Fluorescent tools for the standardized work in Gram-negative bacteria.
Salmonella enterica
Fluorescent proteins
Genetic tools
Microscopy
Protein dynamics
Journal
Journal of biological engineering
ISSN: 1754-1611
Titre abrégé: J Biol Eng
Pays: England
ID NLM: 101306640
Informations de publication
Date de publication:
08 Apr 2024
08 Apr 2024
Historique:
received:
20
01
2024
accepted:
18
03
2024
medline:
9
4
2024
pubmed:
9
4
2024
entrez:
8
4
2024
Statut:
epublish
Résumé
Standardized and thoroughly characterized genetic tools are a prerequisite for studying cellular processes to ensure the reusability and consistency of experimental results. The discovery of fluorescent proteins (FPs) represents a milestone in the development of genetic reporters for monitoring transcription or protein localization in vivo. FPs have revolutionized our understanding of cellular dynamics by enabling the real-time visualization and tracking of biological processes. Despite these advancements, challenges remain in the appropriate use of FPs, specifically regarding their proper application, protein turnover dynamics, and the undesired disruption of cellular functions. Here, we systematically compared a comprehensive set of 15 FPs and assessed their performance in vivo by focusing on key parameters, such as signal over background ratios and protein stability rates, using the Gram-negative model organism Salmonella enterica as a representative host. We evaluated four protein degradation tags in both plasmid- and genome-based systems and our findings highlight the necessity of introducing degradation tags to analyze time-sensitive cellular processes. We demonstrate that the gain of dynamics mediated by the addition of degradation tags impacts the cell-to-cell heterogeneity of plasmid-based but not genome-based reporters. Finally, we probe the applicability of FPs for protein localization studies in living cells using standard and super-resolution fluorescence microscopy. In summary, our study underscores the importance of careful FP selection and paves the way for the development of improved genetic reporters to enhance the reproducibility and reliability of fluorescence-based research in Gram-negative bacteria and beyond.
Identifiants
pubmed: 38589953
doi: 10.1186/s13036-024-00420-9
pii: 10.1186/s13036-024-00420-9
doi:
Types de publication
Journal Article
Langues
eng
Pagination
25Subventions
Organisme : Grand Challenge Initiative Global Health grant of the Berlin University Alliance
ID : 113_MC_GH_MEL-BER_Erhardt_HU
Organisme : Grand Challenge Initiative Global Health grant of the Berlin University Alliance
ID : 113_MC_GH_MEL-BER_Erhardt_HU
Organisme : European Union's Horizon 2020 research and innovation program
ID : 864971
Informations de copyright
© 2024. The Author(s).
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