Biology Reference
In-Depth Information
TABLE 14.2
Experimental Advances in SSMC Studies
Entry
Year Description
References
1
2004 Two-step genetic cascade (T7 RNA polymerase and later GFP)
21
2
2004 Expression of a pore-forming protein (
-hemolysin) and
prolongation of protein synthesis up to 100 h
α
22
3
2008 Self-replication of RNA template by Qβ-replicase, which is
expressed in situ from the RNA template itself. Production of
β-galactosidase after translation of the complementary RNA
strand
23
4
2009 Production of two functional membrane enzymes for the two-step
synthesis of phosphatidic acid (a membrane component)
24
5
2011 DNA amplification (by PCR reaction) and vesicle self-reproduction
by the external addition of a precursor
25
6
2012 Synthesis of cytoskeleton proteins (MreB and MreC)
26
encapsulated. Several lipid vesicle preparation methods shared this strategy. Most of the
studies reported above have been carried out by forming lipid vesicles by hydrating thin
lipid films obtained directly from lipids or by freeze-drying preformed liposomes. The
injection method has also been occasionally used (this method consists of the injection of
lipids, as ethanol solution, in an aqueous solution). These methods are not only classical
methods from the viewpoint of liposome technology, but are also good models of how
lipid vesicles originated in primitive times. One typical scenario, in fact, foresees that lipid
vesicles formed in aqueous solution where a sort of rudimentary metabolism was already
established. Thanks to the encapsulation of these molecules, a primitive cell emerged from
the primitive
It is thought that the confinement into a semipermeable membranous
compartment not only protected the primitive metabolic network from dilution, parasitic
reactions, and interfering reactions, but also allowed the establishment of gradients that
could be ultimately used for generating chemical energy (e.g. ATP), and, fundamentally,
allowed the formation of a unit that could behave autopoietically.
'
soup.
'
268
But what is really known about the physics of solute encapsulation, especially in the case of
multicomponent systems, and in the case of diluted solutions (as it is expected to be the
primitive
)? Until a couple of years ago, it was taken for granted that encapsulation of
solutes did not represent a critical aspect of the emergence of primitive cells
soup
'
'
the argument
was simply not discussed in the literature. Our investigation on the physics of solute
entrapment stemmed from a study on the minimal physical size of primitive cells.
27
Previous studies on protein synthesis
like those reported above (see also
11
for a more
complete review)
were carried out by constructing SSMCs by means of large lipid vesicles,
namely, with diameters above 800 nm. However, together with a minimal biochemical
complexity, a minimal cell could also feature a minimal physical size, which would perform
better than a large one because of the limited volume that would facilitate reactions
between a few components. Moreover, smaller vesicles could be physically more stable than
larger ones, and need less time (and less material) to self-reproduce.
We approached the issue of minimal cellular size by attempting to synthesize a protein
inside conventional lipid vesicles with diameters of 200 nm. We reasoned that the
complexity of the transcription/translation machinery well represents the complexity of
primitive cells. The size of 100 nm was chosen on the basis of theoretical considerations,
elaborated by biophysicists, chemists, and biologists.
28
POPC vesicles were prepared
by
injection methods
in a solution containing all the molecular components (i.e. the PURE
system) needed to synthesize a reporter protein (enhanced GFP, eGFP). As we have
remarked above, the PURE system contains about 80 macromolecules (actually 83) and
several small molecules (nucleotides triphosphate, amino acids, salts, etc.). When needed,
