FORMATION OF TETHERED LIPID BILAYER BY VESICLE
FUSION OF E COLI POLAR LIPID EXTRACT – THE EFFECT
OF SALT CONCENTRATION
In this report, I am going to report the effect of salt concentration(100mM vs 500mM) on the
vesicle fusion of E. coli polar extract(mass ratio PE:PG:cardiolipin = 67:23.2:9.8) vesicles to
self-assembly monolayer.
In brief, E coli vesicles were prepared by sonication + extrusion through 100nm porous
membranes at 5mg/ml in 100mM KCl + 10mM HEPES at pH~7. There are 2 sets of vesicles
in this experiment:
(1) vesicles in 100mM KCl
(2) vesicles in 500mM KCl – this set of vesicles were prepared from vesicles set(1) by
diluting it by half by using 900mM KCl so that finally it contained 500mM KCl
It is hard to say what happened to vesicles set(2) due to osmotic pressure. Dynamic light
scattering showed there might be smaller vesicles in vesicles set(2)(data not shown). However
independent experiment showed that the size of vesicles did not matter for vesicle fusion(data
not shown)
The final concentration of vesicles used for all experiments was 1mg/ml
Vesicles solution was injected onto the SAM coated with FC16:beta-ME = 1:1. The excess
vesicles were rinsed off after 2.5 hours by using 100mM KCl and all impedance
measurements were done in 100mM KCl.
The impedance spectra is fitted to an equivalent circuit in figure 1. The reported values are
without normalization. To obtained normalized values, capacitance has to multiplied by 3
while resistance has to be divided by 3.
Figure 1: Equivalent circuit for fitting of impedance spectra
(1) tBLM formed with vesicles in 100mM KCl(fitting from 30 to 30kHz):
αof CPE
CPE of tBLM
(x 10e-7 Farad)
2.77  0.01
2.41  0.01
2.26  0.01
2.17  0.01
1st sample
2nd sample
3rd sample
4th sample
0.971  0.001
0.987  0.000
0.978  0.000
0.987  0.000
Resistance
(x kilo ohms)
125  2
255  1
185  3
170  3
(2) tBLM formed with vesicles in 500mM KCl(fitting from 1 to 30kHz):
αof CPE
CPE of tBLM
(x 10e-7 Farad)
2.16  0.00
2.19  0.00
2.22  0.00
2.15  0.00
5th sample
6th sample
7th sample
8th sample
0.997  0.000
0.997  0.000
0.997  0.000
0.998  0.000
108
1st sample.z
2nd sample.z
3rd sample.z
4th sample.z
5th sample.z
6th sample.z
7th sample.z
8th sample.z
7
10
|Z|
106
105
104
103
102
10-1
Resistance
(x mega ohms)
5.97  0.08
4.32  0.05
5.01  0.06
6.85  0.10
100
101
102
103
104
105
104
105
Frequency (Hz)
theta
-100
-75
-50
-25
10-1
100
101
102
103
Frequency (Hz)
Figure 2: Impedance spectra of tBLM formed with vesicle fusion of E coli vesicles. The 1 st to 4th
samples were formed in 100mM KCl while the 5th to 8th samples were formed with 500mM KCl