"Experiments & Results"

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Marimo gel

Preparation of spinach thylakoid membranes

All purification procedures were performed at 4 ^oC. Thylakoid membranes were prepared from spinach
leaves by the modified method of Yu et al. The spinach leaves (～160 g) were washed with deionized water
and homogenized in 500 mL of homogenization solution (0.3 M sucrose, 20 mM NaCl, 5 mM MgCl₂, 50 mM Tris-HCl,
pH was adjusted to 7.6) for 40 sec using an AM-10 homogenizer (Nihon Seiki Seisakusho, Japan). The homogenate was filtered
through four-time folded gauze. The flow through was suspended in 400 mL of a high ionic strength buffer (10 mM Hepes-KOH,
150 mM NaCl, pH8). The suspension was centrifuged at 10,000g for 20 min and the precipitate was resuspended
in 15 mL of homogenization solution including 5% DMSO and flash frozen in liquid nitrogen, and stored in liquid nitrogen.

(Yu A. H. C; Hosono K. Biotechnol. Lett. 1991, 13, 411.)

Preparation of Marimo-gel by N₂ blow

Experiment

This procedure is based on the method described by Paul F. et al. and Zekorn T. et al. A 0.3 mL of suspension of
thylakoid membrane containing 1.41 mg protein/mL and 2.7 mL of a 2%(w/v) sodium alginate solution (100 mM K₃PO₄,
100 mM MgCl₂, 100 mM NaCl, 500 mM PIPES, 200 mM ADP, pH was adjusted to 7.6) were placed in a 1 mL syringe.
Using syringe pump (HA2000P, Harvard apparatus, US), the alginate solution including thylakoid membrane was slowly ejected
from the needle and was blown by a nitrogen gas. The tear shaped green droplet firstly encountered mineral oil phase
and transformed into globular shape. Then the droplet sunk into the second phase, which contains 50 mM BaCl₂
and cross-linkage of alginate with barium occurred. Because leak was not observed even after few weeks from the encapsulation,
the cross-linked alginate mesh seemed to be enough small to support thylakoid membranes.

(Paul F.; Vignais P. M. Enzyme Mcrob. Technol. 1980, 2, 281.)
(Zekron T.; Horcher A.; Siebers U.; Schnettler R.; Klock G.; Hering B.; Zimmermann U.; Bretzel R. G.; Federlin K.
Acta Diabetol, 1992, 29, 99.)

Results

From the photograph and histogram, it is evident that Marimo-GEL of large size (>300 µm diameter) could be prepared,
which not small enough by introducing into the flow cell.

Preparation of Marimo-gel by using micro emulsion

Experiment

0.2 g/mL ER290 surfactant in paraffin oil 1000 µL was added on the mixture of 2 wt % alginate 100 µL and
2.1 mg/mL thylakoid membrane 100 µL in the brown bottle. The brown bottle was vortexed to form micell which was composed of
the mixture of alginate and thylakoid membrane. The micell in the dispersion liquid was added on the 50 mM BaCl₂ solution in a
microcentrifuge tube. When the tube was centrifuged, Marimo-GEL was formed due to cross-link between alginate and Ba²⁺ ions.

Results

From the photograph and histogram, it is evident that Marimo-GEL of very small size (<100 µm diameter) could be prepared,
which small enough by introducing into the flow cell.