Publications

Atsushi Sakai, Yoshihiro Murayama, Kei Fujiwara , Takahiro Fujisawa, Saori Sasaki, Satoru Kidoaki, and *Miho Yanagisawa,
Increasing Elasticity through Changes in the Secondary Structure of Gelatin by Gelation in a Microsized Lipid Space,
ACS Central Science 4(4), 477-483 (2018).

[Summary] Even though microgels are used in a wide variety of applications, determining their mechanical properties has been elusive because of the difficulties in analysis. In this study, we investigated the surface elasticity of a spherical microgel of gelatin prepared inside a lipid droplet by using micropipet aspiration. We found that gelation inside a microdroplet covered with lipid membranes increased Young’s modulus E toward a plateau value E* along with a decrease in gel size. In the case of 5.0 wt % gelatin gelled inside a microsized lipid space, the E* for small microgels with R ≤ 50 μm was 10-fold higher (35–39 kPa) than that for the bulk gel (∼3 kPa). Structural analysis using circular dichroism spectroscopy and a fluorescence indicator for ordered beta sheets demonstrated that the smaller microgels contained more beta sheets in the structure than the bulk gel. Our finding indicates that the confinement size of gelling polymers becomes a factor in the variation of elasticity of protein-based microgels via secondary structure changes.

Kei Fujiwara, Takuma Adachi, Nobuhide Doi,
Artificial Cell Fermentation as a Platform for Highly Efficient Cascade Conversion,
ACS Synthetic Biology 7(2), 363-370 (2017).

[Summary] Because of its high specificity and stereoselectivity, cascade reactions using enzymes have been attracting attention as a platform for chemical synthesis. However, the sensitivity of enzymes outside their optimum conditions and their rapid decrease of activity upon dilution are drawbacks of the system. In this study, we developed a system for cascade enzymatic conversion in bacteria-shaped liposomes formed by hypertonic treatment, and demonstrated that the system can overcome the drawbacks of the enzymatic cascade reactions in bulk. This system produced final products at a level equivalent to the maximum concentration of the bulk system (0.10 M, e.g., 4.6 g/L), and worked even under conditions where enzymes normally lose their function. Under diluted conditions, the conversion rate of the artificial cell system was remarkably higher than that in the bulk system. Our results indicate that artificial cells can behave as a platform to perform fermentative production like microorganisms.

*Kei Fujiwara, *Miho Yanagisawa,
Liposomal internal viscosity affects the fate of membrane deformation induced by hypertonic treatment,
Soft Matter 13, 9192-9198 (2017).

Kenji Nishizawa, Kei Fujiwara, Masahiro Ikenaga, Nobushige Nakajo, Miho Yanagisawa, *Daisuke Mizuno,
Universal glass-forming behavior of in vitro and living cytoplasm,
Scientific Reports 7, 15143 (2017).

*Kei Fujiwara, Tsunehito Sawamura, Tatsuya Niwa, Tatsuki Deyama, Shin-ichiro M. Nomura, Hideki Taguchi, Nobuhide Doi,
In vitro transcription–translation using bacterial genome as a template to reconstitute intracellular profile,
Nucleic Acids Research 45(19), 11449-11458 (2017).

[Summary] In vitro transcription–translation systems (TX–TL) can synthesize most of individual genes encoded in genomes by using strong promoters and translation initiation sequences. This fact raises a possibility that TX–TL using genome as a template can reconstitute the profile of RNA and proteins in living cells. By using cell extracts and genome prepared from different organisms, here we developed a system for in vitro genome transcription–translation (iGeTT) using bacterial genome and cell extracts, and surveyed de novo synthesis of RNA and proteins. Two-dimensional electrophoresis and nano LC–MS/MS showed that proteins were actually expressed by iGeTT. Quantitation of transcription levels of 50 genes for intracellular homeostasis revealed that the levels of RNA synthesis by iGeTT are highly correlated with those in growth phase cells. Furthermore, activity of iGeTT was influenced by transcription derived from genome structure and gene location in genome. These results suggest that intracellular profiles and characters of genome can be emulated by TX–TL using genome as a template.

Chikako Kurokawa, Kei Fujiwara, Masamune Morita, Ibuki Kawamata, Yui Kawagishi, Atsushi Sakai, Yoshihiro Murayama, M Nomura Shin-ichiro, Satoshi Murata, *Masahiro Takinoue, *Miho Yanagisawa,
DNA cytoskeleton for stabilizing artificial cells,
Proceedings of the National Academy of Sciences of the United States of America 114, 7228-7233 (2017).

[Summary] Although liposomes and lipid droplets have been used for numerous applications, the fragility of the lipid membrane causes an unintentional collapse, which is problematic for advanced applications. To solve this problem, we constructed an artificial cytoskeleton with DNA nanotechnology (a DNA cytoskeleton). The DNA cytoskeleton is a DNA network formed underneath the membrane of positively charged lipids through electrostatic interactions without the need for special handling. The DNA cytoskeleton significantly improves mechanical stability and, therefore, confers tolerance against osmotic shock to liposomes like the cytoskeleton in live cells. Because of its biocompatibility and the easiness of implementing design changes, the DNA cytoskeleton could become a tool for great stabilizer of liposomes and lipid droplets.