Biology subjects

Ellis, T.

Publications and source records attributed to Ellis, T..

9 recordsLinked to original sources

bioRxiv2018

Engineering a model cell for rational tuning of GPCR signaling

G protein-coupled receptor (GPCR) signaling is the primary method eukaryotes use to respond to specific cues in their environment. However, the relationship between stimulus and response for each GPCR is difficult to predict due to diversity in natural signal transduction architecture and expression. Using genome engineering in yeast, we here constructed an insulated, modular GPCR signal transduction system to study how the response to stimuli can be predictably tuned using synthetic tools. We delineated the contributions of a minimal set of key components via computational and experimental refactoring, identifying simple design principles for rationally tuning the dose-response. Using four different receptors, we demonstrate how this enables cells and consortia to be engineered to respond to desired concentrations of peptides, metabolites and hormones relevant to human health. This work enables rational tuning of cell sensing, while providing a framework to guide reprogramming of GPCR-based signaling in more complex systems.

synthetic biology↗

bioRxiv2018

Horizontal gene flow into Geobacillus is constrained by the chromosomal organization of growth and sporulation

Horizontal gene transfer (HGT) in bacteria occurs in the context of adaptive genome architecture. As a consequence, some chromosomal neighbourhoods are likely more permissive to HGT than others. Here, we investigate the chromosomal topology of horizontal gene flow into a clade of Bacillaceae that includes Geobacillus spp. Reconstructing HGT patterns using a phylogenetic approach coupled to model-based reconciliation, we discover three large contiguous chromosomal zones of HGT enrichment. These zones encompass and connect classically defined genomic islands. Analyzing topological and strand biases of recent and older transfer events, we show that restrictions on entry are rapidly enforced by selection and that restrictive and permissive zones have existed in their current locations for long periods of evolution. The largest zone, characterized by a high influx of metabolic genes, is centred on the terminus. The other two zones flank a narrow non-permissive zone around the origin of replication and extend to delimit the first third of the chromosome - the part of the chromosome that is confined to the forespore during early spore formation. Horizontal transfers into this area are biased towards functions classically controlled by the forespore-specific sigma factor {sigma}F: signal transduction, transcription, and particularly membrane biogenesis. Similar enrichment patterns are present in spore-forming but absent in non-spore-forming Bacilli. Our results suggest that the topology of HGT in Geobacillus, and Bacilli more generally, reflects constraints imposed by chromosomal organization for fast and sporulation, as asymmetric chromosomal entrapment in the forespore during early spore formation restricts where HGT-driven innovation in sporulation can occur.

evolutionary biology↗

bioRxiv2018

Convergent evolution of psilocybin biosynthesis by psychedelic mushrooms

Psilocybin is a psychoactive compound with clinical applications produced by dozens of mushroom species1. There has been a longstanding interest in psilocybin research with regard to treatment for addiction2, depression3, and end-of-life suffering4. However, until recently very little was known about psilocybin biosynthesis and its ecological role. Here we confirm and refine recent findings5 about the genes underpinning psilocybin biosynthesis, discover that there is more than one psilocybin biosynthesis cluster in mushrooms, and we provide the first data directly addressing psilocybins ecological role. By analysing independent genome assemblies for the hallucinogenic mushrooms Psilocybe cyanescens and Pluteus salicinus we recapture the recently discovered psilocybin biosynthesis cluster5,6 and show that a transcription factor previously implicated in its regulation is actually not part of the cluster. Further, we show that the mushroom Inocybe corydalina produces psilocybin but does not contain the established biosynthetic cluster, and we present an alternative cluster. Finally, a meta-transcriptome analysis of wild-collected mushrooms provides evidence for intra-mushroom insect gene expression of flies whose larvae grow inside Psilocybe cyanescens. These larvae were successfully reared into adults. Our results show that psilocybin does not confer complete protection against insect mycophagy, and the hypothesis that it is produced as an adaptive defense compound may need to be reconsidered.

evolutionary biology↗

bioRxiv2018

Engineered cell-to-cell signalling within growing bacterial cellulose pellicles

Bacterial cellulose is a strong and flexible biomaterial produced at high yields by Acetobacter species and has applications in healthcare, biotechnology and electronics. Naturally, bacterial cellulose grows as a large unstructured polymer network around the bacteria that produce it, and tools to enable these bacteria to respond to different locations are required to grow more complex structured materials. Here, we introduce engineered cell-to-cell communication into a bacterial cellulose-producing strain of Komagataeibacter rhaeticus to enable different cells to detect their proximity within growing material and trigger differential gene expression in response. Using synthetic biology tools, we engineer Sender and Receiver strains of K. rhaeticus to produce and respond to the diffusible signalling molecule, acyl-homoserine lactone (AHL). We demonstrate that communication can occur both within and between growing pellicles and use this in a boundary detection experiment, where spliced and joined pellicles sense and reveal their original boundary. This work sets the basis for synthetic cell-to-cell communication within bacterial cellulose and is an important step forward for pattern formation within engineered living materials.

synthetic biology↗

bioRxiv2017

Mid-Infrared Spectroscopy Study of Effects of Neonicotinoids on Forager Honey Bee (Apis mellifera) Fat Bodies and Their Connection to Colony Collapse Disorder

This study investigated the negative effects of neonicotinoid pesticides on honey bees in environment surrounding areas of pesticide use. The aim of the experiment is to identify possible contributors to the sudden decrease in honey bee population over the past 60 years, a phenomenon known as Colony Collapse Disorder. Analysis was performed on three sets of bees: the control group which was not in contact with pesticides, the infected dead group which was a set of bees suspected to have died due to neonicotinoids, and the infected alive group which was suspected to be under the influence of neonicotinoids. After dissecting the bee samples and extracting their fat bodies, the chemical composition and protein structures of the samples were analyzed using Mid-Infrared Beamline at the Canadian Light Source. Results from the spectra of bee samples exposed to neonicotinoids demonstrated possible residual pesticide chemicals within fat bodies. Several spectral peaks were also correlated with a possible change in protein secondary structures from primarily {beta}-sheet to -helix within fat bodies of neonicotinoid-affected bees. It is likely that the pesticides caused the growth of additional -helical structures, which is consistent with consequences of the inhibition of nicotinic acetylcholine receptors (nAChRs) a current pathway of harm of Colony Collapse Disorder as identified in past literature.

pharmacology and toxicology↗

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↗

bioRxiv2017

Rational Design Of RNA Structures That Predictably Tune Eukaryotic Gene Expression

Predictable tuning of gene expression is essential for engineering genetic circuits and for optimising enzyme levels in metabolic engineering projects. In bacteria, gene expression can be tuned at the stage of transcription, by exchanging the promoter, or at stage of translation by altering the ribosome binding site sequence. In eukaryotes, however, only promoter exchange is regularly used, as the tools to modulate translation are lacking. Working in S. cerevisiae yeast, we here describe how hairpin RNA structures inserted into the 5 untranslated region (5UTR) of mRNAs can be used to tune expression levels by altering the efficiency of translation initiation. We demonstrate a direct link between the calculated free energy of folding in the 5UTR and protein abundance, and show that this enables rational design of hairpin libraries that give predicted expression outputs. Our approach is modular, working with different promoters and protein coding sequences, and it outperforms promoter mutation as a way to predictably generate a library where a protein is induced to express at a range of different levels. With this tool, computational RNA sequence design can be used to predictably fine-tune protein production, providing a new way to modulate gene expression in eukaryotes.

synthetic biology↗

bioRxiv2016

Tunable Extracellular Self-Assembly of Multi-Protein Conjugates from Bacillus subtilis

The ability to stably and specifically conjugate recombinant proteins to one another is a powerful in vitro technique for engineering multifunctional enzymes, protein therapeutics and novel biological materials. However, for many applications spontaneous in vivo protein conjugation would be preferable to in vitro methods. Exploiting the recently described SpyTag-SpyCatcher system, we describe here how enzymes and structural proteins can be genetically-encoded to covalently conjugate in culture media following programmable secretion by Bacillus subtilis. Using this novel approach, we demonstrate how self-conjugation of a secreted industrial enzyme, XynA, dramatically increases its resilience to boiling and we show that cellular consortia can be engineered to self-assemble functional multi-protein complexes with tunable composition. This genetically-encoded modular system provides a new, flexible strategy for protein conjugation harnessing the substantial advantages of extracellular self-assembly.

synthetic biology↗