Evaluating single-particle tracking by photo-activation localization microscopy (sptPALM) in Lactococcus lactis

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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.

Original languageEnglish
Number of pages1
JournalPhysical Biology
Volume16
Issue number3
DOIs
Publication statusPublished - 8 Mar 2019

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Lactococcus lactis
Microscopy
CRISPR-Cas Systems
Fermentation
Lactic Acid
Dairying
Clustered Regularly Interspaced Short Palindromic Repeats
Guide RNA
Bacteria
Aptitude
DNA
Nutritive Value
Gene Silencing
Proteins
Food

Cite this

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title = "Evaluating single-particle tracking by photo-activation localization microscopy (sptPALM) in Lactococcus lactis",
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.",
author = "{van Beljouw}, {Sam P.B.} and {van der Els}, Simon and Martens, {Koen J.A.} and Michiel Kleerebezem and Bron, {Peter A.} and Johannes Hohlbein",
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Evaluating single-particle tracking by photo-activation localization microscopy (sptPALM) in Lactococcus lactis. / van Beljouw, Sam P.B.; van der Els, Simon; Martens, Koen J.A.; Kleerebezem, Michiel; Bron, Peter A.; Hohlbein, Johannes.

In: Physical Biology, Vol. 16, No. 3, 08.03.2019.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Evaluating single-particle tracking by photo-activation localization microscopy (sptPALM) in Lactococcus lactis

AU - van Beljouw, Sam P.B.

AU - van der Els, Simon

AU - Martens, Koen J.A.

AU - Kleerebezem, Michiel

AU - Bron, Peter A.

AU - Hohlbein, Johannes

PY - 2019/3/8

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AB - 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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