GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules

Synthetic Biology requires efficient and versatile DNA assembly systems to facilitate the building of new genetic modules/pathways from basic DNA parts in a standardized way. Here we present GoldenBraid (GB), a standardized assembly system based on type IIS restriction enzymes that allows the indefinite growth of reusable gene modules made of standardized DNA pieces. The GB system consists of a set of four destination plasmids (pDGBs) designed to incorporate multipartite assemblies made of standard DNA parts and to combine them binarily to build increasingly complex multigene constructs. The relative position of type IIS restriction sites inside pDGB vectors introduces a double loop (“braid”) topology in the cloning strategy that allows the indefinite growth of composite parts through the succession of iterative assembling steps, while the overall simplicity of the system is maintained. We propose the use of GoldenBraid as an assembly standard for Plant Synthetic Biology. For this purpose we have GB-adapted a set of binary plasmids for A. tumefaciens-mediated plant transformation. Fast GB-engineering of several multigene T-DNAs, including two alternative modules made of five reusable devices each, and comprising a total of 19 basic parts are also described

GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules

Synthetic Biology requires efficient and versatile DNA assembly systems to facilitate the building of new genetic modules/pathways from basic DNA parts in a standardized way. Here we present GoldenBraid (GB), a standardized assembly system based on type IIS restriction enzymes that allows the indefinite growth of reusable gene modules made of standardized DNA pieces. The GB system consists of a set of four destination plasmids (pDGBs) designed to incorporate multipartite assemblies made of standard DNA parts and to combine them binarily to build increasingly complex multigene constructs. The relative position of type IIS restriction sites inside pDGB vectors introduces a double loop (“braid”) topology in the cloning strategy that allows the indefinite growth of composite parts through the succession of iterative assembling steps, while the overall simplicity of the system is maintained. We propose the use of GoldenBraid as an assembly standard for Plant Synthetic Biology. For this purpose we have GB-adapted a set of binary plasmids for A. tumefaciens-mediated plant transformation. Fast GB-engineering of several multigene T-DNAs, including two alternative modules made of five reusable devices each, and comprising a total of 19 basic parts are also described

Regulation of the Fruit-Specific PEP Carboxylase SlPPC2 Promoter at Early Stages of Tomato Fruit Development

The SlPPC2 phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) gene from tomato (Solanum lycopersicum) is differentially and specifically expressed in expanding tissues of developing tomato fruit. We recently showed that a 1966 bp DNA fragment located upstream of the ATG codon of the SlPPC2 gene (GenBank AJ313434) confers appropriate fruit-specificity in transgenic tomato. In this study, we further investigated the regulation of the SlPPC2 promoter gene by analysing the SlPPC2 cis-regulating region fused to either the firefly luciferase (LUC) or the β-glucuronidase (GUS) reporter gene, using stable genetic transformation and biolistic transient expression assays in the fruit. Biolistic analyses of 5′ SlPPC2 promoter deletions fused to LUC in fruits at the 8th day after anthesis revealed that positive regulatory regions are mostly located in the distal region of the promoter. In addition, a 5′ UTR leader intron present in the 1966 bp fragment contributes to the proper temporal regulation of LUC activity during fruit development. Interestingly, the SlPPC2 promoter responds to hormones (ethylene) and metabolites (sugars) regulating fruit growth and metabolism. When tested by transient expression assays, the chimeric promoter:LUC fusion constructs allowed gene expression in both fruit and leaf, suggesting that integration into the chromatin is required for fruit-specificity. These results clearly demonstrate that SlPPC2 gene is under tight transcriptional regulation in the developing fruit and that its promoter can be employed to drive transgene expression specifically during the cell expansion stage of tomato fruit. Taken together, the SlPPC2 promoter offers great potential as a candidate for driving transgene expression specifically in developing tomato fruit from various tomato cultivars

Elucidating mechanisms underlying organ abscission.

Abscission consists in the detachment of entire vegetative and reproductive organs due to cell separation processes occurring at the abscission zones (AZs) at specific positions of the plant body. From an evolutionary point of view, abscission is a highly advantageous process resulting into fruit and seed dispersal as well as the shedding of no longer useful organs. In an agricultural context, however, abscission may become a major limiting factor for crop productivity. Domestication of major crops included the selection of plants that did not naturally shed ripe fruits or seeds. The understanding of abscission is of great importance to control seed and fruit production and to improve breeding and harvesting practices. Thus, advances made on model plants and crops are of major importance since they may provide potential candidate genes for further biotechnological applications. Here, we review the current knowledge of the physiological, genetic and genomic aspects related to abscission including the most recently disclosed putative regulators that appear to be implicated in the development and/or activation of the AZs

Exploiting multisite gateway and pENFRUIT plasmid collection for fruit genetic engineering

El pdf es la versión post-print.MultiSite Gateway cloning techniques based on homologous recombination facilitate the combinatorial assembly of basic genetic pieces (i.e., promoters, CDS, and terminators) into gene expression or gene silencing cassettes. pENFRUIT is a collection of MultiSite Triple Gateway Entry vectors dedicated to genetic engineering in fruits. It comprises a number of fruit-operating promoters as well as C-terminal tags adapted to the Gateway standard. In this way, flanking regulatory/labeling sequences can be easily Gateway-assembled with a given gene of interest for its ectopic expression or silencing in fruits. The resulting gene constructs can be analyzed in stable transgenic plants or in transient expression assays, the latter allowing fast testing of the increasing number of combinations arising from MultiSite methodology. A detailed description of the use of MultiSite cloning methodology for the assembly of pENFRUIT elements is presented.This work was supported by the Spanish Ministry of Science and Education (Ramón y Cajal programme BIO2005-01015 and BIO2008-03434 projects), the European Commission (programme I3P and EU-SOL project) and Fundación Genoma España (ESP-SOL project).Peer reviewe

VIGS: A Tool to Study Fruit Development in Solanum lycopersicum

[EN] A visually traceable system for fast analysis of gene functions based on Fruit-VIGS methodology is described. In our system, the anthocyanin accumulation from purple transgenic tomato lines provides the appropriate background for fruit-specific gene silencing. The tomato Del/Ros1 background ectopically express Delila (Del) and Rosea1 (Ros1) transgenes under the control of fruit ripening E8 promoter, activating specifically anthocyanin biosynthesis during tomato fruit ripening. The Virus-Induced Gene Silencing (VIGS) of Delila and Rosea1 produces a color change in the silenced area easily identifiable. Del/Ros1 VIGS is achieved by agroinjection of an infective clone of Tobacco Rattle Virus (pTRV1 and pTRV2 binary plasmids) directly into the tomato fruit. The infective clone contains a small fragment of Del and Ros1 coding regions (named DR module). The co-silencing of reporter Del/Ros1 genes and a gene of interest (GOI) in the same region enables us to identify the precise region where silencing is occurring. The function of the GOI is established by comparing silenced sectors of fruits where both GOI and reporter DR genes have been silenced with fruits in which only the reporter DR genes have been silenced. The Gateway vector pTRV2_DR_GW was developed to facilitate the cloning of different GOIs together with DR genes. Our tool is particularly useful to study genes involved in metabolic processes during fruit ripening, which by themselves would not produce a visual phenotype.We are grateful to Prof. Dinesh Kumar who kindly provided us with TRV-based silencing vectors pTRV1 and pTRV2. We appreciate Prof. Cathie Martin for providing Del/Ros1 transgenic tomato lines. The work described here was supported by BIO2008-034034 grant from the Spanish Ministry of Science and Technology, FPU fellowship from Spanish MICINN and EUSOL project from EU.Fernández Moreno, JP.; Orzáez Calatayud, DV.; Granell Richart, A. (2013). VIGS a tool to study fruit development in Solanum lycopersicum. Virus-Induced Gene Silencing: Methods and Protocols. 975:183-196. https://doi.org/10.1007/978-1-62703-278-0_14S183196975Ratcliff F, Martin-Hernandez AM, Baulcombe DC (2001) Technical Advance. Tobacco rattle virus as a vector for analysis of gene function by silencing. Plant J 25:237–245Lu R, Martin-Hernandez AM, Peart JR et al (2003) Virus-induced gene silencing in plants. Methods (San Diego, Calif) 30:296–303Fu DQ, Zhu BZ, Zhu HL et al (2005) Virus-induced gene silencing in tomato fruit. Plant J 43:299–308Shao Y, Zhu HL, Tian HQ, Wang XG et al (2008) Virus-Induce Gene Silencing in Plant Species. Rus J Plant Physiol 55:168–174Burch-Smith TM, Anderson JC, Martin GB et al (2004) Applications and advantages of virus-induced gene silencing for gene function studies in plants. Plant J 39:734–746Orzaez D, Medina A, Torre S et al (2009) A visual reporter system for virus-induced gene silencing in tomato fruit based on anthocyanin accumulation. Plant Physiol 150:1122–1134Unver T, Budak H (2009) Virus-induced gene silencing, a post transcriptional gene silencing method. Int J Plant Genomic 2009:198–680Orzaez D, Mirabel S, Wieland WH et al (2006) Agroinjection of tomato fruits. A tool for rapid functional analysis of transgenes directly in fruit. Plant physiol 140:3–11Orzaez D, Granell A (2009) Reverse genetics and transient gene expression in fleshy fruits: overcoming plant stable transformation. Plant Signal Behav 4:864–867Ballester AR, Molthoff J, de Vos R et al (2010) Biochemical and molecular analysis of pink tomatoes: deregulated expression of the gene encoding transcription factor SlMYB12 leads to pink tomato fruit color. Plant Physiol 152:71–84Liu Y, Schiff M, Dinesh-Kumar SP (2002) Virus-induced gene silencing in tomato. Plant J 31:777–786Butelli E, Titta L, Giorgio M et al (2008) Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nat Biotechnol 26:1301–1308Davies KM, Marshall GB, Marie Bradley J, Schwinn KE et al (2006) Characterisation of aurone biosynthesis in Antirrhinum majus. Physiol Plant 128:593–603Marti C, Orzaez D, Ellul P et al (2007) Silencing of DELLA induces facultative parthenocarpy in tomato fruits. Plant J 52:865–876Bugos RC, Chiang VL, Zhang XH, Campbell ER et al (1995) RNA isolation from plant tissues recalcitrant to extraction in guanidine. Biotechniques 19:734–744Estornell LH, Orzaez D, Lopez-Pena L et al (2009) A multisite gateway-based toolkit for targeted gene expression and hairpin RNA silencing in tomato fruits. Plant Biotechnol J 7:298–30