Materials and methods

・Preparation of vesicles [1]

1. W/O emulsions were prepared by mixing emulsification.

Add 2.5ml of hexane solution (3wt% Span 80 and 0.1wt% stearylamine) to 0.5ml of 50mM Tris-HCl (0.01M calcein, pH8.0),

and mix them at 8000 rpm for 5 minutes at room temperature.

2. Freeze the emulsions by liquid nitrogen and keep them at -20°C.

3. Add the mixture of egg PC, cholesterol, stearylamine and rhodamine PE in the mole ratio of 5: 5: 1: 0.005 to hexane.

Import the hexane solution into the emulsion solution and export it three times, and replace surfaces of emulsions from span80 to lipid.

4. Add hexane to the emulsion solution, and keep it at －6℃, 20hPa to remove hexane. We used an evaporator.

5. Add 50mM Tris-HCl to it and produce the vesicle suspension.

・Microscopic observation

The prepared vesicles were detected by a fluorescence microscope (Olympus).

The inside of the vesicle was labeled with calcein that shows green fluorescence and the membrane was labeled with rhodamine that shows red fluorescence.

・Purification of plasmids

We transformed the plasmid pET-lacZ to the E. coli cells (JM109) by heat-shock method according to manufacturer’s protocol.

The E.coli cells contain the plasmids were selected according to the resistance to ampicillin.

The overnight culture were collected and the plasmids were purified with the QIAGEN plasmid purification kit,

and were confirmed by electrophorases with a 0.8% agarose gel.

･Measurement of DNA concentration

The concentration of the purified plasmid was measured using a fluorometer (Quantus, Promega) and the DNAONE kit, according to the manuafacter’s protocol.

・Cell-free translation[2,3]

A component constituted cell free translation system, PURE system (PUREfrex®2.0, Gene Frontier), was used in the present study.

The translation reaction (50 μl) was performed at 37°C for 3 hours, according to the manufacturer’s protocol.

The translation product was checked enzymatic activity assay.

･Enzymatic activity assay

The activity of the gene product of lacZ, β-galactosidase, is evaluated by enzymatic assay, asβ-galactosidase,

catalyzes the substrate ONPG to o- nitrophenol, which can be detected at OD420.

50μl of the translation mixture was added to 60 μl of 4 mg/ml ONPG and 490 ml of water.

The resultant mixture was loaded to to a 96-well plate (corning), and the reaction was performed at the room temperature.

The enzymatic reaction was monitored with a plate reader (Epoca 2, BioTek) and measured every 5 minutes for one hour.

・preparation of the sequence of the switch[4]

The sequence of the gene expression switch was partially from the E. coli genome.

PCR amplification was perform to acquire the regulatory sequence and the rpsO gene,

with the primers of 5’-TTCCAGTGAATTGCTGCCGTC-3’ and 5’-TGGTTGAATACCCGGCGTAATG-3’ (Thermo Fisher Scientific).

The PCR reaction was carried out using Taq DNA polymerase (SIGMA) and with a thermal cycler (Dice Touch, TaKaRa).

The PCR product contained the structural gene rpsO and the self-regulation sequence, which functioned as a switch based on its secondary structures at RNA level.

The PCR products of this reaction was stained by Novel juice, and electrophoresis was performed using 0.7 % agarose gel.

The 100 bp DNA ladder (TaKaRa) was used as electrophoresis ladder.

References

[1] Sugiura S, Kuroiwa T, Kagota T, Nakajima M, Sato S, Mukataka S, Walde P, Ichikawa S: Novel method for obtaining homogeneous giant vesicles from a monodisperse water-in-oil emulsion prepared with a microfluidic device. Langmuir. 2008 May 6;24(9):4581-8

[2] Shimizu Y, Inoue A, Tomari Y, Suzuki T, Yokogawa T, Nishikawa K, Ueda T: Cell-free translation reconstituted with purified components. Nat Biotechnol. 2001 Aug;19(8):751-5

[3] Shimizu Y, Kanamori T, Ueda T: Protein synthesis by pure translation systems. Methods. 2005 Jul;36(3):299-304.

[4] Claude Philippe, Flore Eyermann, Lionel Bmnardt, Claude Portiert, Bernard Ehresmann, and Chantal Ehresmann: Ribosomal protein S15 from Escherichia coli modulates its own translation by trapping the ribosome on the mRNA initiation loading site Proc. Natl. Acad. Sci. USA Vol. 90, pp. 4394-4398, May 1993 Biochemistry