Biology Reference
In-Depth Information
SSMCS AS A BIOTECHNOLOGICAL TOOL
As we have described in the previous sections, great emphasis has been given to the self-
assembly events underlying the formation of minimal cells from separated compounds,
freely available in solution. This somehow reflects the spirit of the pioneers in this research
field, born within the origin-of-life community. However, also considering the observations
described earlier, in order to fully develop an
as a more efficient
(controllable and reproducible) way of producing solute-filled vesicles would be
advantageous. In other words, the fact that only few vesicles are able to encapsulate a
complex reaction mixture is seen as an intriguing phenomenon to be studied by scientists
interested in emergent behaviors and self-organization, but can be seen as a problem by
bioengineers trying to build SSMCs for biotechnology.
SSMC technology
'
'
Inspired by these considerations, we started a thorough investigation on a new and very
interesting vesicle formation method, which was firstly described by the group of Weitz in
2003.
31
The method, that can be referred to as the
method, is particularly
useful for preparing lipid vesicles (giant lipid vesicles, or GVs, with typical diameters from
1 to 100
droplet transfer
'
'
μ
m) filled with a desired content, from simple
one-solute
to complex
'
'
solutions. The produced GVs can be transformed in submicron vesicles by
extrusion. The droplet transfer method starts with the formation of lipid-stabilized water-in-
oil (w/o) droplets. These are easily obtained by dispersion of a small volume (e.g. 10
multisolute
'
'
μ
L) of
a water solution into hydrocarbons (e.g. 500
μ
L). Lipids, which are dissolved in the
hydrocarbon (the
), will stabilize the microscopic w/o droplets by forming an oriented
layer (ideally, a monolayer) between the water and the hydrocarbon. In the dispersion
process, a macrodroplet of the aqueous solution is transformed into millions of
microdroplets by a mechanical shearing force (e.g. pipetting, vortexing, etc.). Typically,
several thousands of w/o droplets/
oil
'
'
L of oil are produced from this fragmentation
mechanism, each containing a portion of the solution of interest. Due to the large volume
of each droplet (of the order of pL), stochastic factors are partially smoothed out (however
they are not eliminated, as evidenced by the fact that the content of each w/o droplet is
unique: there is indeed a distribution of the solute concentration among the droplets,
whereas in the ideal case the solutes should have the same concentration in every droplet).
μ
272
After their formation, w/o droplets can be transformed into GVs by the transfer process. This
consists of letting the w/o droplets cross an oil
water interface where a lipid monolayer is
stratified. In this way, the w/o droplet, covered by a lipid monolayer, becomes coated by
another lipid monolayer and acquires in this way the lipid bilayer that characterizes a vesicle.
In practical terms, the w/o emulsion is stratified over an aqueous phase. W/o droplets,
present in the upper phase tend to sediment toward the bottom of the tube, and after
traveling in the oil, reach and cross the oil
water interface, and become transferred into the
bottom aqueous phase as GVs. Centrifugation is often used to speed up the process. Not
all w/o droplets are successfully transformed in GVs. Actually, most of them break during
the oil
water interface crossing, and release their content in the bottom aqueous phase.
However, when a GV forms, it contains all the solutes initially present in the w/o
droplet. This makes the droplet transfer method so attractive for SSMC technology. We
have recently optimized this method for producing oleate/POPC-containing GVs and
characterized the process from several viewpoints. The droplet transfer method has been
used for producing GVs that contain the complex transcription/translation kit.
22,26
Due to
its superior entrapment efficiency, we believe that the droplet transfer method will
become the method of choice for all those applications where SSMCs are used as a
biotechnological tool.
Thinking of future developments, together with the cell-free and liposome technologies,
which dominate the current approaches to SSMCs, we think that microfluidic technology
will also impact very much on the use of SSMCs for biotechnology. Several microfluidic
