Design and Construction of Multigenic Constructs for Plant Biotechnology Using the GoldenBraid Cloning Strategy

GoldenBraid (GB) is an iterative and standardized DNA assembling system specially designed for Multigene Engineering in Plant Synthetic Biology. GB is based on restriction–ligation reactions using type IIS restriction enzymes. GB comprises a collection of standard DNA pieces named “GB parts” and a set of destination plasmids (pDGBs) that incorporate the multipartite assembly of standardized DNA parts. GB reactions are extremely efficient: two transcriptional units (TUs) can be assembled from several basic GBparts in one T-DNA less than 24 h. Moreover, larger assemblies comprising 4–5 TUs are routinely built in less than 2 working weeks. Here we provide a detailed view of the GB methodology. As a practical example, a Bimolecular Fluorescence Complementation construct comprising four TUs in a 12 kb DNA fragment is presented.Sarrion-Perdigones, A.; Palací, J.; Granell Richart, A.; Orzáez Calatayud, DV. (2014). Design and Construction of Multigenic Constructs for Plant Biotechnology Using the GoldenBraid Cloning Strategy. Methods in Molecular Biology. 1116:133-151. doi:10.1007/978-1-62703-764-8_10S1331511116Haseloff J, Ajioka J (2009) Synthetic biology, history, challenges and prospects. J R Soc Interface 6(Suppl 4):S389–S391Check E (2005) Synthetic biology, designs on life. Nature 438:417–418Kosuri S, Eroshenko N, LeProust EM et al (2010) Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips. Nat Biotechnol 28:1295–1299Ellis T, Adie T, Baldwin GS (2011) DNA assembly for synthetic biology, from parts to pathways and beyond. Integr Biol 3:109–118Gibson DG, Young L, Chuang R-Y et al (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6: 343–345Gibson DG, Glass JI, Lartigue C et al (2010) Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329:52–56Sarrion-Perdigones A, Falconi EE, Zandalinas SI et al (2011) GoldenBraid, an iterative cloning system for standardized assembly of reusable genetic modules. PLoS One 6:e21622Sarrion-Perdigones A, Vilar-Vazquez M et al (2013) GoldenBraid2.0, A comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol 162:1618–1631Engler C, Gruetzner R, Kandzia R (2009) Golden gate shuffling, a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 4:e5553Engler C, Kandzia R, Marillonnet S (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS One 3:e3647Bracha-Drori K, Shichrur K, Katz A et al (2004) Detection of protein-protein interactions in plants using bimolecular fluorescence complementation. Plant J 40:419–427Smaczniak C, Immink RG, Muino JM et al (2012) Characterization of MADS-domain transcription factor complexes in Arabidopsis flower development. Proc Natl Acad Sci U S A 109:1560–1565de Folter S, Immink RG, Kieffer M et al (2005) Comprehensive interaction map of the Arabidopsis MADS Box transcription factors. Plant Cell 17:1424–1433Lorenz WW, McCann RO, Longiaru M et al (1991) Isolation and expression of a cDNA encoding Renilla reniformis luciferase. Proc Natl Acad Sci U S A 88:4438–4442Voinnet O, Pinto YM, Baulcombe DC (1999) Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants. Proc Natl Acad Sci U S A 96: 14147–14152Hellens RP, Edwards EA, Leyland NR et al (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42:819–832Butelli E, Titta L, Giorgio M et al (2008) Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nat Biotechnol 26: 1301–1308Kapila J, DeRycke R, VanMontagu M et al (1997) An Agrobacterium-mediated transient gene expression system for intact leaves. Plant Sci 122:101–10

PaperClip:rapid multi-part DNA assembly from existing libraries

Assembly of DNA ‘parts ’ to create larger constructs is an essential enabling technique for bioengineer-ing and synthetic biology. Here we describe a sim-ple method, PaperClip, which allows flexible assem-bly of multiple DNA parts from currently existing libraries cloned in any vector. No restriction en-zymes, mutagenesis of internal restriction sites, or reamplification to add end homology are required. Order of assembly is directed by double stranded oligonucleotides––‘Clips’. Clips are formed by lig-ation of pairs of oligonucleotides corresponding to the ends of each part. PaperClip assembly can be performed by polymerase chain reaction or by cell extract-mediated recombination. Once multi-use Clips have been prepared, assembly of at least six DNA parts in any order can be accomplished with high efficiency within several hours

Multigene Engineering by GoldenBraid Cloning: From Plants to Filamentous Fungi and Beyond

This is the peer reviewed version of the following article: Vazquez-Vilar, M., Gandía, M., García-Carpintero, V., Marqués, E., Sarrion-Perdigones, A., Yenush, L., Polaina, J., Manzanares, P., Marcos, J. F., & Orzaez, D. (2020). Multigene engineering by goldenbraid cloning: from plants to filamentous fungi and beyond. Current Protocols in Molecular Biology, 130, e116, doi: 10.1002/cpmb.116, which has been published in final form at https://doi.org/10.1002/cpmb.116. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.[EN] Many synthetic biologists have adopted methods based on Type IIS restriction enzymes and Golden Gate technology in their cloning procedures, as these enable the combinatorial assembly of modular elements in a very efficient way following standard rules. GoldenBraid (GB) is a Golden Gate¿based modular cloning system that, in addition, facilitates the engineering of large multigene constructs and the exchange of DNA parts as result of its iterative cloning scheme. GB was initially developed specifically for plant synthetic biology, and it has been subsequently extended and adapted to other organisms such as Saccharomyces cerevisiae, filamentous fungi, and human cells by incorporating a number of host¿specific features into its basic scheme. Here we describe the general GB cloning procedure and provide detailed protocols for its adaptation to filamentous fungi¿a GB variant known as FungalBraid. The assembly of a cassette for gene disruption by homologous recombination, a fungal¿specific extension of the GB utility, is also shown. Development of FungalBraid was relatively straightforward, as both plants and fungi can be engineered using the same binary plasmids via Agrobacterium¿mediated transformation. We also describe the use of a set of web¿based tools available at the GB website that assist users in all cloning procedures. The availability of plant and fungal versions of GB will facilitate genetic engineering in these industrially relevant organisms.This article is dedicated to the memory of our friend and colleague Dr. Alejandro Sarrion-Perdigones, an early developer of GoldenBraid. We acknowledge the excellent technical assistance provided by Marisol Gascón (IBMCP, Valencia, Spain) with the fluorescent images. This work was funded by Grant BIO2013- 42193 and Grant BIO2016-78601-R, Plan Nacional I+D, Spanish Ministry of Economy and Competitiveness, RTI2018-101115-B-C21 from the Ministerio de Ciencia, Innovación y Universidades (Spain) (MICINN/FEDER Funds), and PROMETEO/ 2018/066 from Conselleria d'Educació (Generalitat Valenciana, Comunitat Valenciana, Spain) and SUSPHIRE PCI2018- 092893-ERA CoBioTech (109) (MCIU/FEDER).Vázquez-Vilar, M.; Gandía, M.; García-Carpintero, V.; Marqués, E.; Sarrion-Perdigones, A.; Yenush, L.; Polaina, J.... (2020). Multigene Engineering by GoldenBraid Cloning: From Plants to Filamentous Fungi and Beyond. Current Protocols in Molecular Biology. 130(1):1-31. https://doi.org/10.1002/cpmb.116S1311301Bernabé‐Orts, J. M., Casas‐Rodrigo, I., Minguet, E. G., Landolfi, V., Garcia‐Carpintero, V., Gianoglio, S., … Orzaez, D. (2019). Assessment of Cas12a‐mediated gene editing efficiency in plants. Plant Biotechnology Journal, 17(10), 1971-1984. doi:10.1111/pbi.13113Ballester, A.-R., Marcet-Houben, M., Levin, E., Sela, N., Selma-Lázaro, C., Carmona, L., … Gabaldón, T. (2015). Genome, Transcriptome, and Functional Analyses of Penicillium expansum Provide New Insights Into Secondary Metabolism and Pathogenicity. Molecular Plant-Microbe Interactions®, 28(3), 232-248. doi:10.1094/mpmi-09-14-0261-fiKhang, C. H., Park, S.-Y., Lee, Y.-H., & Kang, S. (2005). A dual selection based, targeted gene replacement tool for Magnaporthe grisea and Fusarium oxysporum. Fungal Genetics and Biology, 42(6), 483-492. doi:10.1016/j.fgb.2005.03.004Chen, C., Liu, J., Duan, C., Pan, Y., & Liu, G. (2020). Improvement of the CRISPR-Cas9 mediated gene disruption and large DNA fragment deletion based on a chimeric promoter in Acremonium chrysogenum. Fungal Genetics and Biology, 134, 103279. doi:10.1016/j.fgb.2019.103279Bai Flagfeldt, D., Siewers, V., Huang, L., & Nielsen, J. (2009). Characterization of chromosomal integration sites for heterologous gene expression inSaccharomyces cerevisiae. Yeast, 26(10), 545-551. doi:10.1002/yea.1705Fräbel, S., Wagner, B., Krischke, M., Schmidts, V., Thiele, C. M., Staniek, A., & Warzecha, H. (2018). Engineering of new-to-nature halogenated indigo precursors in plants. Metabolic Engineering, 46, 20-27. doi:10.1016/j.ymben.2018.02.003Fresquet-Corrales, S., Roque, E., Sarrión-Perdigones, A., Rochina, M., López-Gresa, M. P., Díaz-Mula, H. M., … Cañas, L. A. (2017). Metabolic engineering to simultaneously activate anthocyanin and proanthocyanidin biosynthetic pathways in Nicotiana spp. PLOS ONE, 12(9), e0184839. doi:10.1371/journal.pone.0184839Garrigues, S., Gandía, M., & Marcos, J. F. (2015). Occurrence and function of fungal antifungal proteins: a case study of the citrus postharvest pathogen Penicillium digitatum. Applied Microbiology and Biotechnology, 100(5), 2243-2256. doi:10.1007/s00253-015-7110-3Gibson, D. G., Young, L., Chuang, R.-Y., Venter, J. C., Hutchison, C. A., & Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5), 343-345. doi:10.1038/nmeth.1318Gurgel, I. L. da S., Jorge, K. T. de O. S., Malacco, N. L. S. de O., Souza, J. A. M., Rocha, M. C., Fernandes, M. F., … Soriani, F. M. (2019). The Aspergillus fumigatus Mucin MsbA Regulates the Cell Wall Integrity Pathway and Controls Recognition of the Fungus by the Immune System. mSphere, 4(3). doi:10.1128/msphere.00350-19Hernanz-Koers, M., Gandía, M., Garrigues, S., Manzanares, P., Yenush, L., Orzaez, D., & Marcos, J. F. (2018). FungalBraid: A GoldenBraid-based modular cloning platform for the assembly and exchange of DNA elements tailored to fungal synthetic biology. Fungal Genetics and Biology, 116, 51-61. doi:10.1016/j.fgb.2018.04.010Juarez, P., Huet-Trujillo, E., Sarrion-Perdigones, A., Falconi, E., Granell, A., & Orzaez, D. (2013). Combinatorial Analysis of Secretory Immunoglobulin A (sIgA) Expression in Plants. International Journal of Molecular Sciences, 14(3), 6205-6222. doi:10.3390/ijms14036205Kramer, M. F., & Coen, D. M. (2001). Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization. Current Protocols in Molecular Biology. doi:10.1002/0471142727.mb1501s56Marcet-Houben, M., Ballester, A.-R., de la Fuente, B., Harries, E., Marcos, J. F., González-Candelas, L., & Gabaldón, T. (2012). Genome sequence of the necrotrophic fungus Penicillium digitatum, the main postharvest pathogen of citrus. BMC Genomics, 13(1). doi:10.1186/1471-2164-13-646Michielse, C. B., J Hooykaas, P. J., J J van den Hondel, C. A. M., & J Ram, A. F. (2008). Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori. Nature Protocols, 3(10), 1671-1678. doi:10.1038/nprot.2008.154Müller, K. M., & Arndt, K. M. (2011). Standardization in Synthetic Biology. Synthetic Gene Networks, 23-43. doi:10.1007/978-1-61779-412-4_2Patron, N. J., Orzaez, D., Marillonnet, S., Warzecha, H., Matthewman, C., Youles, M., … Rogers, C. (2015). Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist, 208(1), 13-19. doi:10.1111/nph.13532Pérez-González, A., Kniewel, R., Veldhuizen, M., Verma, H. K., Navarro-Rodríguez, M., Rubio, L. M., & Caro, E. (2017). Adaptation of the GoldenBraid modular cloning system and creation of a toolkit for the expression of heterologous proteins in yeast mitochondria. BMC Biotechnology, 17(1). doi:10.1186/s12896-017-0393-yPérez-Nadales, E., & Di Pietro, A. (2011). The Membrane Mucin Msb2 Regulates Invasive Growth and Plant Infection in Fusarium oxysporum  . The Plant Cell, 23(3), 1171-1185. doi:10.1105/tpc.110.075093Salazar-Cerezo, S., Kun, R. S., de Vries, R. P., & Garrigues, S. (2020). CRISPR/Cas9 technology enables the development of the filamentous ascomycete fungus Penicillium subrubescens as a new industrial enzyme producer. Enzyme and Microbial Technology, 133, 109463. doi:10.1016/j.enzmictec.2019.109463Sarrion-Perdigones, A., Chang, L., Gonzalez, Y., Gallego-Flores, T., Young, D. W., & Venken, K. J. T. (2019). Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying. Nature Communications, 10(1). doi:10.1038/s41467-019-13651-ySarrion-Perdigones, A., Falconi, E. E., Zandalinas, S. I., Juárez, P., Fernández-del-Carmen, A., Granell, A., & Orzaez, D. (2011). GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules. PLoS ONE, 6(7), e21622. doi:10.1371/journal.pone.0021622Sarrion-Perdigones, A., Vazquez-Vilar, M., Palaci, J., Castelijns, B., Forment, J., Ziarsolo, P., … Orzaez, D. (2013). GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology. PLANT PHYSIOLOGY, 162(3), 1618-1631. doi:10.1104/pp.113.217661Selma, S., Bernabé‐Orts, J. M., Vazquez‐Vilar, M., Diego‐Martin, B., Ajenjo, M., Garcia‐Carpintero, V., … Orzaez, D. (2019). Strong gene activation in plants with genome‐wide specificity using a new orthogonal CRISPR /Cas9‐based programmable transcriptional activator. Plant Biotechnology Journal, 17(9), 1703-1705. doi:10.1111/pbi.13138Shendure, J. A., Porreca, G. J., Church, G. M., Gardner, A. F., Hendrickson, C. L., Kieleczawa, J., & Slatko, B. E. (2011). Overview of DNA Sequencing Strategies. Current Protocols in Molecular Biology, 96(1). doi:10.1002/0471142727.mb0701s96Szewczyk, E., Nayak, T., Oakley, C. E., Edgerton, H., Xiong, Y., Taheri-Talesh, N., … Oakley, B. R. (2006). Fusion PCR and gene targeting in Aspergillus nidulans. Nature Protocols, 1(6), 3111-3120. doi:10.1038/nprot.2006.405Vafaee, Y., Staniek, A., Mancheno-Solano, M., & Warzecha, H. (2014). A Modular Cloning Toolbox for the Generation of Chloroplast Transformation Vectors. PLoS ONE, 9(10), e110222. doi:10.1371/journal.pone.0110222Vazquez-Vilar, M., Bernabé-Orts, J. M., Fernandez-del-Carmen, A., Ziarsolo, P., Blanca, J., Granell, A., & Orzaez, D. (2016). A modular toolbox for gRNA–Cas9 genome engineering in plants based on the GoldenBraid standard. Plant Methods, 12(1). doi:10.1186/s13007-016-0101-2Villiers, B. R. M., Stein, V., & Hollfelder, F. (2009). USER friendly DNA recombination (USERec): a simple and flexible near homology-independent method for gene library construction. Protein Engineering, Design and Selection, 23(1), 1-8. doi:10.1093/protein/gzp063Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE, 6(2), e16765. doi:10.1371/journal.pone.0016765Sarrion‐Perdigones et al. (2013). See above.Hernanz‐Koersetal. (2018). See above.https://gbcloning.upv.es/https://benchling.co

A synthetic biology approach for consistent production of plant-made recombinant polyclonal antibodies against snake venom toxins

Antivenoms developed from the plasma of hyperimmunized animals are the only effective treatment available against snakebite envenomation but shortage of supply contributes to the high morbidity and mortality toll of this tropical disease. We describe a synthetic biology approach to affordable and cost-effective antivenom production based on plant-made recombinant polyclonal antibodies (termed pluribodies). The strategy takes advantage of virus superinfection exclusion to induce the formation of somatic expression mosaics in agroinfiltrated plants, which enables the expression of complex antibody repertoires in a highly reproducible manner. Pluribodies developed using toxin-binding genetic information captured from peripheral blood lymphocytes of hyperimmunized camels recapitulated the overall binding activity of the immune response. Furthermore, an improved plant-made antivenom (plantivenom) was formulated using an in vitro selected pluribody against Bothrops asper snake venom toxins and has been shown to neutralize a wide range of toxin activities and provide protection against lethal venom doses in mice.Fil: Julve Parreño, Jose Manuel. Universidad Politécnica de Valencia; EspañaFil: Huet, Estefanía. Universidad Politécnica de Valencia; EspañaFil: Fernández del Carmen, Asun. Universidad Politécnica de Valencia; EspañaFil: Segura, Alvaro. Universidad de Costa Rica; Costa RicaFil: Venturi, Micol. Universidad Politécnica de Valencia; EspañaFil: Gandía, Antoni. Universidad Politécnica de Valencia; EspañaFil: Pan, Wei-Song. Universidad Politécnica de Valencia; EspañaFil: Albaladejo, Irene. Universidad Politécnica de Valencia; EspañaFil: Forment, Javier. Universidad Politécnica de Valencia; EspañaFil: Pla, Davinia. Instituto de Biomedicina de Valencia; EspañaFil: Wigdorovitz, Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; ArgentinaFil: Calvete, Juan J.. Instituto de Biomedicina de Valencia; EspañaFil: Gutiérrez, Carlos. Universidad de Las Palmas de Gran Canaria; EspañaFil: Gutiérrez, José María. Universidad de Costa Rica; Costa RicaFil: Granell, Antonio. Universidad Politécnica de Valencia; EspañaFil: Orzáez, Diego. Universidad Politécnica de Valencia; Españ

GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology

[EN] Plant synthetic biology aims to apply engineering principles to plant genetic design. One strategic requirement of plant synthetic biology is the adoption of common standardized technologies that facilitate the construction of increasingly complex multigene structures at the DNA level while enabling the exchange of genetic building blocks among plant bioengineers. Here, we describe GoldenBraid 2.0 (GB2.0), a comprehensive technological framework that aims to foster the exchange of standard DNA parts for plant synthetic biology. GB2.0 relies on the use of type IIS restriction enzymes for DNA assembly and proposes a modular cloning schema with positional notation that resembles the grammar of natural languages. Apart from providing an optimized cloning strategy that generates fully exchangeable genetic elements for multigene engineering, the GB2.0 toolkit offers an ever-growing open collection of DNA parts, including a group of functionally tested, premade genetic modules to build frequently used modules like constitutive and inducible expression cassettes, endogenous gene silencing and protein-protein interaction tools, etc. Use of the GB2.0 framework is facilitated by a number of Web resources that include a publicly available database, tutorials, and a software package that provides in silico simulations and laboratory protocols for GB2.0 part domestication and multigene engineering. In short, GB2.0 provides a framework to exchange both information and physical DNA elements among bioengineers to help implement plant synthetic biology projects.This work was supported by the Spanish Ministry of Economy and Competitiveness (grant no. BIO2010-15384), by a Research Personnel in Training fellowship to A.S.-P., and by a Junta de Ampliacion de Estudios fellowship to M.V.-V.Sarrion-Perdigones, A.; Vázquez Vilar, M.; Palací Bataller, J.; Castelijns, B.; Forment Millet, JJ.; Ziarsolo Areitioaurtena, P.; Blanca Postigo, JM.... (2013). GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiology. 162(3):1618-1631. https://doi.org/10.1104/pp.113.217661S16181631162

Combinatorial Analysis of Secretory Immunoglobulin A (sIgA) Expression in Plants

Delivery of secretory immunoglobulin A (sIgA) to mucosal surfaces as a passive immunotherapy agent is a promising strategy to prevent infectious diseases. Recombinant sIgA production in plants requires the co-expression of four transcriptional units encoding the light chain (LC), heavy chain (HC), joining chain (JC) and secretory component (SC). As a way to optimize sIgA production in plants, we tested the combinatorial expression of 16 versions of a human sIgA against the VP8* rotavirus antigen in Nicotiana benthamiana, using the recently developed GoldenBraid multigene assembly system. Each sIgA version was obtained by combining one of the two types of HC (alpha 1 and alpha 2) with one of the two LC types (k and lambda) and linking or not a KDEL peptide to the HC and/or SC. From the analysis of the anti-VP8* activity, it was concluded that those sIgA versions carrying HC alpha 1 and LC lambda provided the highest yields. Moreover, ER retention significantly increased antibody production, particularly when the KDEL signal was linked to the SC. Maximum expression levels of 32.5 mu g IgA/g fresh weight (FW) were obtained in the best performing combination, with an estimated 33% of it in the form of a secretory complex.This work has been funded by Grant BIO2010-15384 from Plan Nacional I + D of the Spanish Ministry of Science. Juarez P. is a recipient of a FPU fellowship, and Sarrion-Perdigones A. and Huet-Trujillo E. are recipients of a FPI fellowship. We want to thank Monedero for kindly providing scFv and VP8* clones.Juárez Ortega, P.; Huet Trujillo, E.; Sarrion-Perdigones, A.; Falconi, E.; Granell Richart, A.; Orzáez Calatayud, DV. (2013). Combinatorial Analysis of Secretory Immunoglobulin A (sIgA) Expression in Plants. International Journal of Molecular Sciences. 14(3):6205-6222. https://doi.org/10.3390/ijms14036205S62056222143Reichert, J. M., & Valge-Archer, V. E. (2007). Development trends for monoclonal antibody cancer therapeutics. Nature Reviews Drug Discovery, 6(5), 349-356. doi:10.1038/nrd2241Corthésy, B. (2010). Role of secretory immunoglobulin A and secretory component in the protection of mucosal surfaces. Future Microbiology, 5(5), 817-829. doi:10.2217/fmb.10.39Corthésy, B. (2003). Recombinant Secretory Immunoglobulin A in Passive Immunotherapy: Linking Immunology and Biotechnology. Current Pharmaceutical Biotechnology, 4(1), 51-67. doi:10.2174/1389201033378020Corthësy, B. (2008). Secretory immunoglobulin A: well beyond immune exclusion at mucosal surfaces. Immunopharmacology and Immunotoxicology, 31(2), 174-179. doi:10.1080/08923970802438441Sarrion-Perdigones, A., Juarez, P., Granell, A., & Orzaez, D. (2011). Production of Antibodies in Plants. Cell Engineering, 143-164. doi:10.1007/978-94-007-1257-7_7Paul, M., Dolleweerd, C. van, Drake, P. M. W., Reljic, R., Thangaraj, H., Barbi, T., … Ma, J. K.-C. (2011). Molecular pharming. 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Characterization of a recombinant plant monoclonal secretory antibody and preventive immunotherapy in humans. Nature Medicine, 4(5), 601-606. doi:10.1038/nm0598-601Weintraub, J. A., Hilton, J. F., White, J. M., Hoover, C. I., Wycoff, K. L., Yu, L., … Featherstone, J. D. B. (2005). Clinical Trial of a Plant-Derived Antibody on Recolonization of Mutans Streptococci. Caries Research, 39(3), 241-250. doi:10.1159/000084805Wycoff, K. (2005). Secretory IgA Antibodies from Plants. Current Pharmaceutical Design, 11(19), 2429-2437. doi:10.2174/1381612054367508Nicholson, L., Gonzalez-Melendi, P., Van Dolleweerd, C., Tuck, H., Perrin, Y., Ma, J. K.-C., … Stoger, E. (2004). A recombinant multimeric immunoglobulin expressed in rice shows assembly-dependent subcellular localization in endosperm cells. Plant Biotechnology Journal, 3(1), 115-127. doi:10.1111/j.1467-7652.2004.00106.xWieland, W. H., Lammers, A., Schots, A., & Orzáez, D. V. (2006). 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Rapid, modular and reliable construction of complex mammalian gene circuits

We developed a framework for quick and reliable construction of complex gene circuits for genetically engineering mammalian cells. Our hierarchical framework is based on a novel nucleotide addressing system for defining the position of each part in an overall circuit. With this framework, we demonstrate construction of synthetic gene circuits of up to 64 kb in size comprising 11 transcription units and 33 basic parts. We show robust gene expression control of multiple transcription units by small molecule inducers in human cells with transient transfection and stable chromosomal integration of these circuits. This framework enables development of complex gene circuits for engineering mammalian cells with unprecedented speed, reliability and scalability and should have broad applicability in a variety of areas including mammalian cell fermentation, cell fate reprogramming and cell-based assays.Synthetic Biology Engineering Research Center (SA5284-11210)United States. Defense Advanced Research Projects Agency (HR0011-12-C-0067)United States. Defense Advanced Research Projects Agency (DARPA-BAA-11-23)National Science Foundation (U.S.) (CBET-0939511)National Institutes of Health (U.S.). (5-R01-CA155320-02

GB3.0: a platform for plant bio-design that connects functional DNA elements with associated biological data

This is a pre-copyedited, author-produced version of an article accepted for publication in Nucleic Acids Research following peer review. The version of record Vázquez-Vilar, M.; Quijano-Rubio, A.; Fernandez Del Carmen, MA.; Sarrion-Perdigones, A.; Ochoa-Fernández, R.; Ziarsolo Areitioaurtena, P.; Blanca Postigo, JM.... (2017). GB3.0: a platform for plant bio-design that connects functional DNA elements with associated biological data. Nucleic Acids Research. 45(4):2196-2209. doi:10.1093/nar/gkw1326 is available online at: http://doi.org/10.1093/nar/gkw1326.[EN] Modular DNA assembly simplifies multigene engineering in Plant Synthetic Biology. Furthermore, the recent adoption of a common syntax to facilitate the exchange of plant DNA parts (phytobricks) is a promising strategy to speed up genetic engineering. Following this lead, here, we present a platform for plant biodesign that incorporates functional descriptions of phytobricks obtained under pre-defined experimental conditions, and systematically registers the resulting information as metadata for documentation. To facilitate the handling of functional descriptions, we developed a new version (v3.0) of the GoldenBraid (GB) webtool that integrates the experimental data and displays it in the form of datasheets. We report the use of the Luciferase/Renilla (Luc/Ren) transient agroinfiltration assay in Nicotiana benthamiana as a standard to estimate relative transcriptional activities conferred by regulatory phytobricks, and show the consistency and reproducibility of this method in the characterization of a synthetic phytobrick based on the CaMV35S promoter. Furthermore, we illustrate the potential for combinatorial optimization and incremental innovation of the GB3.0 platform in two separate examples, (i) the development of a collection of orthogonal transcriptional regulators based on phiC31 integrase and (ii) the design of a small genetic circuit that connects a glucocorticoid switch to a MYB/bHLH transcriptional activation module.Spanish Ministry of Economy and Competitiveness [BIO2013-42193-R and BIO2016-78601-R projects to A.G. and D.O.]. Funding for open access charge: Spanish Ministry of Economy and Competitiveness [BIO2013-42193-R and BIO2016-78601-R projects to A.G. and D.O.].Vázquez-Vilar, M.; Quijano-Rubio, A.; Fernández Del Carmen, MA.; Sarrion-Perdigones, A.; Ochoa-Fernández, R.; Ziarsolo Areitioaurtena, P.; Blanca Postigo, JM.... (2017). GB3.0: a platform for plant bio-design that connects functional DNA elements with associated biological data. 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A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard

[EN] Background: The efficiency, versatility and multiplexing capacity of RNA-guided genome engineering using the CRISPR/Cas9 technology enables a variety of applications in plants, ranging from gene editing to the construction of transcriptional gene circuits, many of which depend on the technical ability to compose and transfer complex synthetic instructions into the plant cell. The engineering principles of standardization and modularity applied to DNA cloning are impacting plant genetic engineering, by increasing multigene assembly efficiency and by fostering the exchange of well-defined physical DNA parts with precise functional information. Results: Here we describe the adaptation of the RNA-guided Cas9 system to GoldenBraid (GB), a modular DNA con¿ struction framework being increasingly used in Plant Synthetic Biology. In this work, the genetic elements required for CRISPRs-based editing and transcriptional regulation were adapted to GB, and a workflow for gRNAs construction was designed and optimized. New software tools specific for CRISPRs assembly were created and incorporated to the public GB resources site. Conclusions: The functionality and the efficiency of gRNA¿Cas9 GB tools were demonstrated in Nicotiana benthamiana using transient expression assays both for gene targeted mutations and for transcriptional regulation. The availability of gRNA¿Cas9 GB toolbox will facilitate the application of CRISPR/Cas9 technology to plant genome engineeringThis work has been funded by Grant BIO2013-42193-R from Plan Nacional I + D of the Spanish Ministry of Economy and Competitiveness. Vazquez-Vilar M. is a recipient of a Junta de Ampliacion de Estudios fellowship. Bernabe-Orts J.M. is a recipient of a FPI fellowship. We want to thank Nicola J. Patron and Mark Youles for kindly providing humanCas9 and U6-26 clones. We also want to thank Eugenio Gomez for providing Arabidopsis thaliana genomic DNA and Concha Domingo for providing rice genomic DNA. We also want to thank the COST Action FA1006 for the support in the development of the software tools.Vázquez-Vilar, M.; Bernabé-Orts, JM.; Fernández Del Carmen, MA.; Ziarsolo Areitioaurtena, P.; Blanca Postigo, JM.; Granell Richart, A.; Orzáez Calatayud, DV. (2016). A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard. Plant Methods. 12. https://doi.org/10.1186/s13007-016-0101-2S12Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281–308. doi: 10.1038/nprot.2013.143 .Yang X. Applications of CRISPR-Cas9 mediated genome engineering. Mil Med Res. 2015;2:11. doi: 10.1186/s40779-015-0038-1 .Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013;153(4):910–8. doi: 10.1016/j.cell.2013.04.025 .Bortesi L, Fischer R. The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv. 2015;33(1):41–52. doi: 10.1016/j.biotechadv.2014.12.006 .Belhaj K, Chaparro-Garcia A, Kamoun S, Patron NJ, Nekrasov V. Editing plant genomes with CRISPR/Cas9. Curr Opin Biotechnol. 2015;32:76–84. doi: 10.1016/j.copbio.2014.11.007 .Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol. 2013;31(8):686–8. doi: 10.1038/nbt.2650 .Gao J, Wang G, Ma S, Xie X, Wu X, Zhang X, et al. CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum. Plant Mol Biol. 2015;87(1–2):99–110. doi: 10.1007/s11103-014-0263-0 .Fauser F, Schiml S, Puchta H. Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J. 2014;79(2):348–59. doi: 10.1111/tpj.12554 .Schiml S, Fauser F, Puchta H. The CRISPR/Cas system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny. Plant J. 2014;80(6):1139–50. doi: 10.1111/tpj.12704 .Piatek A, Ali Z, Baazim H, Li L, Abulfaraj A, Al-Shareef S, et al. RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors. Plant Biotechnol J. 2015;13(4):578–89. doi: 10.1111/pbi.12284 .Beerli RR, Barbas CF 3rd. Engineering polydactyl zinc-finger transcription factors. Nat Biotechnol. 2002;20(2):135–41. doi: 10.1038/nbt0202-135 .Bogdanove AJ, Voytas DF. TAL effectors: customizable proteins for DNA targeting. Science. 2011;333(6051):1843–6. doi: 10.1126/science.1204094 .Nielsen AA, Voigt CA. Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks. Mol Syst Biol. 2014;10:763. doi: 10.15252/msb.20145735 .Eeckhaut T, Lakshmanan PS, Deryckere D, Van Bockstaele E, Van Huylenbroeck J. Progress in plant protoplast research. Planta. 2013. doi: 10.1007/s00425-013-1936-7 .Mikami M, Toki S, Endo M. Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice. Plant Mol Biol. 2015. doi: 10.1007/s11103-015-0342-x .Patron NJ, Orzaez D, Marillonnet S, Warzecha H, Matthewman C, Youles M, et al. Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytol. 2015. doi: 10.1111/nph.13532 .Liu W, Stewart CN Jr. Plant synthetic biology. Trends Plant Sci. 2015;20(5):309–17. doi: 10.1016/j.tplants.2015.02.004 .Sarrion-Perdigones A, Vazquez-Vilar M, Palaci J, Castelijns B, Forment J, Ziarsolo P, et al. GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol. 2013;162(3):1618–31. doi: 10.1104/pp.113.217661 .Vazquez-Vilar M, Sarrion-Perdigones A, Ziarsolo P, Blanca J, Granell A, Orzaez D. Software-assisted stacking of gene modules using GoldenBraid 2.0 DNA-assembly framework. Methods Mol Biol. 2015;1284:399–420. doi: 10.1007/978-1-4939-2444-8_20 .Duportet X, Wroblewska L, Guye P, Li Y, Eyquem J, Rieders J, et al. A platform for rapid prototyping of synthetic gene networks in mammalian cells. Nucleic Acids Res. 2014;42(21):13440–51. doi: 10.1093/nar/gku1082 .Guo Y, Dong J, Zhou T, Auxillos J, Li T, Zhang W, et al. YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae. Nucleic Acids Res. 2015;43(13):e88. doi: 10.1093/nar/gkv464 .Engler C, Gruetzner R, Kandzia R, Marillonnet S. Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS ONE. 2009;4(5):e5553. doi: 10.1371/journal.pone.0005553 .Sarrion-Perdigones A, Falconi EE, Zandalinas SI, Juarez P, Fernandez-del-Carmen A, Granell A, et al. GoldenBraid: an iterative cloning system for standardized assembly of reusable genetic modules. PLoS ONE. 2011;6(7):e21622. doi: 10.1371/journal.pone.0021622 .Lei Y, Lu L, Liu HY, Li S, Xing F, Chen LL. CRISPR-P: a web tool for synthetic single-guide RNA design of CRISPR-system in plants. Mol Plant. 2014;7(9):1494–6. doi: 10.1093/mp/ssu044 .Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339(6121):823–6. doi: 10.1126/science.1232033 .Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, et al. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol. 2013;31(8):688–91. doi: 10.1038/nbt.2654 .Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 2013;41(15):7429–37. doi: 10.1093/nar/gkt520 .Xie K, Minkenberg B, Yang Y. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci USA. 2015;112(11):3570–5. doi: 10.1073/pnas.1420294112 .Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. A modular cloning system for standardized assembly of multigene constructs. PLoS ONE. 2011;6(2):e16765. doi: 10.1371/journal.pone.0016765 .Sakuma T, Nishikawa A, Kume S, Chayama K, Yamamoto T. Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system. Sci Rep. 2014;4:5400. doi: 10.1038/srep05400 .Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, et al. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. 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Conservation of thermospermine synthase activity in vascular and non-vascular plants

[EN] In plants, the only confirmed function for thermospermine is regulating xylem cells maturation. However, genes putatively encoding thermospermine synthases have been identified in the genomes of both vascular and non-vascular plants. Here, we verify the activity of the thermospermine synthase genes and the presence of thermospermine in vascular and non-vascular land plants as well as in the aquatic plant Chlamydomonas reinhardtii. In addition, we provide information about differential content of thermospermine in diverse organs at different developmental stages in some vascular species that suggest that, although the major role of thermospermine in vascular plants is likely to be xylem development, other potential roles in development and/or responses to stress conditions could be associated to such polyamine. In summary, our results in vascular and non-vascular species indicate that the capacity to synthesize thermospermine is conserved throughout the entire plant kingdom.This work in the laboratories was funded by grants BFU2016-80621-P and BIO2016-79147-R of the Spanish Ministry of Economy, Industry and Competitiveness. AS-G and JH-G are recipients of Fellowships of the Spanish Ministry of Science, Innovation and Universities BES-2017-080387 and of the Spanish Ministry of Education, Culture and Sport FPU15/01756, respectively. JA holds a Ramón y Cajal Contract RYC-2014-15752.Solé-Gil, A.; Hernández-García, J.; López-Gresa, MP.; Blazquez Rodriguez, MA.; Agusti Feliu, J. (2019). Conservation of thermospermine synthase activity in vascular and non-vascular plants. Frontiers in Plant Science. 10:1-10. https://doi.org/10.3389/fpls.2019.00663S11010Ashton, N. W., & Cove, D. J. (1977). 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Arabidopsis thickvein Mutation Affects Vein Thickness and Organ Vascularization, and Resides in a Provascular Cell-Specific Spermine Synthase Involved in Vein Definition and in Polar Auxin Transport. Plant Physiology, 138(2), 767-777. doi:10.1104/pp.104.055756Darriba, D., Taboada, G. L., Doallo, R., & Posada, D. (2011). ProtTest 3: fast selection of best-fit models of protein evolution. Bioinformatics, 27(8), 1164-1165. doi:10.1093/bioinformatics/btr088De Rybel, B., Adibi, M., Breda, A. S., Wendrich, J. R., Smit, M. E., Novák, O., … Weijers, D. (2014). Integration of growth and patterning during vascular tissue formation in Arabidopsis. Science, 345(6197), 1255215. doi:10.1126/science.1255215De Rybel, B., Möller, B., Yoshida, S., Grabowicz, I., Barbier de Reuille, P., Boeren, S., … Weijers, D. (2013). A bHLH Complex Controls Embryonic Vascular Tissue Establishment and Indeterminate Growth in Arabidopsis. Developmental Cell, 24(4), 426-437. doi:10.1016/j.devcel.2012.12.013Gonzalez, M. 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Current Biology, 25(23), 3144-3150. doi:10.1016/j.cub.2015.10.051Knott, J. M., Römer, P., & Sumper, M. (2007). Putative spermine synthases fromThalassiosira pseudonanaandArabidopsis thalianasynthesize thermospermine rather than spermine. FEBS Letters, 581(16), 3081-3086. doi:10.1016/j.febslet.2007.05.074Marina, M., Sirera, F. V., Rambla, J. L., Gonzalez, M. E., Blázquez, M. A., Carbonell, J., … Ruiz, O. A. (2013). Thermospermine catabolism increases Arabidopsis thaliana resistance to Pseudomonas viridiflava. Journal of Experimental Botany, 64(5), 1393-1402. doi:10.1093/jxb/ert012Michael, A. J. (2016). Polyamines in Eukaryotes, Bacteria, and Archaea. Journal of Biological Chemistry, 291(29), 14896-14903. doi:10.1074/jbc.r116.734780Milhinhos, A., Prestele, J., Bollhöner, B., Matos, A., Vera-Sirera, F., Rambla, J. L., … Miguel, C. M. (2013). Thermospermine levels are controlled by an auxin-dependent feedback loop mechanism inPopulusxylem. 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Scots pine aminopropyltransferases shed new light on evolution of the polyamine biosynthesis pathway in seed plants. Annals of Botany, 121(6), 1243-1256. doi:10.1093/aob/mcy012Wu, H., Min, J., Ikeguchi, Y., Zeng, H., Dong, A., Loppnau, P., … Plotnikov, A. N. (2007). Structure and Mechanism of Spermidine Synthases†. Biochemistry, 46(28), 8331-8339. doi:10.1021/bi602498kYamaguchi, K., Takahashi, Y., Berberich, T., Imai, A., Miyazaki, A., Takahashi, T., … Kusano, T. (2006). The polyamine spermine protects against high salt stress inArabidopsis thaliana. FEBS Letters, 580(30), 6783-6788. doi:10.1016/j.febslet.2006.10.078Yamaguchi, K., Takahashi, Y., Berberich, T., Imai, A., Takahashi, T., Michael, A. J., & Kusano, T. (2007). A protective role for the polyamine spermine against drought stress in Arabidopsis. Biochemical and Biophysical Research Communications, 352(2), 486-490. doi:10.1016/j.bbrc.2006.11.041Yoshimoto, K., Takamura, H., Kadota, I., Motose, H., & Takahashi, T. (2016). 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