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Single plasmid systems for inducible dual protein expression and for CRISPR-Cas9/CRISPRi gene regulation in lactic acid bacterium Lactococcus lactis
A. Berlec, K. Škrlec, J. Kocjan, M. Olenic, B. Štrukelj,
Jazyk angličtina Země Anglie, Velká Británie
Typ dokumentu časopisecké články, práce podpořená grantem
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Directory of Open Access Journals
od 2011
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- MeSH
- antibakteriální látky farmakologie MeSH
- CRISPR-Cas systémy MeSH
- editace genu metody MeSH
- fermentace MeSH
- genetické inženýrství metody MeSH
- genom bakteriální * MeSH
- guide RNA, Kinetoplastida genetika metabolismus MeSH
- imunoglobulin G genetika metabolismus MeSH
- klonování DNA MeSH
- Lactococcus lactis genetika metabolismus MeSH
- lidé MeSH
- methyltransferasy genetika metabolismus MeSH
- nisin farmakologie MeSH
- plazmidy chemie metabolismus MeSH
- promotorové oblasti (genetika) účinky léků MeSH
- proteiny teplotního šoku genetika metabolismus MeSH
- regulace genové exprese u bakterií * MeSH
- rekombinantní fúzní proteiny genetika metabolismus MeSH
- transgeny * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Lactococcus lactis is a food-grade lactic acid bacterium that is used in the dairy industry as a cell factory and as a host for recombinant protein expression. The nisin-controlled inducible expression (NICE) system is frequently applied in L. lactis; however new tools for its genetic modification are highly desirable. In this work NICE was adapted for dual protein expression. Plasmid pNZDual, that contains two nisin promoters and multiple cloning sites (MCSs), and pNZPolycist, that contains a single nisin promoter and two MCSs separated by the ribosome binding site, were constructed. Genes for the infrared fluorescent protein and for the human IgG-binding DARPin were cloned in all possible combinations to assess the protein yield. The dual promoter plasmid pNZDual enabled balanced expression of the two model proteins. It was exploited for the development of a single-plasmid inducible CRISPR-Cas9 system (pNZCRISPR) by using a nisin promoter, first to drive Cas9 expression and, secondly, to drive single guide RNA transcription. sgRNAs against htrA and ermR directed Cas9 against genomic or plasmid DNA and caused changes in bacterial growth and survival. Replacing Cas9 by dCas9 enabled CRISPR interference-mediated silencing of the upp gene. The present study introduces a new series of plasmids for advanced genetic modification of lactic acid bacterium L. lactis.
Citace poskytuje Crossref.org
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- $a Lactococcus lactis is a food-grade lactic acid bacterium that is used in the dairy industry as a cell factory and as a host for recombinant protein expression. The nisin-controlled inducible expression (NICE) system is frequently applied in L. lactis; however new tools for its genetic modification are highly desirable. In this work NICE was adapted for dual protein expression. Plasmid pNZDual, that contains two nisin promoters and multiple cloning sites (MCSs), and pNZPolycist, that contains a single nisin promoter and two MCSs separated by the ribosome binding site, were constructed. Genes for the infrared fluorescent protein and for the human IgG-binding DARPin were cloned in all possible combinations to assess the protein yield. The dual promoter plasmid pNZDual enabled balanced expression of the two model proteins. It was exploited for the development of a single-plasmid inducible CRISPR-Cas9 system (pNZCRISPR) by using a nisin promoter, first to drive Cas9 expression and, secondly, to drive single guide RNA transcription. sgRNAs against htrA and ermR directed Cas9 against genomic or plasmid DNA and caused changes in bacterial growth and survival. Replacing Cas9 by dCas9 enabled CRISPR interference-mediated silencing of the upp gene. The present study introduces a new series of plasmids for advanced genetic modification of lactic acid bacterium L. lactis.
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