| dc.contributor | Federici, Fernán | |
| dc.contributor | PONTIFICIA UNIVERSIDAD CATOLICA DE CHILE | |
| dc.creator | Arce Medina, Anibal Andrés | |
| dc.date | 2021-07-22T15:17:53Z | |
| dc.date | 2022-08-23T12:55:56Z | |
| dc.date | 2021-07-22T15:17:53Z | |
| dc.date | 2022-08-23T12:55:56Z | |
| dc.date | 2021 | |
| dc.date.accessioned | 2023-08-21T23:23:13Z | |
| dc.date.available | 2023-08-21T23:23:13Z | |
| dc.identifier | 21140714 | |
| dc.identifier | https://hdl.handle.net/10533/249981 | |
| dc.identifier.uri | https://repositorioslatinoamericanos.uchile.cl/handle/2250/8295077 | |
| dc.description | Synthetic biology seeks the development of programmable and predictable functions in biological systems. One of the latest advances in the field are the toehold switches: de-novo engineered regulators of gene expression that allow the translation of a gene only after the interaction with a cognate “trigger” RNA of a specific sequence.On the other hand, cell-free protein expression systems have been used for decades facilitating discoveries in molecular biology, and have played a critical role in the elucidation of the genetic code. More recently, cell-free systems have emerged as a tool for the engineering of genetic devices, bio-products, and biosensors, among many other applications. The expression of programmable toehold switch-based RNA sensors in cell-free systems has generated as promising platform for diagnostics. Cell-free toehold sensorscan be freeze-dried for room temperature transport to the point-of-need.These sensors, however, have been implemented using reconstituted PURE(Protein expression Using Reconstituted Elements) cell-free protein expres-sion systems that are difficult to source in Latin America, due to their pro-hibitively expensive commercial cost, and cold-chain shipping requirements(-80oC) from suppliers in the northern hemisphere.Here, we describe the implementation of RNA toehold sensors usingE. coli
cell lysate-based cell-free protein expression systems, which can be pro-duced locally and reduce the cost of sensors by two orders of magnitude, while providing sensor performance comparable to commercial PURE cell-free systems. Further optimization of the cell extracts with a CRISPRistrategy enhanced the stability of linear dsDNAs, enabling the use of PCR products as a substrate for cell-free gene expression reactions including to hold sensors. As a proof-of-concept application, we used the tools de-veloped in this thesis to prototype and screen 8 novel RNA toehold sensors for the potato pathogen Potato Virus Y (PVY virus) that dramatically reduces the yield of this important staple crop. The local implementation of low-cost cell-free toehold sensors could enable biosensing capacity at the regional level and lead to more decentralized models for global surveillance of infectious diseases. | |
| dc.format | application/pdf | |
| dc.relation | instname: Conicyt | |
| dc.relation | reponame: Repositorio Digital RI2.0 | |
| dc.relation | info:eu-repo/grantAgreement//21140714 | |
| dc.relation | info:eu-repo/semantics/dataset/hdl.handle.net/10533/93488 | |
| dc.relation | https://repositorio.uc.cl/handle/11534/57970 | |
| dc.rights | info:eu-repo/semantics/openAccess | |
| dc.rights | Attribution-NonCommercial-NoDerivs 3.0 Chile | |
| dc.rights | info:eu-repo/semantics/openAccess | |
| dc.rights | http://creativecommons.org/licenses/by-nc-nd/3.0/cl/ | |
| dc.subject | Ciencias Naturales | |
| dc.subject | Ciencias de la Tierra y del Medio Ambiente | |
| dc.subject | Biología Molecular | |
| dc.title | Open platform for the implementation of RNA sensing reactions in cell-free systems | |
| dc.type | info:eu-repo/semantics/doctoralThesis | |
| dc.type | info:eu-repo/semantics/publishedVersion | |
| dc.type | Tesis | |
