Biology Reference
In-Depth Information
biohydrogenation of xylose and cellobiose to xylitol.
8
This example, along with others like
it,
9
11
showcases the freedom of design for building metabolic networks from the ground
up using cell-free systems. In another example, cell-free circuits can be built and controlled
by the addition of enzymes to the nucleic-acid-based circuits to control their rates of
synthesis and degradation.
12
In the case of cell-free protein synthesis, the open
environment enables simplified preparations of PCR products for protein expression.
Expression templates can be directly added and the concentration controlled for
optimization. In one example, linear expression templates (LETs) prepared using PCR
have been effectively used in cell-free translation systems by multiple research
groups.
13
15
LETs obviate the need for time-consuming gene-cloning steps, accelerating
process and product development pipelines. The ability to use LETs in the cell-free
system is particularly valuable when the goal is the production of a large array of gene
products, such as in genomic studies.
14
Direct Product Access
In cell-free systems, the lack of cell walls or membranes facilitates online product
monitoring, one-step purification and recovery of DNA, RNA, or protein.
16,17
In the case of
cell-free protein synthesis, several single-step purification processes using affinity
chromatography have been demonstrated.
16,18
These include strep- and his-tag purification
and in situ product purification using magnetic affinity beads.
19
In another illustrative
model, Bujara et al. coupled cell-free metabolic engineering to mass spectrometry analysis
for direct profiling of rate-limiting steps to optimize multienzyme catalysis of
dihydroxyacetone phosphate (DHAP) from glucose.
20
Focusing Biological Machinery on a Single User-Defined Objective
Without the need, or ability, to support ancillary processes required for adaptation and
growth, cell-free systems offer the ability to focus metabolism towards the exclusive
objective of the engineer, and not the cell. This affords efficiency advantages necessary for
producing complex biochemical products.
279
Accelerating DesignBuildTest Cycles
Engineering biology today is a time- and money-intensive process. Therefore, one of the key
objectives of synthetic biology is to accelerate the design
test loops required for
engineering biology. Right now, in vivo approaches take, on average, 3
build
4 months to
complete this cycle (Dr. Alicia Jackson, DARPA
Industry Day 2011,
personal communication). Much of this time is taken to identify and modify potential
genes, and assemble and synthesize the potential pathways in living organisms.
However, the design cycle limit of in vivo synthetic biology projects could at best
potentially scale (in the future) as the growth rate of the organism and time for
transformation. In cell-free systems, the design cycle is not limited by how fast cells
reproduce. Rather, it can be faster, potentially approaching the limit of synthesizing the
components (DNA, RNA, proteins). Ultimately, cell-free systems may therefore speed up
the design cycle for engineering by more than 10-fold relative to in vivo approaches,
and could be used as their own biomanufacturing platforms, or as feedback in the
design of in vivo platforms.
'
Living Foundries
'
Decreased Effects of Toxicity
Due to cell death and hindered cell growth, in vivo production of both cytotoxic products
and products synthesized from cytotoxic substrates is a challenge. Unhindered by cell
viability constraints, cell-free systems address this challenge. Over the last decade,
researchers have demonstrated the use of cell-free systems to produce a growing number of
cytotoxic products.
21
These include proteins with cytotoxic amino acids,
22,23
cytolethal
