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Evaluating single-particle tracking by photo-activation localization microscopy (sptPALM) in Lactococcus lactis.
Phys Biol 2019; 16(3):035001PB

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.

Authors+Show Affiliations

Laboratory of Biophysics, Wageningen University and Research, Stippeneng 4, 6708 WE Wageningen, The Netherlands. These authors contributed equally.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

30673632

Citation

van Beljouw, Sam P B., et al. "Evaluating Single-particle Tracking By Photo-activation Localization Microscopy (sptPALM) in Lactococcus Lactis." Physical Biology, vol. 16, no. 3, 2019, p. 035001.
van Beljouw SPB, van der Els S, Martens KJA, et al. Evaluating single-particle tracking by photo-activation localization microscopy (sptPALM) in Lactococcus lactis. Phys Biol. 2019;16(3):035001.
van Beljouw, S. P. B., van der Els, S., Martens, K. J. A., Kleerebezem, M., Bron, P. A., & Hohlbein, J. (2019). Evaluating single-particle tracking by photo-activation localization microscopy (sptPALM) in Lactococcus lactis. Physical Biology, 16(3), p. 035001. doi:10.1088/1478-3975/ab0162.
van Beljouw SPB, et al. Evaluating Single-particle Tracking By Photo-activation Localization Microscopy (sptPALM) in Lactococcus Lactis. Phys Biol. 2019 03 8;16(3):035001. PubMed PMID: 30673632.
* Article titles in AMA citation format should be in sentence-case
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, Y1 - 2019/03/08/ PY - 2019/1/24/pubmed PY - 2019/4/19/medline PY - 2019/1/24/entrez SP - 035001 EP - 035001 JF - Physical biology JO - Phys Biol VL - 16 IS - 3 N2 - 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. SN - 1478-3975 UR - https://www.unboundmedicine.com/medline/citation/30673632/Evaluating_single_particle_tracking_by_photo_activation_localization_microscopy__sptPALM__in_Lactococcus_lactis_ L2 - https://doi.org/10.1088/1478-3975/ab0162 DB - PRIME DP - Unbound Medicine ER -