Biology Reference
In-Depth Information
FIGURE 15.5C
Creation of pores in the membrane relieves limitation on nutritional and energy requirements: a cell-free transcription and
translation system is encapsulated in a lipid vesicle along with DNA encoding GFP and α-hemolysin. Translated α-hemolysin
protein is then able to form a pore through the lipid membrane, thereby allowing exchange of nutrients and toxic byproducts
with the surrounding feeding solution. Creation of the pore allows for longer expression of synthesized protein as measured
by GFP production. The lipid bylayer is indicated by the black dashed line.
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Cost Reduction
Cell-free biology offers remarkable opportunities for engineering biology. However, until
commercial products (e.g. proteins and metabolites) are made using cell-free systems, these
systems will mainly serve as test-beds for understanding biology and accelerating the design
of synthetic programs. To enable commercial applications, costs must continue to be
reduced. Cells have evolved many pathways to reliably produce energy from inexpensive
raw materials. In contrast, more expensive energy sources are typically used in cell-free
systems. An overarching goal for cell-free biology is to develop strategies for using the same
energy substrate molecules and waste feedstreams that are used to fuel in vivo synthetic
biology efforts. Bottom-up approaches will benefit from more efficient methods for rapid
and cost-effective protein production and purification, while more efficient and cost-
effective extract preparation methods are desired for top-down cell-free systems. With the
E. coli
-based cell-free protein synthesis system, engineering the extract preparation method
decreased the extract preparation time and reagent cost by over 50%,
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139
and similar
advances are expected to improve other top-down cell-free systems. Further advances will
likely couple high-throughput screens to characterize and assess the functions of proteins
found in the extract. In one illustrative example, Swartz and colleagues recently surveyed
endogenous proteins in
E. coli
for their influence on CFPS. Using this information, they
were able to improve CFPS yields up to
4 g/L in an 8-hour batch reaction.
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B
Scale
With the exquisite control the open cell-free system enables, researchers have demonstrated
the scalability of the cell extract-based cell-free protein synthesis reaction from the microliter
