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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,
Language English Country England, Great Britain
Document type Journal Article, Research Support, Non-U.S. Gov't
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- MeSH
- Anti-Bacterial Agents pharmacology MeSH
- CRISPR-Cas Systems MeSH
- Gene Editing methods MeSH
- Fermentation MeSH
- Genetic Engineering methods MeSH
- Genome, Bacterial * MeSH
- RNA, Guide, Kinetoplastida genetics metabolism MeSH
- Immunoglobulin G genetics metabolism MeSH
- Cloning, Molecular MeSH
- Lactococcus lactis genetics metabolism MeSH
- Humans MeSH
- Methyltransferases genetics metabolism MeSH
- Nisin pharmacology MeSH
- Plasmids chemistry metabolism MeSH
- Promoter Regions, Genetic drug effects MeSH
- Heat-Shock Proteins genetics metabolism MeSH
- Gene Expression Regulation, Bacterial * MeSH
- Recombinant Fusion Proteins genetics metabolism MeSH
- Transgenes * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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.
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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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