Tuning gene activity by inducible and targeted regulation of gene expression in minimal bacterial cells
Martínez Mariscal, Ana M. (Universitat Autònoma de Barcelona. Institut de Biotecnologia i de Biomedicina "Vicent Villar Palasí")
Kakizawa, Shigeyuki (J. Craig Venter Institute)
Hsu, Jonathan Y. (J. Craig Venter Institute)
Tanaka, Kazuki (J. Craig Venter Institute)
Gonzalez-Gonzalez, Luis 1987- (Universitat Autònoma de Barcelona. Institut de Biotecnologia i de Biomedicina "Vicent Villar Palasí")
Broto, Alicia (Centre de Regulació Genòmica)
Querol Murillo, Enrique (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Lluch Senar, Maria (Centre de Regulació Genòmica)
Piñero Lambea, Carlos (Centre de Regulació Genòmica)
Sun, Lijie (J. Craig Venter Institute)
Weyman, Philip D. (J. Craig Venter Institute)
Wise, Kim S. (J. Craig Venter Institute)
Merryman, Chuck (J. Craig Venter Institute)
Tse, Gavin (J. Craig Venter Institute)
Moore, Adam J. (J. Craig Venter Institute)
Hutchison, Clyde A. (J. Craig Venter Institute)
Smith, Hamilton O. (J. Craig Venter Institute)
Tomita, Masaru (Keio University. Institute for Advanced Biosciences)
Venter, J. Craig (J. Craig Venter Institute)
Glass, John I. (J. Craig Venter Institute)
Piñol Ribas, Jaume (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Suzuki, Yo (J. Craig Venter Institute)

Date:	2018
Abstract:	Functional genomics studies in minimal mycoplasma cells enable unobstructed access to some of the most fundamental processes in biology. Conventional transposon bombardment and gene knockout approaches often fail to reveal functions of genes that are essential for viability, where lethality precludes phenotypic characterization. Conditional inactivation of genes is effective for characterizing functions central to cell growth and division, but tools are limited for this purpose in mycoplasmas. Here we demonstrate systems for inducible repression of gene expression based on clustered regularly interspaced short palindromic repeats-mediated interference (CRISPRi) in Mycoplasma pneumoniae and synthetic Mycoplasma mycoides, two organisms with reduced genomes actively used in systems biology studies. In the synthetic cell, we also demonstrate inducible gene expression for the first time. Time-course data suggest rapid kinetics and reversible engagement of CRISPRi. Targeting of six selected endogenous genes with this system results in lowered transcript levels or reduced growth rates that agree with lack or shortage of data in previous transposon bombardment studies, and now produces actual cells to analyze. The ksgA gene encodes a methylase that modifies 16S rRNA, rendering it vulnerable to inhibition by the antibiotic kasugamycin. Targeting the ksgA gene with CRISPRi removes the lethal effect of kasugamycin and enables cell growth, thereby establishing specific and effective gene modulation with our system. The facile methods for conditional gene activation and inactivation in mycoplasmas open the door to systematic dissection of genetic programs at the core of cellular life.
Grants:	Ministerio de Economía y Competitividad BIO2013-50176EXP Ministerio de Economía y Competitividad BIO2013-4870R
Note:	Altres ajuts: predoctoral fellowship from the Generalitat de Catalunya (FI-DGR 2014)
Note:	This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes
Rights:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, sempre que no sigui amb finalitats comercials, i sempre que es reconegui l'autoria de l'obra original.
Language:	Anglès
Document:	Article ; recerca ; Versió publicada
Subject:	Inducible promoters ; Riboswitch ; Tetracycline-mediated repression ; Clustered regularly interspaced short palindromic repeats (CRISPR) ; Mycoplasma ; Functional genomics
Published in:	ACS Synthetic Biology, Vol. 7, issue 6 (June 2018) , p. 1538-1552, ISSN 2161-5063

DOI: 10.1021/acssynbio.8b00028

15 p, 2.2 MB

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Research literature > UAB research groups literature > Research Centres and Groups (research output) > Health sciences and biosciences > Institut de Biotecnologia i de Biomedicina (IBB)
Articles > Research articles
Articles > Published articles