Biology Reference
In-Depth Information
disease toxins, 24 cytotoxic proteins, 25 and others. 26,27 In one exemplary approach, hepatitis
researchers studying a cytolethal distending toxin from Helicobacter hepaticus had been
unable to produce sufficient levels of protein in vivo to observe its mechanism of action
due to its high cytotoxicity. Using a cell-free approach, researchers were able to produce
sufficient quantities to examine the toxin
s effect on the liver. 24 In another illustrative case,
cell-free translation of the cytotoxic A2 protein led to yields almost 1000 times higher than
previously reported with in vivo production. 25
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Expanding the Chemistry of Life
The products of biological systems are governed by the chemistry of life, which is limited to
natural building blocks. Cell-free systems offer advantages for using synthetic biology for
nonstandard chemistry. 28 One of the most prominent examples is using cell-free protein
synthesis for site-specific incorporation of nonnatural amino acids. In contrast to in vivo
systems, there are no transport limitations for getting nonnatural amino acids into the cell,
and there is flexibility for reprogramming the genetic code because cellular viability need
not be maintained. By employing cell-free protein synthesis, the putatively transport-limited
p-propargyloxyphenylalanine was incorporated site-specifically into proteins at significantly
higher production yields than in vivo systems. 29 The high-yielding site-specific
incorporation of azidophenylalanine and the global replacement of methionine by an
unnatural analogue have also been demonstrated with cell-free systems. 30,31
EXISTING TECHNOLOGIES AND APPLICATIONS IN CELL-FREE
SYNTHETIC BIOLOGY
Cell-free synthetic biology projects focus on exploiting and harnessing biopolymer synthesis,
replication, and evolution. To this end, we organize our discussion of existing research
frontiers by levels of the biological hierarchy: nucleic acids; proteins; metabolites; and
minimal cells. Focus is given towards recent efforts in designing and constructing
programmable circuits, advances in synthesizing biopolymers and metabolites, and steps
toward enabling self-replication.
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Nucleic Acids
As the field of synthetic biology has emerged, we are now at a position where both writing
(i.e. synthesis) and reading (i.e. sequencing) DNA are not limiting. Indeed, nucleic acid
synthesis forms the basis for synthetic biology. 32,33 DNA synthesis can be achieved in vitro
by PCR, recombinant DNA cloning, or by chemical synthesis. PCR has extensive
applications in medical diagnosis, cloning, forensics, phylogenic analysis, etc. Like DNA,
RNA can be synthesized in vitro by chemical synthesis. A good review detailing the history
and advances in RNA synthesis is available. 34 RNA can also be transcribed in vitro using
purified T7 polymerase, NTPs, and optimized buffers. A timeline of developments in this
area of oligonucleotide-based cell-free synthetic biology is illustrated in Figure 15.2A , and
some of the main developments in the field are listed below.
NUCLEIC ACID CIRCUITS
Nucleic acid circuits involve programming biological circuits, which connect the fields of
molecular biology and computational science. Over the last five years, there has been a
dramatic growth in reports describing synthetic in vitro circuits. 34a This growth underscores
the importance of cell-free systems as a testing ground for our ability to engineer and
analyze even more complex biological circuits; a testing ground where at least we can
clearly see when and how we fail. Rather than encoding ones and zeros into high and
low voltages, nucleic acid computing involves choosing specific base sequences on
synthetic strands of DNA to process information. Nucleic acid circuits are modular,
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