Synthetic biology has the vision to re-design biology in an easier, faster, more robust, efficient,
and reliable fashion by applying engineering principles to living systems. To this end, a
modular design approach enables rapid prototyping and manufacturing of various synthetic
genetic circuit libraries. This transformative approach needs expansion of interchangeable,
standardised, and well-characterised genetic components required for composing higherlevel
functional circuits. Additionally, compatibility of biological parts into modular design
assemblies is highly desirable to streamline fabrication of synthetic genetic circuits. This
project is focused on the in vivo characterisation, standardisation, modularisation, and
implementation of a set of biomolecular regulators in Escherichia coli, specifically at the
transcriptional level through the standardised promoter architecture and the posttranscriptional
level via modular Artificial RNA interference (mARi). These regulatory systems
were rationally developed within a modular design and DNA assembly framework to facilitate
their easy adoption and implementation. The regulatory properties of both controllers were
further characterised towards a range of typical genetic and cellular contexts that are
important for diverse applications. Additionally, extensibility and orthogonality of these
controllers allow for multiplexed and simultaneous regulation of multi-gene systems with
alternative configurations. As a demonstration, a standardised inducible promoter was
employed to express stress-inducing recombinant proteins. Furthermore, the production of
these proteins was improved up to 5-fold by the use of an adaptive and dynamic negative
feedback system, which is governed by mARi and driven by the host-stress response.
Ultimately, this improvement was robustly maintained in different tested perturbations.
Owing to its modularity, this feedback system could potentially improve the production of
any recombinant protein of interest without specifically tuning the system or requiring strain
modification. Collectively, the genetic regulatory platforms presented in this project greatly
provide valuable resources for developing the next-generation of engineered biological
circuits.