The cDNA encoding human wild-type BRI2, and the FBD and FDD mutants forms of BRI2 with the addition of a Kozak sequence and an N-terminal Myc epitope tag were PCR amplified, gel purified, and sub-cloned into pcDNA 3.1 directional TOPO vector (Invitrogen). Plasmids were transformed into DH5alpha cells (Invitrogen), and positive colonies were screened by restriction enzyme analysis and direct DNA sequencing. To introduce an Asp to Ala change at aa 170, site-directed mutagenesis (Agilent Technologies) was done according to the manufacturer’s protocol using primers forward (5′-CTA TGT GAT CCC TCT GGC CAC TTC CAT TGT TAT GC-3′) and reverse (5′-GCA TAA CAA TGG AAG TGG CCA GAG GGA TCA CAT AG-3′).
Site directed mutagenesis
Manufactured by Agilent Technologies
Sourced in United States
Site-directed mutagenesis is a molecular biology technique used to introduce specific mutations into a DNA sequence. It allows for the creation of modified genes or proteins by precisely altering one or more nucleotides in a target DNA sequence.
Automatically generated - may contain errors
Lab products found in correlation
Pgex 4t 1, by GE Healthcare (2 mentions)
Glutathione sepharose beads, by New England Biolabs (2 mentions)
Amylose resin, by New England Biolabs (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Plpcx vector8, by Takara Bio (2 mentions)
Puc57 vector, by GenScript (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Dh5alpha cells, by Thermo Fisher Scientific (1 mentions)
Pcdna3.1 directional topo vector, by Thermo Fisher Scientific (1 mentions)
Pgadt7, by Takara Bio (1 mentions)
Pgbkt7, by Takara Bio (1 mentions)
Pcdna4 to vector, by Thermo Fisher Scientific (1 mentions)
Fugene 6, by Promega (1 mentions)
Psicheck 2, by Promega (1 mentions)
Lipofectamine reagent, by Thermo Fisher Scientific (1 mentions)
Pcdna3.1 expression vector, by Addgene (1 mentions)
Pmirglo, by Promega (1 mentions)
Pnl1.1 plasmid, by Promega (1 mentions)
Nanoluc luciferase, by Promega (1 mentions)
Pcdna3.1 plasmid, by Thermo Fisher Scientific (1 mentions)
55 protocols using site directed mutagenesis
1
Cloning and Mutagenesis of BRI2 Variants
2
Genetically Engineered Mcm Proteins
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The FKBP/FRB insertion sites were chosen based on a structural model of the archaeal M. thermoautotrophicum MCM protein (Fletcher et al. 2003 (link)), the secondary structure and sequence alignments of the MtMCM sequence, and those of Mcm2 to Mcm7 from S. cerevisiae. The construction of the FKBP/FRB constructs was based on the plasmids pESC-Mcm2–Mcm7 (pCS14), pESC-Mcm6–Mcm7 (pCS15), and pESC-HA-Mcm3–Mcm5 (pCS232) (Evrin et al. 2009 (link)). Initially, restriction sites (AsiSI and AscI) were introduced at the sites of insertion using site-directed mutagenesis (Agilent). To maintain the reading frame, an additional nucleotide was inserted after the 8-base-pair (bp) restriction site (AsiSI: a; AscI: c). The FRB domain, including linkers, was fused into Mcm2, Mcm3, and Mcm4 via AsiSI restriction sites (AIAGANTCTPRGSGMLPSGMASR-FRB-TSYPYDVPDYAGANDGAAIA). The FKBP domain, including linkers, was fused into Mcm5, Mcm6, and Mcm7 via AscI (SSYPYDVPDYASLGGPSSPKKKRKVSR-FKBP-TSYISFLNSDLINSRTQRVDGQIGAP). The FKBP and FRB genes, including linkers, were codon-optimized for expression in S. cerevisiae (Genscript).
3
Fission Yeast Constructs for GRα Study
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The Schizosaccharomyces pombe strain JP308 (h+ ura 4-D18), JP305 (h+ leu1-32), JP198 (h− styI:: ura4, ∆leu1-32,∆ura4-D18), Bioneer G418 (h+ ∆HSP104:: kanMX4, ade6-M216, ura4-D18,leu1-32) were using in this study. The Saccharomyces cerevisiae strain Y2hGold (MATa, trp1-901, leu2-3, 112, ura3-52, his3-200, gal4Δ, gal80Δ, LYS2:: GAL1UAS–Gal1TATA–His3, GAL2UAS–Gal2TATA–Ade2AUR1CMEL1) was used for the two hybrid system (Clontech). Yeast transformations were obtained by the lithium acetate method39 (link). Cells were grown in synthetic minimal selective medium.
The human GRα (hGRα) was cloned into Nde1/BamHI site of the pRep41 vector (Leu2) and pRep42 (Ura4) under the control of the medium thiamine-repressible nmt42 promoter40 (link). GRα GFP was constructed by GRα N-terminal fusion with green GFP cloned in pRep 42 vector. The GR deletions were generated by excising a BglII/ BglII fragment for the AF1 fragment and by deleting an EcoRI/EcoRI fragment for the LBD, all within the pRep 42 vector. The DBD deletion was constructed by using Site-Directed mutagenesis (Agilent Technologies) to delete between positions 421-488 (Fig.1A ). Vectors pGBKT7 and pGADT7 (Clontech) were used for the two-hybrid system.
The human GRα (hGRα) was cloned into Nde1/BamHI site of the pRep41 vector (Leu2) and pRep42 (Ura4) under the control of the medium thiamine-repressible nmt42 promoter40 (link). GRα GFP was constructed by GRα N-terminal fusion with green GFP cloned in pRep 42 vector. The GR deletions were generated by excising a BglII/ BglII fragment for the AF1 fragment and by deleting an EcoRI/EcoRI fragment for the LBD, all within the pRep 42 vector. The DBD deletion was constructed by using Site-Directed mutagenesis (Agilent Technologies) to delete between positions 421-488 (Fig.
4
Generation of HSPB1 Viral Vectors
5
Cloning and Purification of E6AP and MASTL Proteins
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A vector expressing HA-tagged human E6AP was obtained from Addgene (Plasmid #8658), E6AP mutants were generated using site-directed mutagenesis (Agilent) following the protocol recommended by the manufacturer. Segments of E6AP, including N (aa 1–280), M (aa 280–497), C (aa 497–770), were inserted to a pEGFP vector for IP, and pMBP vector for pull-down. Additional segments, including N1 (aa 1–99), N2 (aa 100–207), and N3 (108–280) were cloned to pEGFP for IP. Expression vectors for MASTL were previously characterized (Yamamoto et al., 2014 (link)). Additionally, three segments of MASTL (N: aa 1–340; M: aa 335–660; C: aa 656–887) were inserted into pGEX 4T-1(GE Healthcare). The resulting expression vectors were transfected into BL21 bacteria cells for protein expression and purification. For the pulldown experiments, GST-tagged proteins were purified on glutathione-Sepharose beads (New England Biolabs), and MBP-tagged proteins were purified on Amylose resin (New England Biolabs).
6
Cloning and Mutagenesis of 3' UTR Regions
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The 3′ UTR region of GATA3 or IL-4, as well as the 3′ UTR plus partial sequences in the coding region of IL-4, were cloned into psiCheck2 (Promega). miR-23a, miR-24, and miR-27a sequences were respectively cloned into pMDH-PGK-EGFP, similar to what was described previously (Lu et al., 2009 (link)). To generate GATA3 or IL-4 3′ UTR mutants, site-directed mutagenesis was performed (Agilent). 1 d before transfection, HEK293T cells were plated at 6.5 × 104 cells per well on a 24-well plate. psiCheck2 bearing WT 3′ UTR or corresponding mutant 3′ UTR were transfected to HEK293T cells with control vector or miRNA expressing plasmid using Fugene 6 (Promega). Luciferase activities were assessed at 24 h after transfection using Dual-Luciferase Reporter assay system (Promega) according to the manufacturer’s protocol.
7
Removing FLAG Tag from Plasmids
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Site-directed mutagenesis (Agilent) with primers listed in S2 Table was performed to remove the sequence encoding the FLAG tag from plasmids obtained from the H2A and H2B mutant library [27 (link)]. Plasmid sequences were confirmed by DNA sequencing. Plasmid names are given in S3 Table .
8
Transient Expression of HIV-1 Env in HEK293T Cells
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For transient expression of Env in HEK293T cells, we used the Env gene from HIV-LAI in a pcDNA3.1 expression vector (Addgene) [87 (link)]. Env variants were introduced by site-directed mutagenesis (Agilent) and confirmed by Sanger sequencing of the Env gene (S1 Table ). HEK293T cells were plated in 6-well plates at a density of 7 × 105 cells/well and allowed to adhere overnight. The next day, cells were transfected with 1.5 μg of eGFP in pcDNA3.1 (GFP control) or 0.15 μg of eGFP and 1.35 μg of Env plasmid using Lipofectamine reagents (Thermo Fisher). After 16 h, the medium was changed. After another 24 h, cells were harvested for analysis.
9
Lentiviral Cell Line Generation and Transient Transfection
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Lentiviral cell line generation was undertaken as previously described [24 (link)]. Briefly, 293 T cells were transfected with pLKO.1-shCTSV plasmids (Sigma-Aldrich), after which viral supernatant was harvested and used to transduce MCF-7 and ZR-75-1 cells. For transient transfections, cells were transfected using GeneJuice® transfection reagent in accordance to manufacturer’s instructions. pcDNA3.1-CTSV ORF (GeneScript), pCMV-Flag-STYX (MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee) or empty vector control plasmids were transfected and incubated for 48 h before protein extraction and analysis. The catalytic cysteine residue of pcDNA3.1-CTSV ORF was mutated to a serine reside using site-directed mutagenesis (Agilent Technologies) to give rise to the mutant construct (mtCTSV). The shRNA-resistant plasmid (rCTSV) was also generated using site-directed mutagenesis of pcDNA3.1-CTSV ORF, with the targeting sequence of shCTSV-1 mutated to alter codon usage but still produce the same protein sequence.
10
Genetic Modification of Yeast Strains
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Yeast strains used in this study are listed in supplemental Table 1 . Yeast plasmids used: pKA88 carrying GFP tagged Abp126 (link), pKA606 carrying wild type Las1725 (link); mutations in pKA606: pKA1087 with Las17 S554A and pKA1088 with S554D. Unless stated otherwise, cells were grown with rotary shaking at 30 °C in liquid YPD medium (1% yeast extract, 2% Bacto-peptone, 2% glucose supplemented with 40 μg/ml adenine) or in synthetic medium (0.67% yeast nitrogen base, 2% glucose) with appropriate supplements. All strains carrying fluorescent tags have growth properties similar to control strains. Point mutations in LAS17 were generated using site directed mutagenesis (Agilent). DNA cassettes carrying mutations for integration were transformed into KAY1801. Mutant colonies were counter-selected on minimal medium, containing 0.005% uracil and 0.1% 5'-Fluoroorotic Acid (5-FOA; Melford laboratories). Allele exchange, in growing Ura3-5-FOA resistant colonies, was confirmed by PCR and sequencing.
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