Cargando…

Split intein-mediated selection of cells containing two plasmids using a single antibiotic

To build or dissect complex pathways in bacteria and mammalian cells, it is often necessary to recur to at least two plasmids, for instance harboring orthogonal inducible promoters. Here we present SiMPl, a method based on rationally designed split enzymes and intein-mediated protein trans-splicing,...

Descripción completa

Detalles Bibliográficos
Autores principales: Palanisamy, Navaneethan, Degen, Anna, Morath, Anna, Ballestin Ballestin, Jara, Juraske, Claudia, Öztürk, Mehmet Ali, Sprenger, Georg A., Youn, Jung-Won, Schamel, Wolfgang W., Di Ventura, Barbara
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6823396/
https://www.ncbi.nlm.nih.gov/pubmed/31672972
http://dx.doi.org/10.1038/s41467-019-12911-1
_version_ 1783464520712192000
author Palanisamy, Navaneethan
Degen, Anna
Morath, Anna
Ballestin Ballestin, Jara
Juraske, Claudia
Öztürk, Mehmet Ali
Sprenger, Georg A.
Youn, Jung-Won
Schamel, Wolfgang W.
Di Ventura, Barbara
author_facet Palanisamy, Navaneethan
Degen, Anna
Morath, Anna
Ballestin Ballestin, Jara
Juraske, Claudia
Öztürk, Mehmet Ali
Sprenger, Georg A.
Youn, Jung-Won
Schamel, Wolfgang W.
Di Ventura, Barbara
author_sort Palanisamy, Navaneethan
collection PubMed
description To build or dissect complex pathways in bacteria and mammalian cells, it is often necessary to recur to at least two plasmids, for instance harboring orthogonal inducible promoters. Here we present SiMPl, a method based on rationally designed split enzymes and intein-mediated protein trans-splicing, allowing the selection of cells carrying two plasmids with a single antibiotic. We show that, compared to the traditional method based on two antibiotics, SiMPl increases the production of the antimicrobial non-ribosomal peptide indigoidine and the non-proteinogenic aromatic amino acid para-amino-L-phenylalanine from bacteria. Using a human T cell line, we employ SiMPl to obtain a highly pure population of cells double positive for the two chains of the T cell receptor, TCRα and TCRβ, using a single antibiotic. SiMPl has profound implications for metabolic engineering and for constructing complex synthetic circuits in bacteria and mammalian cells.
format Online
Article
Text
id pubmed-6823396
institution National Center for Biotechnology Information
language English
publishDate 2019
publisher Nature Publishing Group UK
record_format MEDLINE/PubMed
spelling pubmed-68233962019-11-04 Split intein-mediated selection of cells containing two plasmids using a single antibiotic Palanisamy, Navaneethan Degen, Anna Morath, Anna Ballestin Ballestin, Jara Juraske, Claudia Öztürk, Mehmet Ali Sprenger, Georg A. Youn, Jung-Won Schamel, Wolfgang W. Di Ventura, Barbara Nat Commun Article To build or dissect complex pathways in bacteria and mammalian cells, it is often necessary to recur to at least two plasmids, for instance harboring orthogonal inducible promoters. Here we present SiMPl, a method based on rationally designed split enzymes and intein-mediated protein trans-splicing, allowing the selection of cells carrying two plasmids with a single antibiotic. We show that, compared to the traditional method based on two antibiotics, SiMPl increases the production of the antimicrobial non-ribosomal peptide indigoidine and the non-proteinogenic aromatic amino acid para-amino-L-phenylalanine from bacteria. Using a human T cell line, we employ SiMPl to obtain a highly pure population of cells double positive for the two chains of the T cell receptor, TCRα and TCRβ, using a single antibiotic. SiMPl has profound implications for metabolic engineering and for constructing complex synthetic circuits in bacteria and mammalian cells. Nature Publishing Group UK 2019-10-31 /pmc/articles/PMC6823396/ /pubmed/31672972 http://dx.doi.org/10.1038/s41467-019-12911-1 Text en © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Palanisamy, Navaneethan
Degen, Anna
Morath, Anna
Ballestin Ballestin, Jara
Juraske, Claudia
Öztürk, Mehmet Ali
Sprenger, Georg A.
Youn, Jung-Won
Schamel, Wolfgang W.
Di Ventura, Barbara
Split intein-mediated selection of cells containing two plasmids using a single antibiotic
title Split intein-mediated selection of cells containing two plasmids using a single antibiotic
title_full Split intein-mediated selection of cells containing two plasmids using a single antibiotic
title_fullStr Split intein-mediated selection of cells containing two plasmids using a single antibiotic
title_full_unstemmed Split intein-mediated selection of cells containing two plasmids using a single antibiotic
title_short Split intein-mediated selection of cells containing two plasmids using a single antibiotic
title_sort split intein-mediated selection of cells containing two plasmids using a single antibiotic
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6823396/
https://www.ncbi.nlm.nih.gov/pubmed/31672972
http://dx.doi.org/10.1038/s41467-019-12911-1
work_keys_str_mv AT palanisamynavaneethan splitinteinmediatedselectionofcellscontainingtwoplasmidsusingasingleantibiotic
AT degenanna splitinteinmediatedselectionofcellscontainingtwoplasmidsusingasingleantibiotic
AT morathanna splitinteinmediatedselectionofcellscontainingtwoplasmidsusingasingleantibiotic
AT ballestinballestinjara splitinteinmediatedselectionofcellscontainingtwoplasmidsusingasingleantibiotic
AT juraskeclaudia splitinteinmediatedselectionofcellscontainingtwoplasmidsusingasingleantibiotic
AT ozturkmehmetali splitinteinmediatedselectionofcellscontainingtwoplasmidsusingasingleantibiotic
AT sprengergeorga splitinteinmediatedselectionofcellscontainingtwoplasmidsusingasingleantibiotic
AT younjungwon splitinteinmediatedselectionofcellscontainingtwoplasmidsusingasingleantibiotic
AT schamelwolfgangw splitinteinmediatedselectionofcellscontainingtwoplasmidsusingasingleantibiotic
AT diventurabarbara splitinteinmediatedselectionofcellscontainingtwoplasmidsusingasingleantibiotic