Biology subjects

Stan, G.-B.

Publications and source records attributed to Stan, G.-B..

4 recordsLinked to original sources

bioRxiv2018

A low-cost biological agglutination assay for medical diagnostic applications

Affordable, easy-to-use diagnostic tests that can be readily deployed for point-of-care (POC) testing are key in addressing challenges in the diagnosis of medical conditions and for improving global health in general. Ideally, POC diagnostic tests should be highly selective for the biomarker, user-friendly, have a flexible design architecture and a low cost of production. Here we developed a novel agglutination assay based on whole E. coli cells surface-displaying nanobodies which bind selectively to a target protein analyte. As a proof-of-concept, we show the feasibility of this design as a new diagnostic platform by the detection of a model analyte at nanomolar concentrations. Moreover, we show that the design architecture is flexible by building assays optimized to detect a range of model analyte concentrations supported using straight-forward design rules and a mathematical model. Finally, we re-engineer E. coli cells for the detection of a medically relevant biomarker by the display of two different antibodies against the human fibrinogen and demonstrate a detection limit as low as 10 pM in diluted human plasma. Overall, we demonstrate that our agglutination technology fulfills the requirement of POC testing by combining low-cost nanobody production, customizable detection range and low detection limits. This technology has the potential to produce affordable diagnostics for both field-testing in the developing world, emergency or disaster relief sites as well as routine medical testing and personalized medicine.

synthetic biology↗

bioRxiv2017

Tools for engineering coordinated system behaviour in synthetic microbial consortia

Advancing synthetic biology to the multicellular level requires the development of multiple orthogonal cell-to-cell communication channels to propagate information with minimal signal interference. The development of quorum sensing devices, the cornerstone technology for building microbial communities with coordinated system behaviour, has largely focused on reducing signal leakage between systems of cognate AHL/transcription factor pairs. However, the use of non-cognate signals as a design feature has received limited attention so far. Here, we demonstrate the largest library of AHL-receiver devices constructed to date with all cognate and non-cognate chemical signal interactions quantified and we develop a software tool that allows automated selection of orthogonal chemical channels. We use this approach to identify up to four orthogonal channels in silico and experimentally demonstrate the simultaneous use of three channels in co-culture. The development of multiple non-interfering cell-to-cell communication channels will facilitate the design of synthetic microbial consortia for novel applications including distributed bio-computation, increased bioprocess efficiency, cell specialisation, and spatial organisation.

synthetic biology↗

bioRxiv2017

Burden-driven feedback control of gene expression

Cells use feedback regulation to ensure robust growth despite fluctuating demands on resources and different environmental conditions. Yet the expression of foreign proteins from engineered constructs is an unnatural burden on resources that cells are not adapted for. Here we combined multiplex RNAseq with an in vivo assay to reveal the major transcriptional changes in two E. coli strains when a set of inducible synthetic constructs are expressed. We identified that native promoters related to the heat-shock response activate expression rapidly in response to synthetic expression, regardless of the construct. Using these promoters, we built a CRISPR/dCas9-based feedback regulation system that automatically adjusts synthetic construct expression in response to burden. Cells equipped with this general-use controller maintain capacity for native gene expression to ensure robust growth and as such outperform unregulated cells at protein yields in batch production. This engineered feedback is the first example of a universal, burden-based biomolecular control system and is modular, tuneable and portable.

synthetic biology↗

bioRxiv2017

Cell-free prediction of protein expression costs for growing cells

Translating heterologous proteins places significant burden on host cells, consuming expression resources leading to slower cell growth and productivity. Yet predicting the cost of protein production for any gene is a major challenge, as multiple processes and factors determine translation efficiency. Here, to enable prediction of the cost of gene expression in bacteria, we describe a standard cell-free lysate assay that determines the relationship between in vivo and cell-free measurements and {gamma}, a relative measure of the resource consumption when a given protein is expressed. When combined with a computational model of translation, this enables prediction of the in vivo burden placed on growing E. coli cells for a variety of proteins of different functions and lengths. Using this approach, we can predict the burden of expressing multigene operons of different designs and differentiate between the fraction of burden related to gene expression compared to action of a metabolic pathway.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC=\"FIGDIR/small/172627_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (26K):\norg.highwire.dtl.DTLVardef@19afa63org.highwire.dtl.DTLVardef@1acafbforg.highwire.dtl.DTLVardef@46443borg.highwire.dtl.DTLVardef@12987e3_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗