Optimized delivery of fluorescently labeled proteins in live bacteria using electroporation

• DOI: 10.1007/s00418-014-1213-2
• Publication Date: 2014-04-02T06:07:31Z
• Authors: Marko Sustarsic; Anne Plochowietz; Louise Aigrain; Yulia Yuzenkova; Nikolay Zenkin; Achillefs Kapanidis
• Keywords: Internalization; Viability assay; Agarose; Live cell imaging

Studying the structure and dynamics of proteins in live cells is essential to understanding their physiological activities mechanisms, validating vitro characterization. Improvements labeling imaging technologies are starting allow such vivo studies; however, a number technical challenges remain. Recently, we developed an electroporation-based protocol for internalization, which allows biomolecules labeled with organic fluorophores be introduced at high efficiency into E. coli (Crawford et al. Biophys J 105 (11):2439–2450, 2013). Here, address important related internalization proteins, optimize our method terms (1) electroporation buffer conditions; (2) removal dye contaminants from stock protein samples; (3) non-internalized molecules cell suspension after electroporation. We illustrate usability optimized by demonstrating high-efficiency 10-kDa protein, ω subunit RNA polymerase. Provided that suggested control experiments carried out, any fluorescently up 60 kDa could internalized using method. Further, probe effect voltage on viability demonstrate that, whilst increases increased voltage, compromised. However, due low damaged samples, major fraction loaded always corresponds non-damaged cells. By taking care include only viable analysis, physiologically relevant studies performed, including measurements diffusion, localization intramolecular via single-molecule Förster resonance energy transfer.