| dc.description.abstract | While Synthetic Biology represents a promising approach to solve real-world
problems, the use of genetically modified organisms (GMO) is still a matter of
legal and environmental concerns. Cell-free systems (CFS) are an emerging
technology where cell extracts are used instead of genetically modified
cells, thus, not presenting a ”living prospect” applicable to current legal regulations.
Since there is a need for development of novel systems using the
cell-free approach and considering that most attempts have been focused on
mimicking normal cell behaviour, this work has as its principal aim to generate
modular cell-free and cell-based systems capable of not only detecting substances
present in the environment, such as heavy metals or antibiotics, but
also analysing them via the use of engineered behaviours, such as logic gates.
In vivo logic gates, for instance, have proven difficult to combine into larger
devices. Here we present a cell-based logic system, ParAlleL, which decomposes
a large genetic circuit into a collection of small subcircuits working in
parallel, each subcircuit responding to a different combination of inputs. A final
global output is then generated by a combination of the responses. Using
ParAlleL, for the first time a completely functional 3-bit full adder and full subtractor
were generated using Escherichia coli cells, as well as a calculator-like
display that shows a numeric result, from 0 to 7, when the proper 3-bit binary
input is introduced into the system. This parallel approach facilitates the design
of cell-based logic gates by the decomposition of complex processes into
their main components, avoiding the need for complex genetic engineering.
Cell-free systems, on the other hand, have emerged as a possible solution but
much work is needed to optimize their functionality and simplify their usage for
Synthetic Biology. Here we present a transcription-only genetic circuit (TXO),
which is independent of translation or post-translational maturation. RNA aptamers
are used as reaction output allowing the generation of fast, reliable and
simple-to-design transcriptional units. TXO cell-free reactions and their possible
applications are shown to be a promising new tool for fast and simple
bench-to-market genetic circuit and biosensor applications.
Additionally, this thesis presents a versatile cell-free system based on
the master survivalist bacteria Cupriavidus metallidurans CH34, capable of
not only sensing environmental variables, such as heavy metals, but also
synthesizing proteins and producing bioplastics. This novel cell-free chassis
follows the discovery of the unstable genome that this bacterium carries, which
is also explained here, offering novel possibilities of development considering
the cell free approach. | en |
