Biology subjects

Papachristodoulou, A.

Publications and source records attributed to Papachristodoulou, A..

4 recordsLinked to original sources

bioRxiv2018

Characterisation and development of aspirin inducible biosensors in E. coli Nissle 1917 and SimCells

A simple aspirin-inducible system has been developed by employing the Psal promoter and SalR regulation system originally from Acinetobacter baylyi ADP1, which has been cloned into E. coli for characterisation of gene circuits and induction of novel SimCells (simple cells). Mutagenesis at the DNA binding domain (DBD) and chemical recognition domain (CRD) of the SalR protein in A. baylyi ADP1 suggests that inactive SalRi can compete with activated SalRa, occupying the binding position of Psal promoter. The induction of the Psal promoter was compared in two different designs in E. coli: simple regulation (SRS) and positive autoregulated system (PAR). Both regulatory systems were induced in a dose-dependent manner in the presence of aspirin in the range of 0.05-10 M. Over-expression of SalR in the SRS system reduces both baseline leakiness and inducible strength of Psal promoter. A weak SalR expression significantly improve the inducible strength, which is in a good agreement of the proposed hypothesis of SalRi/SalRa competitive binding. The PAR system provides a feedback loop that fine-tunes the level of SalR, displaying inducible strength. A mathematical model based on SalRi/SalRa competitive binding hypothesis was developed, which not only reproduces the observed experimental results but also predict the performance of a new gene circuit design. The aspirin-inducible systems were also functional in probiotic strain E.coli Nissle 1917 (EcN) and SimCells produced from E. coli MC1000 {Delta}minD. The well-characterised and modularised aspirin-inducible gene circuits would be useful biobricks for bacterial therapy in environment and medical applications.

microbiology↗

bioRxiv2017

Visual Representation of Experimental Protocols

Using robots to automate laboratory tasks could increase throughput and reproducibility, but requires experimental protocols to be specified in a computer-readable format. We present a new user interface (Lists of Liquids) for specifying experimental protocols by directly manipulating a diagram: rather than having to specify individual liquid handling operations, the user can simply specify that particular lists of liquids should be combined as either a Cartesian product or convolution, and the system will plan a series of liquid handling steps to achieve this. This is intended to provide a higher-level interface in order to make the creation of protocols faster and less error prone.

synthetic biology↗

bioRxiv2017

Synthetic negative feedback circuits using engineered small RNAs

Negative feedback is known to endow biological and man-made systems with robust performance in the face of uncertainties and disturbances. To date, synthetic biological feedback circuits have relied upon protein-based, transcriptional regulation to control circuit output. Small RNAs (sRNAs) are non-coding RNA molecules which can inhibit translation of target messenger RNAs (mRNAs). In this paper, we designed, modelled and built two synthetic negative feedback circuits that use rationally-designed sRNAs for the first time. The first circuit builds upon the well characterised tet-based autorepressor, incorporating an externally-inducible sRNA to tune the effective feedback strength. This allows more precise fine-tuning of the circuit output in contrast to the sigmoidal input-output response of the autorepressor alone. In the second circuit, the output is a transcription factor that induces expression of an sRNA which negatively regulates the translation of the mRNA encoding this output, creating direct, closed-loop, negative feedback. Analysis of the noise profiles of both circuits showed that the use of sRNAs did not result in large increases in noise. Stochastic and deterministic modelling of both circuits agreed well with experimental data. Finally, simulations using fitted parameters allowed dynamic attributes of each circuit such as response time and disturbance rejection to be investigated.

synthetic biology↗

bioRxiv2016

Ribo-attenuators: novel elements for reliable and modular riboswitch engineering

Riboswitches are structural genetic regulatory elements that directly couple the sensing of small molecules to gene expression. They have considerable potential for applications throughout synthetic biology and bio-manufacturing as they are able to sense a wide range of small molecules and regulate gene expression in response. Despite over a decade of research they have yet to reach this considerable potential as they cannot yet be treated as modular components. This is due to several limitations including sensitivity to changes in genetic context, low tunability, and variability in performance. To overcome the associated difficulties with riboswitches, we have designed and introduced a novel genetic element called a Ribo-attenuator in Bacteria. This genetic element allows for predictable tuning, insulation from contextual changes, and a reduction in expression variation. Ribo-attenuators allow riboswitches to be treated as truly modular and tunable components, and thus increases their reliability for a wide range of applications.

synthetic biology↗