Gateway technology

Manufactured by Thermo Fisher Scientific

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Gateway technology is a cloning system that enables the transfer of DNA sequences between multiple vectors. It provides a rapid and efficient method for constructing recombinant DNA molecules by facilitating the movement of DNA segments between different vector systems.

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Lab products found in correlation

472 protocols using gateway technology

Cloning and Tagging of Mouse Nlrp1b and SMAC

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cDNA encoding the full-length mouse Nlrp1b gene was cloned from RAW 264.7 macrophages. The full-length gene was shuttled into a pLEX_307 vector (Addgene) with a C-terminal V5 tag using Gateway technology (Thermo Fisher Scientific). Two C-terminal constructs of Nlrp1b starting at S984 and M986 were generated by PCR amplification using specific primers, which contained a 5’ sequence overlap with ubiquitin. The ubiquitin (Ub) sequence was PCR amplified (Addgene 12647) with primers containing a 3’ overlap with corresponding Nlrp1b C-termini. The partially overlapping Ub and Nlrp1b C-termini products were mixed and assembled by PCR to yield the ubiquitin fused constructs. These products were then shuttled into a pLEX_307 vector (Addgene) with a C-terminal V5 tag using Gateway technology (Thermo Fisher Scientific). cDNA encoding SMAC was cloned from The Broad Institute’s ORFeome (Yang et al., 2011 (link)), and similarly amplified to generate the ubiquitin fused constructs. These products were then shuttled into a modified pLEX_307 vector (Addgene) with a C-terminal FLAG tag and a pET-DEST42 vector (Addgene) using Gateway technology (Thermo Fisher Scientific).

Griswold A.R., Cifani P., Rao S.D., Axelrod A.J., Miele M.M., Hendrickson R.C., Kentsis A, & Bachovchin D.A. (2019). A chemical strategy for protease substrate profiling. Cell chemical biology, 26(6), 901-907.e6.

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Recombinant Expression of BRD4 Constructs

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The His-tagged human BRD4 BD1 expression construct pNIC28-Bsa4 (construct ID BRD4A-c002) and BRD4 BD2 expression construct pNIC28-Bsa4 (construct ID BRD4A-c011) have been described (17 (link)). For the photobleaching experiments we used full-length human BRD4 cDNA (RefSeq NM_058243.2) cloned into the pcDNA6.2/N-EmGFP-DEST plasmid (Invitrogen) by the Gateway technology (Invitrogen) to create a chimeric green fluorescent protein (GFP)/wild-type BRD4 expression construct. For the cellular stabilization assays, the codon-optimized sequence of the wild-type and mutant versions of BRD4 BD1 (amino acids 44–166) flanked by an N-terminal His₆ tag and a C-terminal FLAG tag and by 5′ and 3′ site-specific attachment sites for recombination using the Gateway technology (Invitrogen) were prepared by total DNA synthesis (GeneArt). The synthesized DNA fragments were cloned into the pTT5 (49 (link)) destination vector using a lambda site-specific recombination system (LR-recombination, Gateway technology, Invitrogen). Plasmid DNA was prepared using the QIAprep Spin Miniprep kit (Qiagen).

Jung M., Philpott M., Müller S., Schulze J., Badock V., Eberspächer U., Moosmayer D., Bader B., Schmees N., Fernández-Montalván A, & Haendler B. (2014). Affinity Map of Bromodomain Protein 4 (BRD4) Interactions with the Histone H4 Tail and the Small Molecule Inhibitor JQ1. The Journal of Biological Chemistry, 289(13), 9304-9319.

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CARD8 and DPP9 Cloning and Mutagenesis

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Full-length CARD8 (T60 isoform, Uniprot ID Q9Y2G2-5) was cloned into pcDNA3.1 LIC 6A (Addgene plasmid #30124) with a C-terminal FLAG tag and a modified pcDNA3.1 LIC 6D (Addgene plasmid #30127) construct (C-terminal TEV-GFP-FLAG tag). The short isoform of DPP9 (DPP9S, Uniprot ID Q86TI2-1) was also cloned into pcDNA3.1 LIC 6A (N-terminal FLAG-TEV tag or N-terminal His-TEV tag). CARD8 (with several synonymous mutations to avoid CRISPR/Cas9 editing) was also shuttled into pInducer20 vector (Addgene, #44012) using Gateway technology (Thermo Fischer Scientific) and pLEX_305-N-dTAG (Addgene, #91797). CARD8-ZUC containing the ZU5, UPA and CARD was cloned as previously described (Chui et al., 2020 (link)) and shuttled to pLEX_305-N-dTAG (Addgene, #91797). CARD8-CT constructs were synthesized (GenScript) with an N-terminal ubiquitin sequence followed by CARD8 (S297-L537), cloned into the pcDNA3.1 vector (Ub-CARD8-CT) and shuttled into the pLEX307 vector using Gateway technology (Thermo Fischer Scientific). Point mutations were introduced with Q5 site-directed mutagenesis (NEB) or QuikChange site-directed mutagenesis (Agilent). Most constructs will be made available on Addgene.

Sharif H., Hollingsworth L.R., Griswold A.R., Hsiao J.C., Wang Q., Bachovchin D.A, & Wu H. (2021). Dipeptidyl Peptidase 9 Sets a Threshold for CARD8 Inflammasome Formation by Sequestering Its Active C-terminal Fragment. Immunity, 54(7), 1392-1404.e10.

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Cloning and Tagging of Mouse Nlrp Genes

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cDNA encoding the mouse Nlrp1b1 gene was cloned from RAW 264.7 macrophages. cDNA encoding the mouse Nlrp1b3, Nlrp1b3-D927V, and Nlrp1b5 genes were obtained from R. Vance and J. Mogridge. cDNA encoding the full-length mouse Nlrp1a and Nlrp1b2 were purchased from Genscript (OMu19634 and OMu00866D, respectively). As the Nlrp1a alleles in different inbred strains are highly similar between inbred mouse strains^{13 (link)}, we only studied the C57BL/6J Nlrp1a sequence here. All Nlrp1 cDNAs were subcloned into modified pLEX_307 vectors with N-terminal V5-GFP or C-terminal FLAG tags using Gateway technology (Thermo Fisher Scientific). cDNAs for mouse Gsdmd, mouse Casp1, and mouse Pycard were purchased from Origene. Casp1 was subcloned into a modified pLEX_307 vector with a hygromycin resistance marker, Gsdmd was subcloned into a modified pLEX_307 vector with a C-terminal HA tag, and Pycard was subcloned into a modified pLEX_307 vector containing N-terminal V5-GFP and C-terminal FLAG tags using Gateway technology (Thermo Fisher Scientific).

Gai K., Okondo M.C., Rao S.D., Chui A.J., Ball D.P., Johnson D.C, & Bachovchin D.A. (2019). DPP8/9 inhibitors are universal activators of functional NLRP1 alleles. Cell Death & Disease, 10(8), 587.

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BiFC and Subcellular Localization of AthBG2

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For the BiFC experiments, AthBG2 and Y14 CDS were cloned into pENTR/D-TOPO vector and mobilized respectively to pSAT4A-DEST-n(1-174)EYFP-N1 and pSAT5-DEST-c(175-END)EYFP-C1 (Tzfira et al., 2005 (link)) vectors using Gateway technology (Invitrogen, Carlsbad, CA, United States). Primers used for cloning are given in Supplementary Table S4. The clones were cotransformed in onion peel using PDS-1000 Helios Gene Gun (Biorad). The transformed onion peels were incubated at 22°C in darkness for 24 h and fluorescence was analyzed in TCS SP2 (AOBS) laser confocal scanning microscope after the incubation (Leica Microsystems). Negative control experiments were conducted along with this experiment.
For the subcellular localization of AthBG2, the CDS cloned into pENTR/D-TOPO vector was mobilized to pEG104 (Earley et al., 2006 (link)) vector using Gateway technology (Invitrogen, Carlsbad, CA, United States). The vector and YFP-CDS construct were transformed individually in onion peels and fluorescence was analyzed as described above.

Mishra B.S., Jamsheer K M., Singh D., Sharma M, & Laxmi A. (2017). Genome-Wide Identification and Expression, Protein–Protein Interaction and Evolutionary Analysis of the Seed Plant-Specific BIG GRAIN and BIG GRAIN LIKE Gene Family. Frontiers in Plant Science, 8, 1812.

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Cloning and Tagging of S2L Proteins

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The p35S::GFP-S2La construct was generated by inserting the entire coding sequence of S2La (including stop codon) amplified from wild-type Col-0 cDNA downstream of the GFP coding sequence in the pB7WGF2 plasmid (Ghent plasmids collection, https://gateway.psb.ugent.be) via Gateway technology (Invitrogen). The same was done for the p35S::GFP-S2Lb construct, except that the entire coding sequence of S2Lb was obtained from the U16729 pENTR-D-TOPO plasmid (ABRC). The pS2Lb::S2Lb-GFP and pS2Lb::S2Lb constructs were generated by inserting a PCR-amplified 3.1 kb S2Lb genomic fragment (entire genomic coding sequence and 1 kb of promoter region) in frame with a downstream GFP reporter gene in the pB7FWG,0 plasmid or in the pB7WG plasmid, respectively (Ghent plasmids collection, https://gateway.psb.ugent.be) via Gateway technology (Invitrogen). As the S2Lb fragment was cloned without STOP codon, a TAG codon was then introduced in the pS2Lb::S2Lb construct by changing one nucleotide using a site-specific mutagenesis kit (QuikChange XL Site-directed mutagenesis kit, Agilent).

Fiorucci A.S., Bourbousse C., Concia L., Rougée M., Deton-Cabanillas A.F., Zabulon G., Layat E., Latrasse D., Kim S.K., Chaumont N., Lombard B., Stroebel D., Lemoine S., Mohammad A., Blugeon C., Loew D., Bailly C., Bowler C., Benhamed M, & Barneche F. (2019). Arabidopsis S2Lb links AtCOMPASS-like and SDG2 activity in H3K4me3 independently from histone H2B monoubiquitination. Genome Biology, 20, 100.

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Localization of AtNAA10 and AtNAA15

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For localization, AtNAA10 and AtNAA15 were PCR amplified by using the NAA10_L_f, NAA10_L_r, NAA15_L_f and NAA15_L_r primers, and cloned via Gateway technology (Invitrogen) in the binary vector pK7YWG2.0, which contains the coding sequence for YFP (yellow fluorescent protein). YFP-fusion proteins were expressed in tobacco cells 2 days after transient transformation with the respective pK7YWG2.0 construct. Fluorescence was analysed by confocal laser scanning microscopy36 (link). Nicotiana tabacum cells were transiently transformed37 (link). For complementation, AtNAA10 was amplified using the primers NAA10_C_f and NAA10_C_r and cloned into the binary expression vector pK2GW7 by using the Gateway technology (Invitrogen). Correct cloning was verified by DNA sequencing. Primer sequences are listed in Supplementary Table 1.

Linster E., Stephan I., Bienvenut W.V., Maple-Grødem J., Myklebust L.M., Huber M., Reichelt M., Sticht C., Geir Møller S., Meinnel T., Arnesen T., Giglione C., Hell R, & Wirtz M. (2015). Downregulation of N-terminal acetylation triggers ABA-mediated drought responses in Arabidopsis. Nature Communications, 6, 7640.

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Molecular Biology Construct Generation

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All DNA constructs used in the study were generated by following classic molecular biology protocols or using Gateway technology (Invitrogen). For ligase-independent ligation, the constructs were based on ligation-free cloning MasterMix (Applied Biological Materials, E011-5-A) according to the manufacturer's instructions. For ligase-dependent cloning, the endonuclease digested vectors and PCR fragments were separately purified using a PCR cleanup kit (Axygen, AP-PCR-250) and ligated at 16°C with T4 DNA ligase (New England Biolabs, M2020) for at least 8 h. For Gateway cloning, all gene sequences were cloned into the pQBV3 vector (Gateway) and then recombined with the specific destination vectors using Gateway technology (Invitrogen). All the primers used for the generation of constructs are shown in supplemental Table 1.

Yan B., Yang Z., He G., Jing Y., Dong H., Ju L., Zhang Y., Zhu Y., Zhou Y, & Sun J. (2021). The blue light receptor CRY1 interacts with GID1 and DELLA proteins to repress gibberellin signaling and plant growth. Plant Communications, 2(6), 100245.

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Gateway Cloning for Protein Studies

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All plasmid constructions were performed using Gateway technology (Invitrogen, USA). For the PPI study, genes of interest (GOI) were cloned into Gateway destination vectors, pEarleyGate201-YN and pEarleyGate202-YC^{51 (link)}. For Co-IP, the GmMYB176 was cloned into pEarleyGate101 and pEarleyGate104^{52 (link)} to obtain pEG101-GmMYB176-YFP and pEG104-YFP-GmMYB176, respectively. For RNAi, GmbZIP genes were cloned into pK7GWIWG2D (II). For overexpression, GmMYB176–GmbZIP gene fusion was created using an 18 bp linker (AGCACAACATTTCAACCA) by fusion PCR using the primers listed in Supplementary Data 4. The PCR products were cloned into the Gateway vector pK7WG2D. All the plasmid constructs were transformed individually into Agrobacterium tumefaciens GV3101 by electroporation.
For Y1H assay, 30 bp GmCHS8 promoter fragments in three tandem repeats (107 bp) were synthesized (Supplementary Table 4) and cloned into a pAbAi vector to obtain p30bpTR-AbAi. The prey GOI were cloned into pGADT7 using Gateway technology (Invitrogen, USA).

Anguraj Vadivel A.K., McDowell T., Renaud J.B, & Dhaubhadel S. (2021). A combinatorial action of GmMYB176 and GmbZIP5 controls isoflavonoid biosynthesis in soybean (Glycine max). Communications Biology, 4, 356.

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Yeast Two-Hybrid Assays for MiEFF and SmD1

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For pairwise Y2H assays, the CDS of the MiEFF16 MiEFF18a and MeEFF18a were amplified (Supplemental Table S2) and inserted into pB27 (by restriction/ligation using SfiI restriction site for MiEFF16 and MiEFF18a, and into pB27-GW using Gateway technology (Invitrogen) for MeEFF18a), as C-terminal fusions with LexA. Full-length SmD1 CDS sequences (SlSmD1, NbSmD1b, and AtSmD1b) were amplified (Supplemental Table S2) and inserted into pP6-GW using Gateway technology as C-terminal fusions with Gal4-AD. The pB27 and pP6 constructs were verified by sequencing and used to transform the L40ΔGal4 (MATa) and Y187 (MATα) yeast strains, respectively. Y187 and L40ΔGal4 were crossed and diploids were selected on medium lacking tryptophan and leucine. Interactions were investigated on medium lacking tryptophan, leucine, and histidine and supplemented with 0.5-mM 3-aminotriazole (3-AT).

Mejias J., Chen Y., Bazin J., Truong N.M., Mulet K., Noureddine Y., Jaubert-Possamai S., Ranty-Roby S., Soulé S., Abad P., Crespi M.D., Favery B, & Quentin M. (2022). Silencing the conserved small nuclear ribonucleoprotein SmD1 target gene alters susceptibility to root-knot nematodes in plants. Plant physiology, 189(3).

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