-T, served as a positive or negative control, respectively.

Li Y., Liu W., Sun L.P, & Zhou Z.G. (2017). Evidence for PII with NAGK interaction that regulates Arg synthesis in the microalga Myrmecia incisa in response to nitrogen starvation. Scientific Reports, 7, 16291.

+ Open protocol

+ Expand

Yeast Two-Hybrid Interaction Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

Saccharomyces cerevisiae Y2HGold (Clontech) was used for the Y2H assay. pGBKT7‐ and pGADT7‐derived constructs were transformed into Y2HGold and the Y2H experiment was performed according to the protocols provided by the manufacturer. DDO (minimal synthetic defined base with added double dropout supplement − Leu/−Trp) culture plate was used for positive transformation screening and QDO (SD base with added quadruple dropout supplement − Ade/−His/−Leu/−Trp)/X‐α‐Gal culture plate was used for protein interaction verification. Vectors pGBKT7‐53 and pGADT7‐T (Clontech) were used as positive control because the murine p53 protein (53) can interact with SV40 large T‐antigen (T) in yeast, while pGBKT7‐Lam (Clontech) and pGADT7‐T were used as negative control because human lamin C protein (Lam) cannot interact with T in yeast.

Liu M., Li Y., Zhu Y., Sun Y, & Wang G. (2021). Maize nicotinate N‐methyltransferase interacts with the NLR protein Rp1‐D21 and modulates the hypersensitive response. Molecular Plant Pathology, 22(5), 564-579.

+ Open protocol

+ Expand

Yeast Two-Hybrid Assay for Protein-Protein Interactions

Check if the same lab product or an alternative is used in the 5 most similar protocols

The yeast two-hybrid assay was carried out using a GAL4 DNA-binding domain-encoding bait vector (pGBKT7) and a GAL4 activation domain-encoding prey vector (pGADT7) in the yeast strain AH109, as described by Liu et al. (30 (link)). All fusion constructs were made as full-length fusions. The interaction between human lamin C bait fusion (pGBKT7-Lam) and simian virus 40 (SV40) prey fusion (pGADT7-T) (Clontech) was used as a negative control, and the interaction between murine p53 bait fusion (pGBKT7-53) and pGADT7-T served as a positive control.

Zeng Z.X., Liu L.Y., Xiao S.B., Lu J.F., Liu Y.L., Li J., Zhou Y.Z., Liao L.J., Li D.Y., Zhou Y., Nie P, & Xie H.X. (2022). Secreted in a Type III Secretion System-Dependent Manner, EsaH and EscE Are the Cochaperones of the T3SS Needle Protein EsaG of Edwardsiella piscicida. mBio, 13(4), e01250-22.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!