Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Record number 549344
Title Evaluating single-particle tracking by photo-activation localization microscopy (sptPALM) in Lactococcus lactis
Author(s) Beljouw, Sam P.B. van; Els, Simon van der; Martens, Koen J.A.; Kleerebezem, Michiel; Bron, Peter A.; Hohlbein, Johannes
Source Physical Biology 16 (2019)3. - ISSN 1478-3967 - 1 p.
DOI https://doi.org/10.1088/1478-3975/ab0162
Department(s) Biophysics
Host-Microbe Interactomics
VLAG
WIAS
Microbiology
Publication type Refereed Article in a scientific journal
Publication year 2019
Abstract

Lactic acid bacteria (LAB) are frequently used in food fermentation and are invaluable for the taste and nutritional value of the fermentation end-product. To gain a better understanding of underlying biochemical and microbiological mechanisms and cell-to-cell variability in LABs, single-molecule techniques such as single-particle tracking photo-activation localization microscopy (sptPALM) hold great promises but are not yet employed due to the lack of detailed protocols and suitable assays. Here, we qualitatively test various fluorescent proteins including variants that are photoactivatable and therefore suitable for sptPALM measurements in Lactococcus lactis, a key LAB for the dairy industry. In particular, we fused PAmCherry2 to dCas9 allowing the successful tracking of single dCas9 proteins, whilst the dCas9 chimeras bound to specific guide RNAs retained their gene silencing ability in vivo. The diffusional information of the dCas9 without any targets showed different mechanistic states of dCas9: freely diffusing, bound to DNA, or transiently interacting with DNA. The capability of performing sptPALM with dCas9 in L. lactis can lead to a better, general understanding of CRISPR-Cas systems as well as paving the way for CRISPR-Cas based interrogations of cellular functions in LABs.

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