Tuning Gene Activity by Inducible and Targeted Regulation of Gene Expression in Minimal Bacterial Cells

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• dc.contributor.author Mariscal, Ana M.
• dc.contributor.author Broto, Alicia
• dc.contributor.author Lluch-Senar, Maria 1982-
• dc.contributor.author Piñero-Lambea, Carlos
• dc.contributor.author Suzuki, Yo
• dc.date.accessioned 2019-02-28T08:45:03Z
• dc.date.available 2019-02-28T08:45:03Z
• dc.date.issued 2018
• dc.description.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.
• dc.description.sponsorship This work was supported by internal funding from the J. Craig Venter Institute to H.O.S. and C.A.H., as well as grants BIO2013-4870R and BIO2013-50176EXP from the Ministerio de Economía y Competitividad to E.Q. and J.P., respectively, and Japan Society for the Promotion of Science KAKENHI grants JP26710015, JP15KK0266, and JP26106004 to S.K.A.M.M. is a recipient of a predoctoral fellowship from the Generalitat de Catalunya (FI-DGR 2014).
• dc.format.mimetype application/pdf
• dc.identifier.citation Mariscal AM, Kakizawa S, Hsu JY, Tanaka K, González-González L, Broto A et al. Tuning Gene Activity by Inducible and Targeted Regulation of Gene Expression in Minimal Bacterial Cells. ACS Synth Biol. 2018;7(6):1538-52. DOI: 10.1021/acssynbio.8b00028
• dc.identifier.doi http://dx.doi.org/10.1021/acssynbio.8b00028
• dc.identifier.issn 2161-5063
• dc.identifier.uri http://hdl.handle.net/10230/36692
• dc.language.iso spa
• dc.publisher American Chemical Society (ACS)
• dc.relation.ispartof ACS Synthetic Biology. 2018;7(6):1538-52
• dc.relation.projectID info:eu-repo/grantAgreement/ES/1PE/BIO2013-4870R
• dc.relation.projectID info:eu-repo/grantAgreement/ES/1PE/BIO2013-50176-EXP
• dc.rights Copyright © 2018 American Chemical Society. 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.
• dc.rights.accessRights info:eu-repo/semantics/openAccess
• dc.subject.keyword Clustered regularly interspaced short palindromic repeats (CRISPR)
• dc.subject.keyword Functional genomics
• dc.subject.keyword Inducible promoters
• dc.subject.keyword Mycoplasma
• dc.subject.keyword Riboswitch
• dc.subject.keyword Tetracycline-mediated repression
• dc.title Tuning Gene Activity by Inducible and Targeted Regulation of Gene Expression in Minimal Bacterial Cells
• dc.type info:eu-repo/semantics/article
• dc.type.version info:eu-repo/semantics/publishedVersion