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

Autor: Mariscal AM; Departament de Bioquímica i Biologia Molecular and Institut de Biotecnologia i Biomedicina , Universitat Autònoma de Barcelona , Cerdanyola del Vallès, Barcelona 08193 , Spain., Kakizawa S; Synthetic Biology Group , J. Craig Venter Institute , La Jolla , California 92037 , United States.; National Institute of Advanced Industrial Science and Technology , Tsukuba , Ibaraki 305-8560 , Japan., Hsu JY; Synthetic Biology Group , J. Craig Venter Institute , La Jolla , California 92037 , United States.; Department of Bioengineering , University of California, San Diego , La Jolla , California 92093 , United States., Tanaka K; Synthetic Biology Group , J. Craig Venter Institute , La Jolla , California 92037 , United States.; Institute for Advanced Biosciences , Keio University , Tsuruoka , Yamagata 997-0035 , Japan.; Faculty of Environment and Information Studies , Keio University , Fujisawa , Kanagawa 252-0882 , Japan., González-González L; Departament de Bioquímica i Biologia Molecular and Institut de Biotecnologia i Biomedicina , Universitat Autònoma de Barcelona , Cerdanyola del Vallès, Barcelona 08193 , Spain., Broto A; EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG) , The Barcelona Institute of Science and Technology , Barcelona 08036 , Spain.; Universitat Pompeu Fabra (UPF) , Barcelona 08002 , Spain., Querol E; Departament de Bioquímica i Biologia Molecular and Institut de Biotecnologia i Biomedicina , Universitat Autònoma de Barcelona , Cerdanyola del Vallès, Barcelona 08193 , Spain., Lluch-Senar M; EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG) , The Barcelona Institute of Science and Technology , Barcelona 08036 , Spain.; Universitat Pompeu Fabra (UPF) , Barcelona 08002 , Spain., Piñero-Lambea C; EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG) , The Barcelona Institute of Science and Technology , Barcelona 08036 , Spain.; Universitat Pompeu Fabra (UPF) , Barcelona 08002 , Spain., Sun L; Synthetic Biology Group , J. Craig Venter Institute , La Jolla , California 92037 , United States., Weyman PD; Synthetic Biology Group , J. Craig Venter Institute , La Jolla , California 92037 , United States., Wise KS; Synthetic Biology Group , J. Craig Venter Institute , La Jolla , California 92037 , United States., Merryman C; Synthetic Biology Group , J. Craig Venter Institute , La Jolla , California 92037 , United States., Tse G; Synthetic Biology Group , J. Craig Venter Institute , La Jolla , California 92037 , United States.; Department of Bioengineering , University of California, San Diego , La Jolla , California 92093 , United States., Moore AJ; Synthetic Biology Group , J. Craig Venter Institute , La Jolla , California 92037 , United States.; Department of Bioengineering , University of California, San Diego , La Jolla , California 92093 , United States., Hutchison CA 3rd; Synthetic Biology Group , J. Craig Venter Institute , La Jolla , California 92037 , United States., Smith HO; Synthetic Biology Group , J. Craig Venter Institute , La Jolla , California 92037 , United States., Tomita M; Institute for Advanced Biosciences , Keio University , Tsuruoka , Yamagata 997-0035 , Japan., Venter JC; Synthetic Biology Group , J. Craig Venter Institute , La Jolla , California 92037 , United States., Glass JI; Synthetic Biology Group , J. Craig Venter Institute , La Jolla , California 92037 , United States., Piñol J; Departament de Bioquímica i Biologia Molecular and Institut de Biotecnologia i Biomedicina , Universitat Autònoma de Barcelona , Cerdanyola del Vallès, Barcelona 08193 , Spain., Suzuki Y; Synthetic Biology Group , J. Craig Venter Institute , La Jolla , California 92037 , United States.
Jazyk: angličtina
Zdroj: ACS synthetic biology [ACS Synth Biol] 2018 Jun 15; Vol. 7 (6), pp. 1538-1552. Date of Electronic Publication: 2018 May 22.
DOI: 10.1021/acssynbio.8b00028
Abstrakt: 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.
Databáze: MEDLINE
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