Biology subjects

Stoof, R.

Publications and source records attributed to Stoof, R..

3 recordsLinked to original sources

bioRxiv2020

FlowScatt: enabling volume-independent flow cytometry data by decoupling fluorescence from scattering

MotivationMeasuring fluorescence by flow cytometry is fundamental for characterising single-cell performance. While it is known that fluorescence and scattering values tend to positively correlate, the impact of cell volume on fluorescence is typically overlooked. This makes of fluorescence values alone an inaccurate measurement for high-precision characterisations. ResultsWe developed FlowScatt, an open-source software package that removes volume-dependency in the fluorescence channel. Using FlowScatt, flourescence values are re-calculated based on the unified volume per cell that arises from scattering decomposition. AvailabilityFlowScatt is openly available as a Python package on https://github.com/rstoof/FlowScatt. Experimental data for validation is available online. Contactangel.goni-moreno@newcastle.ac.uk

bioinformatics↗

bioRxiv2020

Contextual dependencies expand the re-usability of genetic inverters

The design and implementation of Boolean logic functions in living cells has become a very active field within synthetic biology. By controlling networks of regulatory proteins, novel genetic circuits are engineered to generate predefined output responses. Although many current implementations focus solely on the genetic components of the circuit, the host context in which the circuit performs is crucial for its outcome. Here, we characterise 20 genetic NOT logic gates (inverters) in up to 7 bacterial-based contexts each, to finally generate 135 different functions. The contexts we focus on are particular combinations of four plasmid backbones and three hosts, two Escherichia coli and one Pseudomonas putida strains. Each NOT logic gate shows seven different logic behaviours, depending on the context. That is, gates can be reconfigured to fit response requirements by changing only contextual parameters. Computational analysis shows that this range of behaviours improves the compatibility between gates, because there are considerably more possibilities for combination than when considering a unique function per genetic construct. Finally, we address the issue of interoperability and portability by measuring, scoring, and comparing gate performance across contexts. Rather than being a limitation, we argue that the effect of the genetic background on synthetic constructs expand the scope of the functions that can be engineered in complex cellular environments, and advocate for considering context as a fundamental design parameter for synthetic biology.

synthetic biology↗

bioRxiv2019

A model for the spatio-temporal design of gene regulatory circuits

The design of increasingly complex gene regulatory networks relies upon mathematical modelling to link the gap that goes from conceptualisation to implementation. An overarching challenge is to update modelling abstractions and assumptions as new mechanistic information arises. Although models of bacterial gene regulation are often based on the assumption that the role played by intracellular physical distances between genetic elements is negligible, it has been shown that bacteria are highly ordered organisms, compartmentalizing their vital functions in both time and space. Here, we analysed the dynamical properties of regulatory interactions by explicitly modelling spatial constraints. Key to the model is the combined search by a regulator for its target promoter via 1D sliding along the chromosome and 3D diffusion through the cytoplasm. Moreover, this search was coupled to gene expression dynamics, with special attention to transcription factor-promoter interplay. As a result, promoter activity within the model depends on its physical separation from the regulator source. Simulations showed that by modulating the distance between DNA components in the chromosome, output levels changed accordingly. Finally, previous experimental results with engineered bacteria in which this distance was minimized or enlarged were successfully reproduced by the model. This suggests that the spatial specification of the circuit alone can be exploited as a design parameter to select programmable output levels.

synthetic biology↗