Why and How?
a cell-free transcription-translation (TXTL) platform to construct
biochemical systems in vitro by executing synthetic gene circuits.
Unlike the other
cell-free expression systems, our platform is based on an E. coli
that uses the endogenous TX and TL machineries. The cicruits (plasmids
or linear DNA) are executed in a cell-free TXTL mix entirely prepared
our lab. This cell-free system is available under the name myTXTL
at Arbor Biosciences
research is based on this unique system and includes: (I) prototyping
regulatory elements and
circuits, (II) quantitative biology of self-assembly with
phages as models, (III) bottom-up
contruction of a minimal cell, (IV) application to
biotechnologies and medicine. Our work is both fundamental and
applied and covers the
research areas of synthetic biology and quantitative biology such as
protein synthesis was developed in the 60s to understand the
process of protein synthesis in living organisms. In vitro protein
synthesis had an immediate impact with the elucidation of the genetic
code (1). In the 70s, DNA-dependent cell-free expression became
a research tool to analyze gene products and to unravel the regulation
of natural genetic elements such as the E. coli lactose (2) and
tryptophan (3) operons. The development of highly efficient hybrid
cell-free expression systems in the early 90s marked a turning point
for this technology (4). Cell-free TXTL
systems, optimized for large-scale protein synthesis as an
alternative to the recombinant protein technology (5, 6), are used in
an increasing number of applications in biotechnology, industry and
the emergence of synthetic biology, a new generation of cell-free TXTL
systems has been engineered. The construction of biological systems in
test tubes using DNA programs provides a means to study biochemical
processes in isolation, with a greater level of control and a greater
freedom of design compared to in vivo. In addition to increasing our
knowledge of the molecular repertoire found in biology, constructing
information-based biochemical systems in vitro offers the possibility
of expanding the capabilities of existing biological systems (10).
Elementary gene circuits (11, 12), pattern formation (13) and
prototypes of artificial cells (14, 15) have been engineered with
cell-free TXTL systems. Cell-free synthetic biology is a rapidly
demonstrated that cell-free TXTL using the E. coli endogenous
TX machinery (core RNAP + housekeeping sigma factor 70) is as
efficient as the conventional T7 bacteriophage systems (16). Methods
to tune the mRNA and protein
degradation rates were added to this system (17) so as to change the
dynamics of expression. A model of cell-free protein synthesis was
published (18), in collaboration with the Bar-Ziv lab at the
Weizamnn Institute of Sciences. We then developed a
that recapitulates the entire transcription scheme of E. coli (12). The
primary sigma factor 70 is used to cascade any of the
six other sigma factors 19, 24, 28, 32, 38, 54-NtrC, as well the T7 and
T3 RNA polymerases. Hundreds of
circuit parts are available from E. coli to design, build and test
synthetic circuits in vitro. We developed new metabolisms to energize
TXTL up to 2 mg/ml in batch mode (19) and published an improved TXTL
toolbox 2.0 (20). The
cell-free TXTL toolbox 2.0 is used to prototype regulatory elements
as riboregulators or CRISPR guide RNAs) and gene circuits in vitro,
such as the multiple stage cascade shown in Figure 1.
1: Left: a switch was
bacteriophage promoters using a commercial kit. The circuit is composed
of 3 plasmids and two stages. In the first stage, the phage T7 RNA
polymerase and the E. coli lac repressor are expressed from SP6
promoters (The phage SP6 RNA polymerase is added to the TXTL
reaction). In the second stage, the expression of the luminescent
reporter protein firefly Luciferase is activated by the T7 RNA
polymerase and/or repressed by the lac repressor. The repression can be
inhibited by addition of IPTG, an inhibitor of the lac
repressor. This circuit was published in (11). Right: a multiple stage cascade
composed of 5 stages. This circuit was constructed with the new all
E. coli cell-free TXTL system specifically developed for cell-free
synthetic biology. The solid arrows show the cascades, the dotted lines
represent the negative feedback due to the competition between sigma
circuit was published in (12) and (20).
we challenged the system by testing large natural DNA programs. First,
determined that the maximum size of the genetic DNA program that can be
a test tube is about 150-250 genes, based on the toolbox 2.0
performances (20). We
expressed the T7 phage (40 kbp,
about 60 genes) and
observed its complete synthesis. In addition we showed that the phage
genome is replicated (21). It is the first time that a living entity
was entirely synthesized in vitro from the expression of its genome.
This work is not limited to the bacteriophage T7, the phages MS2 and
also be synthesized in vitro (20).
2: Left: a schematic
recapitulates the steps of phage expression and synthesis in a test
tube. The phage T7 has its own RNA polymerase and its own DNA
polymerase. We demonstrated that the phage T7 genome was replicated
concurrently with its synthesis. This work was published
in (20) and (21). Right:
microscopy image of T7 phages synthesized in cell-free TXTL reactions
one of the most challenging goals in cell-free synthetic biology is the
construction minimal cells. Different types of cell analogs have
been proposed. Our approach consists of encapsulting the TXTL system
into cell-sized synthetic liposomes. The liposomes are programmed with
gene circuits towards self-reproduction, by achieving cell functions
membrane permeability (14) and
cytoskeleton (22). This approach helps us understand the links
between information, self-assembly and metabolism (23).
3: Left: cell-free TXTL
was used to express the toxin
alpha-hemolysin into cell-sized liposomes. The reporter protein eGFP
was fused to the toxin to visualized the interaction of the
pore-forming protein with
the phospholipid membrane. The toxin forms a membrane channel of 1.3 nm
diameter that allows exchanges of small nutrients and reaction
byproducts between the liposome and the external medium, which results
the extension of TXTL expression inside the liposomes. This work
was described in (14) and (20). Right:
the all E. coli toolbox was used to express the MreB and MreC
cytoskeletal protein inside liposomes at the same time. MreB
polymerizes at the membrane
through its interaction with MreC, a membrane protein. These
observations were published in (19).
Biotechnology and Medicine
collaboration with other labs and companies, we are using our TXTL
system to develop molecules and devices, such as solid state probes,
for application in
biotechnlogies and medicine. We use the fast prototyping capabilities
of TXTL to test and select peptides and proteins with therapeutic
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platform for cell-free synthetic biology. ACS Synthetic Biology DOI:
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synthesis and assembly of the bacteriophage T7 in a single cell-free
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lab is or was sponsored by: DARPA, ONR, NSF, BSF, HFSP and UMN.