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dc.contributor.author | Cordero-Cucart, María Teresa | es_ES |
dc.contributor.author | Aragonés, V. | es_ES |
dc.contributor.author | Daros Arnau, Jose Antonio | es_ES |
dc.date.accessioned | 2019-09-05T20:04:31Z | |
dc.date.available | 2019-09-05T20:04:31Z | |
dc.date.issued | 2018 | es_ES |
dc.identifier.uri | http://hdl.handle.net/10251/125104 | |
dc.description.abstract | [EN] With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts of recombinant RNAs are limited. Here, we describe a protocol to produce large amounts of recombinant RNA in Escherichia coli based on co-expression of a chimeric molecule that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. Viroids are relatively small, non-coding, highly base-paired circular RNAs that are infectious to higher plants. The host plant tRNA ligase is an enzyme recruited by viroids that belong to the family Avsunviroidae, such as Eggplant latent viroid (ELVd), to mediate RNA circularization during viroid replication. Although ELVd does not replicate in E. coli, an ELVd precursor is efficiently transcribed by the E. coli RNA polymerase and processed by the embedded hammerhead ribozymes in bacterial cells, and the resulting monomers are circularized by the co-expressed tRNA ligase reaching a remarkable concentration. The insertion of an RNA of interest into the ELVd scaffold enables the production of tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. A main fraction of the RNA product is circular, a feature that facilitates the purification of the recombinant RNA to virtual homogeneity. In this protocol, a complementary DNA (cDNA) corresponding to the RNA of interest is inserted in a particular position of the ELVd cDNA in an expression plasmid that is used, along the plasmid to coexpress eggplant tRNA ligase, to transform E. coli. Co-expression of both molecules under the control of strong constitutive promoters leads to production of large amounts of the recombinant RNA. The recombinant RNA can be extracted from the bacterial cells and separated from the bulk of bacterial RNAs taking advantage of its circularity. | es_ES |
dc.description.sponsorship | This work was supported by grants BIO2017-83184-R and BIO2017-91865-EXP from the Spanish Ministerio de Ciencia, Innovacion y Universidades (co-financed FEDER funds). | es_ES |
dc.language | Inglés | es_ES |
dc.publisher | Journal of Visualized Experiments | es_ES |
dc.relation.ispartof | Journal of Visualized Experiments | es_ES |
dc.rights | Reserva de todos los derechos | es_ES |
dc.subject | Biochemistry | es_ES |
dc.subject | Issue 141 | es_ES |
dc.subject | Recombinant RNA | es_ES |
dc.subject | Circular RNA | es_ES |
dc.subject | Viroid,tRNA ligase | es_ES |
dc.subject | RNA aptamer | es_ES |
dc.subject | Escherichia coli | es_ES |
dc.title | Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli | es_ES |
dc.type | Artículo | es_ES |
dc.identifier.doi | 10.3791/58472 | es_ES |
dc.relation.projectID | info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/BIO2017-83184-R/ES/VIRUS DE PLANTAS: PATOGENOS Y TAMBIEN VECTORES PARA LA PRODUCCION DE PROTEINAS, METABOLITOS, RNAS Y NANOPARTICULAS/ | es_ES |
dc.relation.projectID | info:eu-repo/grantAgreement/AEI//BIO2017-91865-EXP/ES/VIROIDES CONTRA INSECTOS/ | es_ES |
dc.rights.accessRights | Abierto | es_ES |
dc.contributor.affiliation | Universitat Politècnica de València. Instituto Universitario Mixto de Biología Molecular y Celular de Plantas - Institut Universitari Mixt de Biologia Molecular i Cel·lular de Plantes | es_ES |
dc.description.bibliographicCitation | Cordero-Cucart, MT.; Aragonés, V.; Daros Arnau, JA. (2018). Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli. Journal of Visualized Experiments. (141). https://doi.org/10.3791/58472 | es_ES |
dc.description.accrualMethod | S | es_ES |
dc.relation.publisherversion | http://doi.org/10.3791/58472 | es_ES |
dc.type.version | info:eu-repo/semantics/publishedVersion | es_ES |
dc.description.issue | 141 | es_ES |
dc.identifier.eissn | 1940-087X | es_ES |
dc.identifier.pmid | 30582599 | |
dc.relation.pasarela | S\382332 | es_ES |
dc.contributor.funder | Agencia Estatal de Investigación | es_ES |
dc.contributor.funder | Ministerio de Ciencia, Innovación y Universidades |