Self-organization Assay for Min Proteins of Escherichia coli in Micro-droplets Covered with Lipids

Shunshi Kohyama,Kei Fujiwara,Natsuhiko Yoshinaga

doi:10.21769/bioprotoc.3561

Shunshi Kohyama, Kei Fujiwara + Show 1 more

Open Access

https://doi.org/10.21769/bioprotoc.3561

Copy DOI

Abstract

The Min system determines the cell division plane of bacteria. As a cue of spatiotemporal regulation, the Min system uses wave propagation of MinD protein (Min wave). Therefore, the reconstitution of the Min wave in cell-sized closed space will lead to the creation of artificial cells capable of cell division. The Min waves emerge via coupling between the reactions among MinD, MinE, and ATP and the differences in diffusion rate on the cell membrane and in the cytoplasm. Because Min waves appear only under the balanced condition of the reaction-diffusion coupling, special attentions are needed towards several technical points for the reconstitution of Min waves in artificial cells. This protocol describes a technical method for stably generating Min waves in artificial cells.

Highlights

[Background] Min system, which determines the cell center for symmetric cell division, is one of the most striking examples of intracellular organization systems in bacteria (Rothfield et al, 2005; Rowlett and Margolin, 2013)
Min system uses pattern formation of time-dependent protein gradient inside cells known as Min waves (Loose et al, 2008; Halatek and Frey, 2012; Bonny et al, 2013; Zieske et al, 2016; Kohyama et al, 2019)
The Min wave is emerged by a reaction-diffusion coupling of two proteins: MinD and MinE

Summary

Introduction

[Background] Min system, which determines the cell center for symmetric cell division, is one of the most striking examples of intracellular organization systems in bacteria (Rothfield et al, 2005; Rowlett and Margolin, 2013). The Min wave is emerged by a reaction-diffusion coupling of two proteins: MinD and MinE. Sodium chloride (NaCl) (Nacalai Tesque, catalog number: 31320-05) 31. HEPES (Nacalai Tesque, catalog number: 17546-05) 35. Glycerol (Nacalai Tesque, catalog number: 17018-25) 36.

Results

Conclusion