Biology Reference
In-Depth Information
The first step in SSMC research was done in 1991, when extension of the initial studies on
vesicle self-reproduction
15
to phospholipid vesicles was attempted. The biochemical
machinery for phosphatidylcholine production, composed of four enzymes working
sequentially, was inserted inside phosphatidylcholine vesicles, aiming at producing lipids
from internal reactions at the expense of lipid building blocks.
18
The low chemical yield did
not allow the observation of the desired behavior. Next, it was demonstrated by our group
that RNA and DNA could be replicated inside lipid vesicles, by means of an RNA template
and Q
-replicase, and a DNA template and DNA polymerase, respectively. Moreover, the
intravesicle production of poly(adenylic acid) was obtained from ADP and polynucleotide
phosphorylase. Finally, in 1999 we reported for the first time the ribosomal production of a
polypeptide (poly(phenylalanine)) inside lipid vesicles. Details and references to these and
other studies on biopolymerization reactions inside lipid vesicles can be found in recently
published reviews.
9,11
β
Time was ripe for the first experimental report on the synthesis of a functional protein (the
green fluorescent protein, GFP) inside lipid vesicles, which was carried out in 2001.
19
After
about 10 years, several advancements have been recorded in this field, but the synthesis of
proteins remains the key ingredient in SSMC research. This can be understood from two
points of view. The first is related to the complexity of this multistep reaction that models
well the complexity of a minimal genetic/metabolic system. The approximately 80 different
macromolecules needed to perform such a reaction (DNA, RNA polymerase, ribosome and
translation factors, tRNAs, aminoacyl-tRNA synthases) already represent an important part
of an autopoietic SSMC. As a consequence, physical and biochemical considerations
obtained by studying the synthesis of proteins inside compartments have a sort of general
implication for the emergence of functional compartments from separated molecules.
Second, the control of protein synthesis allows the development of the next generation of
SSMC, where more complex functions are developed thanks to the in situ production of
proteins (e.g. enzymes, transcription factors, etc.).
265
The construction of SSMCs relies on cell-free technology. Initial reports on protein synthesis
were carried out by encapsulating cell extracts (e.g. typically from
E. coli
) inside lipid vesicles.
Cell extracts, however, although they contain all required molecules for transcription and
translation, are not well characterized in terms of molecular composition, and are not very
attractive from an SB point of view. An important breakthrough in the field of protein
synthesis was achieved by the group of Takuya Ueda, who invented in 2001 the PURE system
(Protein synthesis Using Recombinant Elements), a fully reconstituted molecular system
containing the minimal number of macromolecules required to carry out protein
biosynthesis in vitro, starting from the corresponding coding DNA (or RNA).
20
The PURE
system consists of four subsystems, namely: (1) transcription; (2) translation; (3) amino acid
charging onto tRNA (aminoacylation); and (4) energy regeneration. The composition of the
PURE system is shown in
Table 14.1
. As can be seen, the PURE system is composed of 36
individually purified
E. coli
proteins, highly purified 70 S ribosome, tRNAs (for a total of
about 80 macromolecules), and low-molecular-weight compounds such as NTPs, 20 amino
acids, DTT, spermidine, formyl-tetrahydrofolate, salts, and creatine phosphate as ultimate
phosphate donor. In contrast to cell-extract-based systems, the PURE system realizes a totally
defined condition concerning the number and concentration of its components. This system
can be considered as a standard chassis for synthetic biology (
http://partsregistry.org/
Main_Page
).
There are six significant examples of
systems created by the semisynthetic
approach. In all these examples, the desired function has been achieved by expressing one
or more proteins inside lipid vesicles, and therefore realizing the first steps toward the
construction of an SSMC. The first one
21
deals with the control of genetic expression and
consists of the construction of a two-step genetic cascade inside a lipid vesicle. In particular,
advanced
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