Membrane-Embedded Channel of Bacteriophage Phi29 DNA-Packaging Motor for Translocation and Sensing of Double-Stranded DNA - Nanopores: Sensing and Fundamental Biological Interactions - page 82

Fig. 4.2 Purified reengineered connector formed arrays, which was observed by EM (a, b) and
AFM (c). (d) Coomasie-blue stained SDS-gel showing native and reengineered connectors.
Figures reproduced with permissions from: (a, b, d) Ref. [ 42 ], # Nature Publishing Group;
(c) Ref. [ 43 ], # The Royal Society of Chemistry

and authentic motor configuration was verified through its competency to package
the double stranded DNA after incorporation into procapsid (Sect. 6.1 , Fig. 4.15 )and
to assemble the resulting DNA-filled capsid into infectious phi29 virion.
To prepare the connector reconstituted lipid vesicles, lipid stock solutions of
1,2-dioleoyl- sn -glycero-3-phosphocholine (DOPC) or 1,2-diphytanoyl- sn glycerol-
3-phosphocholine (DPhPC) in chloroform (Avanti Polar Lipids) were syringed
into a glass vial, dried under a stream of nitrogen, and placed under vacuum
overnight to generate a lipid film [ 42 ]. After removal of chloroform, the lipids
were rehydrated in a buffer containing 200-300 mM sucrose (to create a high
osmotic pressure) and co-incubated with the connector. Such incubation provides
an opportunity for the hydrophobic layer to interact with the hydrophobic domain
of individual lipid molecules by hydrophilic-hydrophobic two-layer alignment.
The dehydration-hydration method led to the production of giant liposome up
to 50 m[ 47 ]. Small unilamellar vesicles (100 nm or 400 nm) were then gene-

rated by extruding through polycarbonate membrane filters for single channel
measurements.

m