Biology Reference
In-Depth Information
between cell growth (catalyst synthesis) and production
(catalyst utilization) through the use of cell-free systems
provides efficiency benefits for how we use synthetic
biology for synthetic chemistry. In short, cell-free
systems provide an unprecedented freedom of design to
modify and control biological systems.
Cell-free systems can be generated from crude extracts or
purified components. Crude extracts are prepared by lysing
cells (typically harvested in the exponential growth phase)
and are cheaper, as well as simpler to produce than purified
systems. While crude extract-based systems are prominent
in the field and have now been commercialized, 2 purified
systems also have their merits. Specifically, the fact that
every component of a purified system is well-characterized
and can be tuned provides advantages for engineering
design. Furthermore, purified systems lack unwanted
activities (such as proteases and nucleases) that might be
deleterious to crude extract systems. An example of a
well-utilized purified system is the PURE system for
cell-free translation, pioneered by the Ueda group. 3
FIGURE 15.1
Cell-free synthetic biology
applications: The various
applications of cell-free
synthetic biology covered in
this chapter are indicated.
Scale has long been considered a technological limitation of cell-free systems. Due to recent
advances in cost-effective high-level protein synthesis, 4 E. coli -based cell-free protein
synthesis systems have been successfully scaled-up to the manufacturing scale. 2 Production
rates, production yields, and product activity were maintained over a volumetric expansion
of 6 orders of magnitude. 2,5 This pioneering advance has transformed cell-free systems from
a foundational bench-top tool into an enabling technology with promise for commercial
scale applications.
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ADVANTAGES OF CELL-FREE BIOLOGY
Cell-free technologies simplify biological systems and remove unnecessary overheads for
engineering efforts. In this section, we highlight some of the many advantages gained by
removing physical barriers and decoupling cellular catalysts from the genetic architecture of
the cell.
Direct Environment Control of an Open System
Cell-free biology enables direct access to and control of the biological environment. In
living cells, the organism must maintain an environment conducive to every biocatalytic
mechanism necessary for survival. In contrast, the cell-free environment can be directly
optimized for a user-defined objective. The indispensable microbiology workhorse of
polymerase chain reaction (PCR) is a simple but powerful cell-free synthetic biology
example where the DNA polymerase, template, primer, and salt concentrations are directly
controlled and optimized in a nuclease-free environment. 6 In another useful but simple cell-
free example, RNA transcription can also be directly optimized while maintaining an RNase-
free environment. 7 In cell-free protein synthesis, the capability to directly control variables
such as ionic strength, pH, redox potential, hydrophobicity, and enzyme and reactant
concentrations has provided extraordinary advantages.
Direct Influence of Reaction Networks
Open cell-free biology enables the addition or removal of catalysts and/or reagents to
directly influence reaction networks. In one example, Zhang and coworkers have
demonstrated the optimized combination of 12 enzymes for 99.6% energy-efficient
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