A chimeric vector for dual use in cyanobacteria and Escherichia coli, tested with cystatin, a nonfluorescent reporter protein

Background Developing sustainable autotrophic cell factories depends heavily on the availability of robust and well-characterized biological parts. For cyanobacteria, these still lag behind the more advanced E. coli toolkit. In the course of previous protein expression experiments with cyanobacteria...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Mojca Juteršek, Marko Dolinar
Formato:	article
Lenguaje:	EN
Publicado:	PeerJ Inc. 2021
Materias:	Cyanobacteria Synechocystis sp. PCC 6803 Reporter protein Cystatin Biosafety Synthetic biology Medicine R
Acceso en línea:	https://doaj.org/article/d593f8359a2648baa8d43fb3446fcc1d
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:d593f8359a2648baa8d43fb3446fcc1d
record_format	dspace
spelling	oai:doaj.org-article:d593f8359a2648baa8d43fb3446fcc1d2021-11-05T15:05:16ZA chimeric vector for dual use in cyanobacteria and Escherichia coli, tested with cystatin, a nonfluorescent reporter protein10.7717/peerj.121992167-8359https://doaj.org/article/d593f8359a2648baa8d43fb3446fcc1d2021-11-01T00:00:00Zhttps://peerj.com/articles/12199.pdfhttps://peerj.com/articles/12199/https://doaj.org/toc/2167-8359Background Developing sustainable autotrophic cell factories depends heavily on the availability of robust and well-characterized biological parts. For cyanobacteria, these still lag behind the more advanced E. coli toolkit. In the course of previous protein expression experiments with cyanobacteria, we encountered inconveniences in working with currently available RSF1010-based shuttle plasmids, particularly due to their low biosafety and low yields of recombinant proteins. We also recognized some drawbacks of the commonly used fluorescent reporters, as quantification can be affected by the intrinsic fluorescence of cyanobacteria. To overcome these drawbacks, we envisioned a new chimeric vector and an alternative reporter that could be used in cyanobacterial synthetic biology and tested them in the model cyanobacterium Synechocystis sp. PCC 6803. Methods We designed the pMJc01 shuttle plasmid based on the broad host range RSFmob-I replicon. Standard cloning techniques were used for vector construction following the RFC10 synthetic biology standard. The behavior of pMJC01 was tested with selected regulatory elements in E. coli and Synechocystis sp. PCC 6803 for the biosynthesis of the established GFP reporter and of a new reporter protein, cystatin. Cystatin activity was assayed using papain as a cognate target. Results With the new vector we observed a significantly higher GFP expression in E. coli and Synechocystis sp. PCC 6803 compared to the commonly used RSF1010-based pPMQAK1. Cystatin, a cysteine protease inhibitor, was successfully expressed with the new vector in both E. coli and Synechocystis sp. PCC 6803. Its expression levels allowed quantification comparable to the standardly used fluorescent reporter GFPmut3b. An important advantage of the new vector is its improved biosafety due to the absence of plasmid regions encoding conjugative transfer components. The broadhost range vector pMJc01 could find application in synthetic biology and biotechnology of cyanobacteria due to its relatively small size, stability and ease of use. In addition, cystatin could be a useful reporter in all cell systems that do not contain papain-type proteases and inhibitors, such as cyanobacteria, and provides an alternative to fluorescent reporters or complements them.Mojca JuteršekMarko DolinarPeerJ Inc.articleCyanobacteriaSynechocystis sp. PCC 6803Reporter proteinCystatinBiosafetySynthetic biologyMedicineRENPeerJ, Vol 9, p e12199 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Cyanobacteria Synechocystis sp. PCC 6803 Reporter protein Cystatin Biosafety Synthetic biology Medicine R
spellingShingle	Cyanobacteria Synechocystis sp. PCC 6803 Reporter protein Cystatin Biosafety Synthetic biology Medicine R Mojca Juteršek Marko Dolinar A chimeric vector for dual use in cyanobacteria and Escherichia coli, tested with cystatin, a nonfluorescent reporter protein
description	Background Developing sustainable autotrophic cell factories depends heavily on the availability of robust and well-characterized biological parts. For cyanobacteria, these still lag behind the more advanced E. coli toolkit. In the course of previous protein expression experiments with cyanobacteria, we encountered inconveniences in working with currently available RSF1010-based shuttle plasmids, particularly due to their low biosafety and low yields of recombinant proteins. We also recognized some drawbacks of the commonly used fluorescent reporters, as quantification can be affected by the intrinsic fluorescence of cyanobacteria. To overcome these drawbacks, we envisioned a new chimeric vector and an alternative reporter that could be used in cyanobacterial synthetic biology and tested them in the model cyanobacterium Synechocystis sp. PCC 6803. Methods We designed the pMJc01 shuttle plasmid based on the broad host range RSFmob-I replicon. Standard cloning techniques were used for vector construction following the RFC10 synthetic biology standard. The behavior of pMJC01 was tested with selected regulatory elements in E. coli and Synechocystis sp. PCC 6803 for the biosynthesis of the established GFP reporter and of a new reporter protein, cystatin. Cystatin activity was assayed using papain as a cognate target. Results With the new vector we observed a significantly higher GFP expression in E. coli and Synechocystis sp. PCC 6803 compared to the commonly used RSF1010-based pPMQAK1. Cystatin, a cysteine protease inhibitor, was successfully expressed with the new vector in both E. coli and Synechocystis sp. PCC 6803. Its expression levels allowed quantification comparable to the standardly used fluorescent reporter GFPmut3b. An important advantage of the new vector is its improved biosafety due to the absence of plasmid regions encoding conjugative transfer components. The broadhost range vector pMJc01 could find application in synthetic biology and biotechnology of cyanobacteria due to its relatively small size, stability and ease of use. In addition, cystatin could be a useful reporter in all cell systems that do not contain papain-type proteases and inhibitors, such as cyanobacteria, and provides an alternative to fluorescent reporters or complements them.
format	article
author	Mojca Juteršek Marko Dolinar
author_facet	Mojca Juteršek Marko Dolinar
author_sort	Mojca Juteršek
title	A chimeric vector for dual use in cyanobacteria and Escherichia coli, tested with cystatin, a nonfluorescent reporter protein
title_short	A chimeric vector for dual use in cyanobacteria and Escherichia coli, tested with cystatin, a nonfluorescent reporter protein
title_full	A chimeric vector for dual use in cyanobacteria and Escherichia coli, tested with cystatin, a nonfluorescent reporter protein
title_fullStr	A chimeric vector for dual use in cyanobacteria and Escherichia coli, tested with cystatin, a nonfluorescent reporter protein
title_full_unstemmed	A chimeric vector for dual use in cyanobacteria and Escherichia coli, tested with cystatin, a nonfluorescent reporter protein
title_sort	chimeric vector for dual use in cyanobacteria and escherichia coli, tested with cystatin, a nonfluorescent reporter protein
publisher	PeerJ Inc.
publishDate	2021
url	https://doaj.org/article/d593f8359a2648baa8d43fb3446fcc1d
work_keys_str_mv	AT mojcajutersek achimericvectorfordualuseincyanobacteriaandescherichiacolitestedwithcystatinanonfluorescentreporterprotein AT markodolinar achimericvectorfordualuseincyanobacteriaandescherichiacolitestedwithcystatinanonfluorescentreporterprotein AT mojcajutersek chimericvectorfordualuseincyanobacteriaandescherichiacolitestedwithcystatinanonfluorescentreporterprotein AT markodolinar chimericvectorfordualuseincyanobacteriaandescherichiacolitestedwithcystatinanonfluorescentreporterprotein
_version_	1718444196268867584

A chimeric vector for dual use in cyanobacteria and Escherichia coli, tested with cystatin, a nonfluorescent reporter protein

Ejemplares similares