Gateway recombination cloning technology

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

Gateway recombination cloning technology is a molecular biology tool that enables the rapid and efficient transfer of DNA sequences between different vector systems. It utilizes site-specific recombination to facilitate the movement of DNA fragments, allowing for the creation of various expression constructs. The core function of this technology is to provide a streamlined method for DNA manipulation and gene expression studies.

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35 protocols using gateway recombination cloning technology

Conditional Expression of hADAR2 in Yeast

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The relevant plasmids used in this study are presented in Table S1. The Gateway recombination cloning technology (Invitrogen, Waltham, MA) was used to clone the hADAR2 into the URA3 marked plasmid (pYES2-DEST52 Gateway destination vector [catalog #12286-019]; Thermo Fisher Scientific, Waltham, MA). The URA3 marker was later swapped with the LEU2 marker to create BSB641. This plasmid enables the conditional expression of the hADARs in yeast, under a galactose-inducible promoter (GAL1p-hADAR2).
The plasmid carrying the URA3 reporter gene (BSB656) was created using in vivo homologous recombination in yeast, by co-transforming the yeast URA3 gene (including its 5′ promoter region and 3′ UTR) into a linearized HIS3-marked plasmid (pRS313⁴⁹ (link)) digested with XhoI/XbaI. In vivo homologous recombination was used to insert the target and reverse complement “tail” sequences (shown in Table S2) by linearizing BSB656 with either EcoRI (target insertion) or BamHI (“tail” insertion). Site-directed mutagenesis was used to insert the EcoRI (right before the URA3 start codon) and BamHI (right after the URA3 stop codon). The target and “tail” insertions were validated by PCR and Sanger sequence using OSB2366/OSB207 and OSB1202/OSB502, respectively (Table S3).

Schneider N., Steinberg R., Ben-David A., Valensi J., David-Kadoch G., Rosenwasser Z., Banin E., Levanon E.Y., Sharon D, & Ben-Aroya S. (2024). A pipeline for identifying guide RNA sequences that promote RNA editing of nonsense mutations that cause inherited retinal diseases. Molecular Therapy. Nucleic Acids, 35(1), 102130.

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Cloning Human IL-4 cDNA into Lentiviral Vector

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For cloning of human IL-4 complimentary DNA into the lentiviral vector pGLTR-X-PURO, the Invitrogen Gateway Recombination Cloning Technology (Life Technologies Ltd) was used. pGLTR-X-PURO was kindly provided by Dr Stephan Geley.^{59 (link)} IL-4 was re-cloned from pcD-human-IL-4(clone 125) (ATCC, Rockville, MD, USA) using a pair of specific primers (5′-CAAAAAAGCAGGCTCCATGGGTCTCACCTCCCAAC-3′ and 5′-CAAGAAAGCTGGGTCTCAGCTCGAACACTTTGAATATTTC-3′), which incorporated specific overhangs for the gateway reaction into the pDONOR221. The pDONOR221-Escherichiacoli-β-glucuronidase (GUS) was used for the control vector pGLTR-GUS-PURO and was provided by Invitrogen (Life Technologies Ltd).

Nappo G., Handle F., Santer F.R., McNeill R.V., Seed R.I., Collins A.T., Morrone G., Culig Z., Maitland N.J, & Erb H.H. (2017). The immunosuppressive cytokine interleukin-4 increases the clonogenic potential of prostate stem-like cells by activation of STAT6 signalling. Oncogenesis, 6(5), e342-.

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Fluorescent Protein Localization in Plants

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Sequences encoding full-length and truncated (Trp-rich regions only) ESV1 and LESV protein versions were amplified using constructs from (14 (link)) as templates. The resulting PCR products were first cloned into the pDONR221 vector and subsequently recombined into pB7WGY2 (for the truncated versions; in-frame with N-terminal sequence encoding the Arabidopsis Rubisco small subunit chloroplast transit peptide followed by sequence encoding YFP) or pB7YWG2 (for the full-length versions; in-frame with a C-terminal sequence encoding YFP) via gateway recombination cloning technology (Invitrogen), as described previously (45 (link)). The resulting constructs were transformed into Agrobacterium tumefaciens, infiltrated into tobacco (N. benthamiana), and the fluorescence was imaged as described previously (14 (link)).

Liu C., Pfister B., Osman R., Ritter M., Heutinck A., Sharma M., Eicke S., Fischer-Stettler M., Seung D., Bompard C., Abt M.R, & Zeeman S.C. (2023). LIKE EARLY STARVATION 1 and EARLY STARVATION 1 promote and stabilize amylopectin phase transition in starch biosynthesis. Science Advances, 9(21), eadg7448.

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Construction of a Fusion Transgene for Metabolic Engineering

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The coding DNA sequences of Ta1SST and Ta6SFT [13 (link)] were designed as one transcriptional fusion sequence in silico (Vector NTI 11, Thermo Fisher Scientific, USA). This fusion sequence was regulated by a TaRbcS promoter and terminator [19 (link)] (Fig. 1a). The transgene sequence (TaRbcS-p_Ta1SST:Ta6SFT_TaRbcS-t) was synthesized using the Invitrogen™ GeneArt™ Gene Synthesis service (Thermo Fisher Scientific, USA), and was then transferred into the transfer DNA (T-DNA) region of a binary expression vector using GATEWAY® recombination cloning technology (Invitrogen, USA). The T-DNA region of the binary vector also contained a selectable marker cassette (OsActin-p_bar_CaMV35s-t) that comprised a synthetically constructed bar gene [50 (link)], that encoded for phosphinothricin acetyltransferase (PAT), and regulatory sequences from the rice actin promoter and Cauliflower Mosaic Virus Terminator (Fig. 1b). The final T-DNA vectors were confirmed using Sanger sequencing.

Chen T., Hayes M., Liu Z., Isenegger D., Mason J, & Spangenberg G. (2024). Modified fructan accumulation through overexpression of wheat fructan biosynthesis pathway fusion genes Ta1SST:Ta6SFT. BMC Plant Biology, 24, 352.

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Cloning and Stable Transformation of OsISA Genes

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OsISA1 and OsISA2 coding sequences were amplified by PCR using attB-site containing primers (Table S1). The templates were pGEM-T Easy vectors containing OsISA1 and OsISA2 cDNA sequences [25] (link). The PCR fragments were cloned into pDONR 221 via the BP reaction of the Gateway recombination cloning technology (Invitrogen, LuBioScience GmbH. Lucerne, Switzerland). After sequence conformation, inserts were cloned into the plant vector pB7WG2 [27] (link) using the Gateway LR reaction. Stable Arabidopsis thaliana transgenic lines were generated through Agrobacterium tumefaciens-mediated transformation using the floral dip method [34] (link). Independent transformants were selected on soil by spraying two-week-old plants with 0.1% (v/v) BASTA herbicide.

Streb S, & Zeeman S.C. (2014). Replacement of the Endogenous Starch Debranching Enzymes ISA1 and ISA2 of Arabidopsis with the Rice Orthologs Reveals a Degree of Functional Conservation during Starch Synthesis. PLoS ONE, 9(3), e92174.

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Cloning of dTAG-tagged KRAS and LACZ

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Cloning of pLEX_305-N-dTAG-KRAS^G12V was previously described.^{26 (link)} pLEX_305-C-dTAG-LACZ was generated by cloning LACZ (Addgene, #25893) into pLEX_305-C-dTAG using gateway recombination cloning technology (Invitrogen) as previously described.^{26 (link)} In addition to an N- or C-terminal FKBP12^F36V tag, respectively, these plasmids contain tandem HA tags for monitoring of protein expression and a puromycin selectable marker.

Ferguson F.M., Nabet B., Raghavan S., Liu Y., Leggett A.L., Kuljanin M., Kalekar R.L., Yang A., He S., Wang J., Ng R.W., Sulahian R., Li L., Poulin E.J., Huang L., Koren J., Dieguez-Martinez N., Espinosa S., Zeng Z., Corona C.R., Vasta J.D., Ohi R., Sim T., Kim N.D., Harshbarger W., Lizcano J.M., Robers M.B., Muthaswamy S., Lin C.Y., Look A.T., Haigis K.M., Mancias J.D., Wolpin B.M., Aguirre A.J., Hahn W.C., Westover K.D, & Gray N.S. (2020). Discovery of a selective inhibitor of Doublecortin Like Kinase 1. Nature chemical biology, 16(6), 635-643.

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Cloning of dTAG-tagged KRAS and LACZ

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Heterologous Expression of Lipid Biosynthetic Genes

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All the genes, including: CpFATB2, AtrΔ11,OnuΔ11, YroFAR, HarFAR, OnuFAR_E/Z and EaDAcT were amplified from cDNA with primers spanning from the start codon to the stop codon of the ORF, except AveΔ11, CroΔ11 and CpaE11, which were synthesized and codon optimized for the N. benthamiana codon bias (Invitrogen, Life Technologies). Truncation, gene fusion, ER targeting signal and retention signal attachments were achieved by PCR and fusion PCR34 (link), as presented in the Methods.
All genes and their modified versions were cloned into plant expression vector pXZP393 by Gateway recombination cloning technology (Invitrogen). After confirming the integrity of the constructs by sequencing, the expression clones were introduced into Agrobacterium tumefaciens strain GV3101 (MP90RK) by electroporation (1500 V mm⁻¹, 5 ms, Eppendorf 2510).
In order to inhibit the host cells’ transgene-silencing apparatus and extend transgene expression over a longer period of time with a higher degree of expression, the viral silencing suppressor protein P19 was co-introduced into the Agrobacterium mixtures21 .

Ding B.J., Hofvander P., Wang H.L., Durrett T.P., Stymne S, & Löfstedt C. (2014). A plant factory for moth pheromone production. Nature Communications, 5, 3353.

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Amplification of M. tuberculosis membrane proteins

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All of the coding sequences for H37Rv were obtained from NCBI (NC_000962.3)^{29 (link)}. Gateway Recombination Cloning Technology (Invitrogen) was used to amplify the membrane proteins according to the manufacturer’s instructions. Briefly, the target genes were amplified via attB PCR and constructed into the entry vector (pDONR221) and expression clone (pDEST17) using Gateway® BP and LR enzyme cloning.

Li H., Liu L., Zhang W.J., Zhang X., Zheng J., Li L., Zhu X., Yang Q., Zhang M., Liu H., Chen X, & Jin Q. (2019). Analysis of the Antigenic Properties of Membrane Proteins of Mycobacterium tuberculosis. Scientific Reports, 9, 3042.

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Generation of TRIM8 dTAG Cell Lines

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To generate the TRIM8 dTAG cell lines, TC32 and TC71 cells were co-infected with FKBP12F36V-2XHA tagged TRIM8 and sgRNAs targeting TRIM8 to replace endogenous TRIM8 with FKBP12F36V-2XHA tagged TRIM8. Cells were then selected with both puromycin (FKBP12F36V-2XHA tagged TRIM8) and blasticidin (sgRNA). PAM sequences in the FKBP12F36V-2XHA tagged TRIM8 construct were mutated by Gibson assembly (New England Biolabs) using the manufacturer’s instructions to prevent cutting from infected sgRNAs. pLEX_305-TRIM8-dTAG were generated by cloning TRIM8 into pLEX_305-N-dTAG and pLEX_305-C-dTAG using gateway recombination cloning technology (Invitrogen) as previously described (Nabet et al., 2020 (link)). dTAG^V-1 was synthesized as previously described (Nabet et al., 2020 (link)).

Seong B.K., Dharia N.V., Lin S., Donovan K.A., Chong S., Robichaud A., Conway A., Hamze A., Ross L., Alexe G., Adane B., Nabet B., Ferguson F.M., Stolte B., Wang E.J., Sun J., Darzacq X., Piccioni F., Gray N.S., Fischer E.S, & Stegmaier K. (2021). TRIM8 modulates the EWS/FLI oncoprotein to promote survival in Ewing sarcoma. Cancer Cell, 39(9), 1262-1278.e7.

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