International Workshop on Soft Matter Physics (ISSP/SOFT 2010, 24th Aug 2010)

Cascade Dynamics Observed in
Self-division of Giant Vesicle
Containing Amplified DNA
T. Sugawara1, 2, K. Kurihara1, T. Toyota1, 2, K. Suzuki1, 2
1. Dept. of Basic Sciences, Graduated School of Arts and Sciences
2. Research Center of Life Sciences as Complex Systems
the University of Tokyo, Japan

Missing Link in History of Life
3.5 billion years

present

Inorganic
Material

Inanimate

Amino Acid

Missing Link

Coacervate

Prokaryote
Bacteria

OrganicMolecule
A. Oparin
S.L. Miller

Ancient Earth?

AnimateLipid

RNA / DNAArchaeaEukaryote

Search for the Missing Link
RNA world

Mysterious
Evolution
Jump(the Missing Link)

Lipid world
Protein world

LifeDNA(MW: 107-1013)

VesicleProtein(MW: 5000-150000)

Constructive Approach to Origin of Life・Prebiotic : Construct a “Minimal Cell” using prebiotic materials
・Semi-synthetic : Reconstruct a “Minimal Cell” using purified biomaterials
obtained from a living cell

“Synthesizing Life” : J. W. Szostak, D. P. Bartel, P. L. Luisi, Nature 2001

Giant Vesicle-based Artificial Cell

Giant Vesicle-based Artificial Cell

Self-reproduction and Non Self-reproduction

Non Self-reproduction

Up-takes the same membrane
molecule as the one whichconstructs itself

Self-reproduction

Up-takes a membrane precursor
and converts it to the membrane
molecule of itself

P. L. Luisi, "The Emergence of Life”, Cambridge University Press

Membrane Molecule with Imine Bondhydrophobic

Imine Bond

H2 O

H2 O

hydrophilicCondensation

Hydrolysis- Morphological Change- Spontaneous Movement

・Bond formation in hydrophobic environment
・Hydrolysis in hydrophilic environment

Robust Self-reproducing Giant Vesicles

V*: Bolaamphiphile

E: Electrolytehydrolysis
CV: Lipid

C: Catalyst

Giant Multi Lamellar Vesicle

K. Takakura, et al., Langmuir 20, 3832 (2004)

Movie of Self-reproduction of Giant Vesicles

Dynamics of Robust Self-reproducing Giant Vesicle

Plausible mechanism of quasi-equal division
Prof. T. Umeda (Kobe Univ.)

A GMV has the following energy:

wherem : number of lamellas
Ai : area of the i-th lamella
Ni : number of molecules in the i-th lamellak : elastic constanta : area per molecule

Theoretical model of Self-reproducing Giant Vesicle
Assumptions
1. A GMV has the following energy:(1)wherem : number of lamellas
Ai : area of the i-th lamella
Ni : number of molecules in the i-th lamellak : elastic constanta : area per molecule

2. Amphiphiles are added to the surface of
the GMV at a constant rate (p) per area,and diffuse into inner lamellas.
3. The distance between two adjacentlamellas is fixed when the shape of theGMV changes.
4. Energy minimization determines thedirection of the shape and topology
changes of the GMV.

MovieIntrusion of Membrane Precursor into Giant Multi
Lamellar Vesicle

Role of “ E” in Self-division
Hydrolysis of Membrane Precursor+Bolaamphiphile Membrane Precursor (V*)

E0 min.

1.5 min.

CMembrane Molecule (V)Electrolyte (E)

EVE

10 µm

6 min.

3 min.

A Large Number of GVs are Self-reproducing !
Initial Sample

After self-reproduction

V*
10 µm

10 µm

Mass-scale analysis of self-reproducing GVs is important.
FCM : Flow Cytometry

Flow Cytometer

FCM
Fluorescence
Detector

FLFS

Forward Scatter Detector

Ar Laser
488 nmFL (Amount of Catalyst) Frequency of FS

Fluorophore containing GV(Number of GVs)

FCM Analysis on Self-reproducing GVs

10 mol% Catalyst containing GVDetecting Number = 104
Frequency of FL(Number of GVs)

104103102
101
100100101102103

104

FS (size of GV) [1-10 µm]

FS: Size of GV, FL: Florescence intensity of fluorophore per GV

T. Toyota, et al., Langmuir 24, 3037 (2008)

Size-Calibration of GVs by Filtration
Membrane Filter

Initial Sample

Amount of Catalyst (au)

Initial Sample

12 µm Filtration

3 µm Filtration

1.2 µm Filtration

104104104104103103103

103

102

102

102

102

101

101

101

101

100
100

101

102

Size (au)

103

104

100
100

101

102

Size (au)

103

104

100
100

101

102

Size (au)

103

104

100
100

101

102
103
Size (au)104Particle Size Distribution: 0.2 – 1.0 µm(DLS analysis)

Calibration of Amounts of Catalysts
C = Cn + 0.1Cf

Cf: Fluorescence Catalyst

Cn: Non-fluorescence Catalyst

Fluorescence Intensity (au)[Cf] @GV = 1 mol%

0.1 mol%

104

104

104

103

103

103

102

102

102

101

101

101

100
100

101

102

Size (au)

103

104

100
100

101

102

Size (au)

103

104

100
100

0.01 mol%

101

102
Size (au)

103

104

Distribution of fluorescence intensity depends on the amounts of Cf.
Distribution of size dose not depend on the amounts of Cf.

How to read 2D plot

Amount of contained catalyst per GV

Size: Const.
Cat.: Up.

Size: Up
Cat.: Up.

Fusion ?
Size: Down
Cat.: Const.

InitialShrinkSize: Down
Cat.: Down

Size: Const.
Cat.: Down

Fission ?
Size of GV

Size: UpCat.: Const.

104

Initial Sample

103

4 mL

102

V*: 5 mM
E: 25 mM

101
100
100

Amount of Catalyst (au)Amount of Catalyst (au)Self-reproduction after 1st Feeding

1 mL
101102Size (au)

103

104

V: 5 mM
C: 10 mol%
E: 25 mM

Stirring 2 h104
103
102
101
100100101

102
103
Size (au)Distribution of size:
almost the same but larger part disappeared
Distribution of catalyst:
one eighth, (1/2)3104

Robustly Self-reproducing GV is Realized !
Initial StageAfter

1st Feeding

Precursor Solution

After Recharging
of Catalyst

Catalyst Solution
2 hr

After

2nd Feeding

Precursor Solution

4 hr102-fold呍 !
Increased呍呍呍呍呍

Construction of Self-reproducing GVs
Tolerant to a Highly Ionic MediumHydrolysis of membrane precursor V*
hydrolysis+CV*

VE

phase-contrast microscopic image
before addition of V*

Excess size
Amount of C

Addition of V*

50 µm50 µm

4h after addition of V*

Size of GV

22

Vesicular Size(b)

Addition ofprecursor

Vesicular Size

Amount of Catalyst

SortingAmount of Catalyst

Amount of Catalyst(a)Sorting of Self-reproducing GVs
Tolerant to a Highly Ionic Medium(c)

Vesicular Size

Irregular ensemble of giant
vesicles with diameter of over
20 µm, which was always arisen
at swelling, was eliminated bysorting.

Distribution after addition ofmembrane precursor was
New vesicular ensemble R grew !
discrete and similar to that
K. Kurihara, et al., Soft Matter 6, 1888 (2010)of original one.
→ P202 (25th Aug)

Giant Vesicle-based Artificial Cell

Giant Vesicle-based Artificial Cell

Enzymatic Reaction in Vesicle

・Polyadenine Synthesis (LV): P. Walde et al., JACS, 1994.
・Polymerase Chain Reaction (LV): T. Oberholzer et al., Chem. Biol., 1995.
・Protein Synthesis (LV): P. L. Luisi et al., BBRC, 1999.
・Transcription Reaction (GV): K. Yoshikawa et al., Langmuir, 2001.
・Genetic Network (GV): T. Yomo et al., FEBS Lett., 2004.

Amplification of Information Molecule in GV
DNA polymerase
Mg2+PCR

DNA as an Information Molecule

Competition between sufficient andinsufficient vesicles in respect ofcontents of replication reagents.
DNA can be amplified by PCR method easily.

Tunings for PCR in GV
Stabilization of Vesicular Membrane

Distribution of Template DNA in GVs“Size Effect”

5%

3 µm

1 µm

~1 DNA~0.03 DNA
10µm10µm[Template DNA] = 0.1 nM

in highly ionic aqueous dispersion

Encapsulation by a freezing-thawing method

Purification of template DNA

Two-Step PCR

1229 bps expresses GFP

denaturex20

94 ºC

annealing elongation

Nested PCR

plasmid

68 ºC

RT

Purified template DNAGeneral(3 steps)

68 ºC

RT

Hydrothermal vent in the deep sea

Replication of DNA in Giant Vesicles

DNA amplification
by PCR

POPC, DSPE-PEG5000, Cholesterol(95 : 5 : 10)

Template DNA

denature @ 98℃

Mg2+

KOD –plus(DNA polymerase)

Template DNA(1229bp)

annealing @ 68℃, 15 sec
PrimerPrimers

SYBR® Green I(dsDNA indicator)

DNase Ielongation @ 68℃, 60 sec
Polymerase

Polymerase Chain Reaction

DNA & Ions
EthanolPrecipitation

After PCR Solution
Ca2+DNA polymerase

Mg2+

Lipids&Enzymes
1882 bp1489 bp

Negative Control II(without PCR)

hydrolyzed by DNase, DNA in GV isprotected by membranes.
Phenol & Chloroform
extraction

Negative Control I(without Template)

Ca2+(Ladder Marker)

DNase I / Buffer Although DNA in outer water phase isTemplate & PCR

Extraction of Replicated DNA for Gel Electrophoresis

Length ofthe template
DNA is 1229 bp.

925 bp

Condensed DNA Solution

PAGE

200 mA, 50 V60 min.
SYBR Green I

DNase I

Template DNA was amplified accurately.

Fluorescence intensity of a vesicle

Before PCR

PCR cycles

Size of a vesicle (FS)1

1

FL = 120

Fluorescence intensity of a vesicle

Population Analysis of the Performance of PCR in GVs
After PCR (20cycles)

Size of a vesicle (FS)・A group having a local maximum at
FL=120 appeared after 20-PCR cycles.
・Fluorescence intensity of GV (DNA)increased by ca.100 times (average)
・Ratio of vesicles to replicate DNA is
9.2% (FL>25)

Size of Giant Vesicle vs. Ratio of PCR-Performed GV
Fluorescence intensity of a vesicle

104
103

20 times

102

The survival race has already started !

ex) Mean number of capsulated DNA in GV
10 µm GV
30 DNAs
5 µm GV
4 DNAs0 times
1 µm GV
0.3 DNAs
FL = 40

101
100
100

101

102

103

104

Ratio of PCR-performed GV: Av. 10%

Influence of Lamellarity of GVs on the ratio of PCR-PerformedGVs

10 µm

K. Sato, K. Obinata, T. Sugawara, I. Urabe,T. Yomo, J. Biosci. Bioeng., 102, 171-178 (2006).
Even a similar sized vesicle can have various
effective volumes due to the internal structure.

Meaning of Enzymatic Reaction in GV

1) Entrapment of precious component
2) Effective volume in GV –Lamella structure3) Electrostatic interaction between membrane
and ingredients
4) Lamella structure and PCR performance