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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

The result's identifiers

  • Result code in IS VaVaI

    <a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216208%3A11160%2F18%3A10393247" target="_blank" >RIV/00216208:11160/18:10393247 - isvavai.cz</a>

  • Result on the web

    <a href="http://www.nature.com/articles/s41598-018-19402-1" target="_blank" >http://www.nature.com/articles/s41598-018-19402-1</a>

  • DOI - Digital Object Identifier

    <a href="http://dx.doi.org/10.1038/s41598-018-19402-1" target="_blank" >10.1038/s41598-018-19402-1</a>

Alternative languages

  • Result language

    angličtina

  • Original language name

    Single plasmid systems for inducible dual protein expression and for CRISPR-Cas9/CRISPRi gene regulation in lactic acid bacterium Lactococcus lactis

  • Original language description

    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.

  • Czech name

  • Czech description

Classification

  • Type

    J<sub>imp</sub> - Article in a specialist periodical, which is included in the Web of Science database

  • CEP classification

  • OECD FORD branch

    30104 - Pharmacology and pharmacy

Result continuities

  • Project

  • Continuities

    I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Others

  • Publication year

    2018

  • Confidentiality

    S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Data specific for result type

  • Name of the periodical

    Scientific Reports

  • ISSN

    2045-2322

  • e-ISSN

  • Volume of the periodical

    8

  • Issue of the periodical within the volume

    January

  • Country of publishing house

    GB - UNITED KINGDOM

  • Number of pages

    11

  • Pages from-to

  • UT code for WoS article

    000422716900143

  • EID of the result in the Scopus database

    2-s2.0-85040775746