The full-length CDS regions of CsAP3, CUM26, AP3, and PI were cloned into pCold™ TF vector (TaKaRa #3365) and the verified constructs were transformed into BL21 (DE3) E. coli to express the corresponding proteins. IPTG was added into the LB medium when the OD600 of the BL21 cells was 0.4 to 0.6, and then the cultures were incubated at 16°C for 8 to 10 h. The proteins were purified with Ni-NTA Agarose (QIAGEN #30210) according to the user manual after the induction by IPTG. The purified proteins were analyzed by SDS-PAGE and quantified with the Bradford methods (Kruger, 1994 (link)). DNA fragments labeled with the Biotin 3′ End DNA Labeling Kit (Thermo #89818) were used as probes and unlabelled DNA fragments were used as competitor. CArG boxes were designed to be in the middle of the probes. EMSA assays were performed according to the instructions of the LightShift® Chemiluminescent EMSA Kit (Thermo #20148). The probe sequences for EMSA are listed in Supplementary Table S2 .
Pcold tf vector
Manufactured by Takara Bio
Sourced in Japan, China
The PCold-TF vector is a tool for the expression of recombinant proteins in Escherichia coli. It contains a cold-shock promoter that can induce protein expression at lower temperatures, which may help improve the solubility and folding of some proteins.
Automatically generated - may contain errors
Lab products found in correlation
Lightshift chemiluminescent emsa kit, by Thermo Fisher Scientific (4 mentions)
Ni nta agarose, by Qiagen (3 mentions)
Glutathione sepharose 4b beads, by GE Healthcare (2 mentions)
Restriction endonuclease, by Takara Bio (2 mentions)
β d thiogalactopyranoside, by Tiangen Biotech (2 mentions)
E coli bl21, by Tiangen Biotech (2 mentions)
Biotin 3 end dna labeling kit, by Thermo Fisher Scientific (1 mentions)
Ni sepharose 6 fast flow beads, by GE Healthcare (1 mentions)
Rnasealert lab test kit, by Thermo Fisher Scientific (1 mentions)
Synergy 2, by Agilent Technologies (1 mentions)
Ni tna agarose, by Qiagen (1 mentions)
Smart race cdna amplification kit, by Takara Bio (1 mentions)
Primestar hs dna polymerase, by Takara Bio (1 mentions)
Pgex 4t 1 vector, by GE Healthcare (1 mentions)
Pact vector, by Promega (1 mentions)
Gateway bp clonase enzyme mix, by Thermo Fisher Scientific (1 mentions)
Gateway lr clonase enzyme mix, by Thermo Fisher Scientific (1 mentions)
Hissep ni nta agarose resin, by Yeasen (1 mentions)
Lightshift emsa optimization control kit, by Thermo Fisher Scientific (1 mentions)
Pcold 1, by Takara Bio (1 mentions)
18 protocols using pcold tf vector
1
Cloning, Expression, and EMSA Analysis of Floral Organ Identity Genes
2
Cloning and Expression of Xylanase Genes
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The recombinant pCold TF-mbaxA plasmid and pCold I-tfxA_cd plasmid were stored at −80°C in laboratory. The pCold TF vector, DNase I (RNase-free), T4 DNA ligase, PCR kit, and restriction endonuclease were purchased from Takara (Dalian, China). Oat spelt xylan (X0627), birchwood xylan (X0502), and beechwood xylan (X4252) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. (Shanghai, China). Xylose (X) was obtained from Merck (Darmstadt, Germany). Standard xylooligosaccharides (X2 to X6) were procured from Megazyme (Wicklow, Ireland). Protein molecular weight markers and antibodies were supplied by Songon Biotechnology Co., Ltd. (Shanghai, China). High-affinity Ni-charged resin was provided by GenScript Biotechnology Co., Ltd. (Nanjing, China). The primers were synthesized at Shanghai Sunny Biotechnology Co., Ltd. (Shanghai, China).
3
Purification and Activity Assay of RNase Enzymes
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The coding sequences of PhS3‐RNase (without signal peptide) and PhS3‐RNase (M) were separately cloned into the pCold‐TF vector (TaKaRa, Kusatsu, Japan). Relevant primer sequences used are listed in Table S1 . Trigger Factor (TF) is a 48 kDa soluble tag located at the N‐terminus of His. The His‐fused proteins were expressed in Escherichia coli Trans BL21 (DE3) plysS (Transgen) at 16°C for 24 h at 180 rpm and purified using Ni Sepharose 6 Fast Flow beads (10249123; GE Healthcare, Waukesha, WI, USA) according to the manufacturer’s instructions. Protein concentration was determined by Bradford protein assays. The relative fluorescence units (RFU) of the recombinant proteins were monitored according to the manufacturer’s instructions of RNase Alert Lab Test Kit (Ambion) using Synergy 2 (Biotek, Winooski, VT, USA).
4
Protein-DNA Binding Assay with OsGIF1 and SLR1
5
Protein-DNA Binding Assay with OsGIF1 and SLR1
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EMSA was performed as previously described with minor modifications39 (link). Full-length OsGIF1 and SLR1 cDNAs were amplified and cloned into the pCold-TF vector (Takara). His-OsGIF1 and His-SLR1 recombinant proteins were purified using Ni-NTA agarose (QIAGEN, 30210), following the manufacturer’s instructions. GST (Glutathione S-transferase) and GST-OsGRF4 recombinant protein were expressed in the Escherichia coli BL21 (DE3) strain and then purified using Glutathione Sepharose 4B beads (GE Healthcare, 17-0756-01). 42 bp DNA probes were artificially amplified and labelled using a biotin label kit (Biosune). DNA gel shift assays were performed using the LightShift Chemiluminescent EMSA kit (Thermo Fisher Scientific, 20148). Relevant primer sequences are given in Supplementary Information Table 8 .
6
Recombinant TF-RDR2-RRM Fusion Protein Production
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To produce recombinant the TF-RDR2-RRM fusion protein, the DNA sequence encoding RDR amino acids 1 to 100 (or a mutant version containing five alanine substitutions as described in Fig. 3 ), was codon-optimized for E. coli, synthesized by GenScript, and cloned into the pCold-TF vector (Takara) BamHI and HindIII site. The resulting construct was transformed into ArcticExpress competent cells (Agilent). Protein expression was induced by cold shock. The fusion protein was affinity purified using Ni-TNA agarose (Qiagen). Details are provided in SI Appendix, Detailed Methods .
7
Cloning and Expression of CcTPS1
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Based on the specific fragments obtained from the transcriptome data, the full-length sequence of the candidate TPS was cloned according to the manufacturer’s instructions for the SMART RACE cDNA amplification kit (Clontech). The full-length ORF of CcTPS1 was amplified by PCR using PrimeSTAR HS DNA Polymerase (Takara) and then cloned into a pCold-TF vector (Takara) containing an N-terminal six-histidine tag at the NdeI and BamHI restriction sites, resulting in the plasmid pCold-TF/CcTPS1. The recombinant plasmid was transformed into E. coli TOP10 cells for sequence verification, then transferred into E. coli strain Rosetta (DE3) for heterologous expression. All primers used are listed in Supplemental Table 6 .
8
Cloning Chimeric RAR and RXR Receptors
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The hinge region and LBD of RAR and RXR were isolated from human and penis worm by PCR with specific primers (Table S3 ) and cloned into pBIND and/or pACT vectors (Promega, accession numbers AF264722 and AF264723.1), to produce “chimeric” receptors with the yeast transcriptional activator GAL4 (RAR-LBD-GAL4) or the viral enhancer, VP16 (RXR-LBD-VP16), which acts on proximal downstream promoters, respectively [44 (link),45 (link)]. The priapulid RAR LBD was amplified by PCR from pGEM-pCauRAR with specific primer (FP: 5′-ACTGGATCCTCGATTATGTCTATGCAACAGCGA-3′, RP: 5′-GATTCTAGAACTAGTGATTTCACGGTATGCAG-3′) and the product was digested with BamHI and XbaI. The digested fragment was subcloned into BamHI–XbaI site of pCold-TF vector (TAKARA bio, accession number AB213654), for the priapulid RAR LBD−His6-tagged trigger factor hybrid protein. Plasmid sequences were confirmed using automated Sanger sequencing (Eurofins GATC). The human RARα LBD was previously cloned into pGEX-4T-1 vector (GE Healthcare Life Sciences, accession number U13853), for the human RARα LBD-Glutathione S-transferase hybrid protein [22 (link)].
9
Cloning and Expression of BBX11 Constructs
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The full-length BBX11 coding sequence (CDS) was cloned into the pDONR223 vector using the Gateway BP Clonase Enzyme Mix (Invitrogen). CDSs were introduced into the pEarly Gate-104 or pEarly Gate-203 plant binary vector using the Gateway LR Clonase Enzyme Mix (Invitrogen) to generate 35S:YFP-BBX11 and 35S:myc-BBX11 constructs, respectively (Earley et al., 2006 (link)). To generate constructs for transient luciferase transfection assays, BBX11, HY5, and BBX21 CDSs were cloned into the EcoRI/XhoI sites of the pGreenII 62-SK vector (Hellens et al., 2005 (link)). The 2540-bp BBX11 promoter upstream of ATG was cloned into the HindIII/NcoI sites of the pGreen II 0800-LUC vector. The generation of pGreen II 0800-HY5pro-LUC (Lin et al., 2018 (link)), pET28a-HY5 (Heng et al., 2019a (link)), and pCold-TF-BBX21 (Xu et al., 2016 (link)) has been previously described. To produce the construct for prokaryotic expression, the BBX11 CDS was cloned into the EcoRI/HindIII sites of the pCold-TF vector (Takara). Primers used for plasmid construction are listed in Supplemental Table 1 .
10
Purification and EMSA of AaMYB15
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Full-length coding sequence of AaMYB15 was cloned and inserted into pCold -TF vector (Takara, Japan) for protein purification. Empty pCold -TF vector was used as negative control. The plasmids were transferred into E. coli strain Rosetta (DE3) (TransGen Biotech, China). HisSep Ni-NTA Agarose Resin and 5 mL Gravity Chromatography Colimns (Yeasen, China) were used to purify fusion proteins.
EMSA was performed according to instructions of LightShiftTM EMSA Optimization& Control Kit (Thermo Fisher Scientific, China). Biotin-labeled probes of ORAM6 and ORAM6-mutated and unlabeled probes as competitor were synthesized by Sangon, China. All primers and probes related were listed in Table S1.
EMSA was performed according to instructions of LightShiftTM EMSA Optimization& Control Kit (Thermo Fisher Scientific, China). Biotin-labeled probes of ORAM6 and ORAM6-mutated and unlabeled probes as competitor were synthesized by Sangon, China. All primers and probes related were listed in Table S1.
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