Biology subjects

Ellis, T.

Publications and source records attributed to Ellis, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Strain handling exposes IS-mediated recurrent genome reduction at a dynamic accessory locus in Komagataeibacter rhaeticus

Bacterial genomes are dynamic and harbour accessory regions that can be gained, lost, or rearranged in response to selective pressures. Members of the genus Komagataeibacter are used for bacterial cellulose production and researched as platforms for engineered living materials. However, their genomes are rich in mobile genetic elements (MGEs), including insertion sequences (ISs), genomic islands and prophage remnants, which drive genome rearrangements under agitated cultivation conditions. Whether this genome architecture contributes to genomic changes during routine strain maintenance and distribution remains poorly understood. Comparative genome sequencing of independent Komagataeibacter rhaeticus iGEM stocks revealed recurrent loss of a 210 kbp chromosomal region flanked by homologous IS5 sequences. This deletion was identified in a stock received as an agar stab and reproduced from an intact-genome population subjected to simulated agar-stab handling. Phylogenomic analysis of the affected locus revealed gene-tree/species-tree discordance, heterogeneous GC content and multiple ISs, supporting its characterization as a dynamic accessory region. Populations carrying the larger genome reduction showed improved growth and cellulose production relative to the intact genome reference, whereas populations carrying a smaller nested deletion showed reduced performance. These findings demonstrate that routine strain handling is not necessarily genomically neutral and can expose structural genomic variabilities that generate population-level genomic heterogeneity with phenotypic consequences. For bacteria with MGE-rich genomes, handling history may represent an underappreciated source of genomic and phenotypic variation between laboratories. More broadly, identifying unstable accessory regions may highlight genomic content that is dispensable under laboratory conditions, providing candidates for genome streamlining and strain improvement.

microbiology↗

bioRxiv2026

Synthetic Combinatorial Minimisation of Cell Cycle Control in Yeast

The eukaryotic cell cycle, with its inherent regulatory redundancy, provides an ideal target for exploring genome modularisation and minimisation through synthetic genomics. Building upon principles established by the Synthetic Yeast Genome (Sc2.0) project, we used CRISPR-mediated genome engineering to relocate nine key cell cycle genes into a synthetic gene cluster in the S. cerevisiae genome, to allow combinatorial study of these genes in yeast. We employed Cre/loxP recombination to rapidly generate hundreds of strains with different gene deletion combinations in the module, with the objective of identifying minimal gene sets that permit robust cell cycle function. Using FACS-sorting and POLAR (Pool of Long Amplified Reads) sequencing, we conducted high-throughput analysis of gene deletion combinations in large cell pools, detecting approximately 80% of theoretically possible gene combinations, including those predicted from prior mathematical modelling studies. Our findings demonstrate that the cell cycle gene set can be minimised while maintaining viability, though only select combinations of gene deletions ensure robust fitness across different conditions. This work establishes a framework for genome minimisation, opening the door to the design of simplified, modular synthetic genomes for diverse applications.

synthetic biology↗

bioRxiv2026

Unlocking Bacterial Cellulose Functionalisation: Comparative Genetic and Co-Culture Strategies in Komagataeibacter

Bacterial cellulose (BC) is produced by diverse bacterial species, including members of the genus Komagataeibacter, and has emerged as a potential sustainable alternative to conventional materials such as plastics and leather. Its production by bacteria is rapid and easily scalable, and via genetic modifications or culturing the bacteria with other microbes, it is possible to rationally enhance the BC that is produced, for example by adding functional components. Here, we systematically evaluated BC functionalization strategies, including genetic engineering and co-culturing with other engineered microbes across the most widely used Komagataeibacter species: K. rhaeticus, K. xylinus, K. medellinensis and K. sucrofermentans. We established that all tested strains are amenable to DNA transformation and capable of expressing heterologous genes from identical genetic constructs. However, we identified species-specific differences in heterologous gene expression, cellulose production in varying environmental conditions and in the abilities of the strains to co-culture with Escherichia coli. Additionally, we demonstrated that all these cellulose-producing bacteria can establish functional symbiotic co-cultures with yeast. While inter-species variations in heterologous gene expression and co-culture dynamics are evident, BC modification through genetic engineering and co-culturing strategies remains achievable across all tested Komagataeibacter species. Our work provides a comparative framework to guide researchers in selecting optimal species based on their specific application requirements.

synthetic biology↗

bioRxiv2025

Engineering signal peptide-mediated recombinant protein secretion in Komagataeibacter rhaeticus for biological Engineered Living Material applications

BackgroundBacterial nanocellulose (BC), produced by Komagataeibacter species, is an ideal scaffold for biological Engineered Living Materials (bioELMs) research. Current BC functionalization strategies often rely on secondary microbial hosts or post-production enzyme immobilization, limiting the scalability and modularity required for programmable bioELMs. Establishing a single-chassis system capable of simultaneous biopolymer synthesis and in situ functionalization remains a primary objective in bioELM research. This study addresses the need by benchmarking signal peptide-mediated protein translocation in K. rhaeticus iGEM, a model bacterium for BC-based bioELMs, enabling a synthetic biology framework for single-chassis based biomaterial functionalization. ResultsGenome-wide analysis confirmed the presence of a complete Sec translocation machinery in K. rhaeticus. Through liquid chromatography-tandem mass spectrometry and SignalP 5.0 prediction, native signal peptides were identified and evaluated alongside previously characterized heterologous signal peptides using {beta}-lactamase and mScarlet as cargo proteins. Protein translocation was found to depend on signal peptide identity, cargo type, and expression mode. Fluorescence imaging revealed cytoplasmic, polar, and peripheral localization patterns, confirming functional engagement with the native translocation machinery. A key limitation identified was the retention of recombinant proteins within the periplasm, restricting extracellular availability. Despite this, signal peptide-mediated translocation enabled the incorporation of enzymatic activity into BC during biosynthesis. A post-growth osmotic shock-release strategy increased measurable enzymatic activity by 30%, demonstrating a practical route to overcome this physiological bottleneck while maintaining the biomaterial production capacity. ConclusionsThis study benchmarks signal peptide-dependent protein translocation in K. rhaeticus and identifies periplasmic retention as a key constraint for extracellular protein release. By linking protein translocation to in situ BC functionalization, this work establishes a synthetic biology framework that supports the development of K. rhaeticus as a single-chassis platform for the scalable production of functionalized bioELMs.

bioengineering↗

bioRxiv2025

Quantitative profiling of millions of nucleotides reveals sequence-encoded interactions that govern plasmid propagation

Plasmids are central to modern biotechnology, especially therapeutic development, yet their propagation in Escherichia coli remains difficult to predict. Although expression-induced burden is well understood and can be mitigated, the impact of foreign DNA segments that do not function in bacteria on plasmid propagation and stability remains largely unknown. Here we developed a pooled, sequencing-based framework that performs quantitative profiling across millions of bases, enabling high-resolution assessment of plasmid fitness at scale and revealing cryptic, sequence-encoded interactions between foreign DNA elements and bacterial hosts. Promoter-like motifs, transcription factor binding site homology, and recombination-prone architectures emerge as major determinants of propagation efficiency, with context dependent effects demonstrating that plasmid behaviour arises from higher-order interactions between parts rather than isolated elements. Extending this framework, we introduce TRACE, a neural network model trained on degenerate sequence libraries that predicts plasmid propagation directly from sequence. TRACE generalises across plasmid architectures and can be fine-tuned on experimental datasets to improve predictions of manufacturability and host compatibility. These advances establish a generalisable, data-driven framework for understanding and designing host-aware plasmids, transforming plasmid production from an empirical process into a predictable property of DNA sequence.

synthetic biology↗

bioRxiv2025

Generating long deletions across the genome with pooled paired prime editing screens

Engineered deletions are a powerful probe for studying genome architecture, function, and regulation. Yet, the lack of effective methods to create them in large numbers and at multi-kilobase scale has rendered interrogating all the gigabases of human DNA elusive. Prime editing based approaches are promising contenders to fill this gap, but have not been adopted for large-scale pooled screening so far. Here, we performed the first high-throughput paired prime editing deletion screens. To establish the technology, we generated 3,802 deletions of up to 1.2 Mb in a reporter locus in two human cell lines, and identified key determinants of paired prime editing efficiency to be deletion length, predicted pegRNA efficacy, and contact frequency. We used these learnings to generate therapeutically important deletions at rates that are expected to give clinical benefits for retinitis pigmentosa and Duchenne muscular dystrophy. Combining several technological advances, we then performed the first pooled paired prime editing screen to delete over 10,471 sequences of median length 1.1 kb that target 149 genes across the genome. Our screens were high quality, recovering 75% of positive controls. We discovered 462 non-coding regions with fitness effects, and validated deletions with impact as well as tolerated ones in regions with functional annotations. Our screens are the largest pooled characterization of precise deletions to date, demonstrating that prime deletion screens can perturb the genome at scale, and highlighting that most non-coding DNA around essential genes is dispensable for cell growth in one condition.

genomics↗

bioRxiv2025

Yorzoi: Predicting RNA-seq coverage from DNA sequence in yeast

Yeast is one of the principal model organisms in synthetic biology and a widely used chassis for testing heterologous DNA sequences. However, designing DNA constructs correctly is currently limited by our incomplete quantitative understanding of how sequences are transcribed and translated, especially non-native sequences. Here, we present Yorzoi, a sequence-to-expression model for bakers yeast that predicts RNA-seq coverage for a 5 kilobase, multi-gene window of DNA at 10 base pair (bp) resolution. To extend its predictive ability beyond native DNA, Yorzoi has been pretrained on a comprehensive dataset not only including native yeast sequences but also human sequences expressed in yeast and structurally rearranged synthetic yeast chromosomes. We demonstrate that our model has learned general rules of yeast transcription by achieving high predictive performance on various downstream tasks. Yorzoi is a powerful tool for in-silico testing of DNA sequences and directly applicable for sequence design. A web application to use our model is available at yorzoi.eu and the code open source on GitHub.

synthetic biology↗

bioRxiv2025

Molecular mimicry of plant cell-surface immune receptors by fungal secreted leucine-rich repeat proteins

O_LILeucine-rich repeat (LRR) receptor-like kinases (LRR-RLKs) are important plant immunity proteins. The wheat pathogen Zymoseptoria tritici produces many virulence effectors during infection; however, most remain uncharacterised. We identified a secreted protein from Z. tritici (ZtLRR) that consists of a single LRR domain and hypothesised that it mimics host LRR-RLKs to suppress plant immunity. C_LIO_LIWe used transient expression to probe ZtLRR function related to production of reactive oxygen species (ROS) and cell death, two important immune processes. We used AlphaFold structural predictions with targeted yeast two-hybrid to demonstrate protein-protein interactions. Transgenic wheat allowed assessment of effector function in the natural host. C_LIO_LIZtLRR suppressed ROS production and cell death in N. benthamiana with high potency. Structural predictions suggested high similarity to several plant LRR-RLKs and interaction with TaBAK1 was confirmed by targeted yeast two-hybrid. Transgenic wheat expressing ZtLRR was impaired in PAMP-triggered immunity (PTI) and had increased susceptibility to infection with compatible Z. tritici. We identified structural orthologs of ZtLRR in diverse fungal lineages and demonstrated that several of these proteins have similar immune-suppressing properties as ZtLRR. C_LIO_LIOur work demonstrates that molecular mimicry of host LRR-RLKs by phytopathogen sLRR effectors is effective at disrupting host immune pathways. C_LI

molecular biology↗

bioRxiv2025

Cultivating Future Materials: Artificial Symbiosis for Bulk Production of Bacterial Cellulose Composites

Symbiotic relationships between micro-organisms are the basis of all global ecosystems. Here we extrapolate this concept for novel material fabrication by creating artificial symbiotic relationships between species that are usually not grown synergistically in nature. Specifically, we combine the cellulose-producing bacterium Komagataeibacter hansenii and the green microalga Chlamydomonas reinhardtii to obtain bulk growth of bacterial cellulose. Usually, bacterial cellulose is produced as floating pellicles at the air-liquid interface of the growing media, because free oxygen, together with the nutrients in the culture medium, is required for the bacteria to synthesise the cellulose fibres. In the co-culture, bacterial cellulose production can be achieved in bulk beyond the restriction of the liquid/air interface as the motile microalgae act as oxygen-generating sites within the culture medium. In exchange, the highly porous and mechanically robust scaffold provided by the cellulose allows the algal community to form a healthy biofilm, which can reach several centimetres thick. We demonstrate that our growing platform allows the simultaneous production of bulk bacterial cellulose in static incubation conditions, taking up an arbitrary and yet tunable 3D shape, dependent on the geometry of the culture vessel and with a relatively high biomass output.

microbiology↗

bioRxiv2024

Iterative SCRaMbLE for Engineering Synthetic Genome Modules and Chromosomes

Synthetic biology offers the possibility of synthetic genomes with customised gene content and modular organisation. In eukaryotes, building whole custom genomes is still many years away, but work in Saccharomyces cerevisiae yeast is closing-in on the first synthetic eukaryotic genome with genome-wide design changes. A key design change throughout the synthetic yeast genome is the introduction of LoxPsym site sequences. These enable inducible genomic rearrangements in vivo via expression of Cre recombinase via SCRaMbLE (Synthetic Chromosome Recombination and Modification by LoxPsym-mediated Evolution). When paired with selection, SCRaMbLE can quickly generate strains with phenotype improvements by diversifying gene arrangement and content in LoxPsym-containing regions. Here, we demonstrate how iterative cycles of SCRaMbLE can be used to reorganise synthetic genome modules and synthetic chromosomes for improved functional performance under selection. To achieve this, we developed SCOUT (SCRaMbLE Continuous Output and Universal Tracker), a reporter system that allows SCRaMbLEd cells to be sorted into a high diversity pool. When coupled with long-read sequencing, SCOUT enables high-throughput mapping of genotype abundance and correlation of gene content and arrangement with growth-related phenotypes. Iterative SCRaMbLE was applied here to yeast strains with a full synthetic chromosome, and to strains with synthetic genome modules encoding the gene set for histidine biosynthesis. Five synthetic designs for HIS modules were constructed and tested, and we investigated how SCRaMbLE reorganised the poorest performing design to give improved growth under selection. The results of iterative SCRaMbLE serve as a quick route to identify genome module designs with optimised function in a selected condition and offer a powerful tool to generate datasets that can inform the design of modular genomes in the future.

synthetic biology↗

bioRxiv2024

Engineered Transcription Factor Binding Arrays for DNA-based Gene Expression Control in Mammalian Cells

Manipulating gene expression in mammalian cells is critical for cell engineering applications. Here we explore the potential of transcription factor (TF) recognition element arrays as DNA tools for modifying free TF levels in cells and thereby controlling gene expression. We first demonstrate proof-of-concept, showing that Tet TF-binding recognition element (RE) arrays of different lengths can tune gene expression and alter gene circuit performance in a predictable manner. We then open-up the approach to interface with any TF with a known binding site by developing a new method called Cloning Troublesome Repeats in Loops (CTRL) that can assemble plasmids with up to 256 repeats of any RE sequence. Transfection of RE array plasmids assembled by CTRL into mammalian cells show potential to modify host cell gene regulation at longer array sizes by sequestration of the TF of interest. RE array plasmids built using CTRL were demonstrated to target both synthetic and native mammalian TFs, illustrating the ability to use these tools to modulate genetic circuits and instruct cell fate. Together this work advances our ability to assemble repetitive DNA arrays and showcases the use of TF-binding RE arrays as a method for manipulating mammalian gene expression, thus expanding the possibilities for mammalian cell engineering.

synthetic biology↗

bioRxiv2024

Modulating Bacterial Nanocellulose Crystallinity through Post-Transcriptional Repression in Komagataeibacter xylinus

Bacterial nanocellulose (BC), a versatile and biodegradable polymer, has been extensively studied as an alternative biomaterial for various applications. For biomedical and packaging uses, where precise control over nanocellulose structure is essential, existing literature describes BC structural modification processes that involve additives or additional steps. With the aim to develop a programmable method to control the bulk microstructure, we developed sRNA-based post-transcriptional repression cassettes that allows precise regulation of the crystalline phase of BC. Before investigating the effects of post-transcriptional repression of bcsD, bcsZ, and ccpA genes, known to influence BC crystallinity, we validated the specificity of the sRNA repression cassette by targeting a genome-integrated red fluorescent protein, mRFP1. The observed growth inhibition (>80%) caused by overexpressed Hfq RNA chaperone in Komagataeibacter xylinus was alleviated ([≤] 23%) by its removal, resulting in a 43% reduction in relative mRFP1 expression. By varying the design of the repression cassette and the inducer concentrations, we successfully modulated the repression of the target genes [with relative expression reductions of 6%-34% for bcsD, 8%-24% for bcsZ, and 2%-20% for ccpA, as confirmed by qRT-PCR]. These gene repression levels led to statistically significant changes in the amorphous content of the BC microstructure, as demonstrated by X-ray diffraction and wide-angle X-ray scattering analysis.

bioengineering↗

bioRxiv2024

SubtiToolKit - a bioengineering kit for Bacillus subtilis and Gram-positive bacteria.

Building DNA constructs of increasing complexity is key to synthetic biology. Golden Gate methods led to the creation of cloning toolkits - collections of modular standardized DNA parts hosted on hierarchic plasmids, developed for yeast, plants, Gram-negative bacteria, and human cells. However, Gram-positive bacteria have been neglected. Bacillus subtilis is a Gram-positive model organism and a workhorse in the bioindustry. Here, we present the SubtiToolKit, a high- efficiency cloning toolkit for B. subtilis and Gram-positive bacteria. Its design permits DNA constructs for transcriptional units, operons, knock-in and knock-out applications. It contains libraries of promoters, RBSs, fluorescent proteins, protein tags, terminators, genome integration parts, a no-leakage genetic device to control the expression of toxic products during E. coli assembly, and a toolbox for industrially relevant strains of Geobacillus and Parageobacillus as an example of SubtiToolKit versatility for other Gram-positive bacteria and its future perspective as a reference toolkit.

synthetic biology↗

bioRxiv2024

Engineered yeast multicellularity via synthetic cell-cell adhesion and direct-contact signalling

Coordination of behaviour in multicellular systems is one the main ways that nature increases the complexity of biological function in organisms and communities. While Saccharomyces cerevisiae yeast is used extensively in research and biotechnology, it is a unicellular organism capable of only limited multicellular states. Here we expand the possibilities for engineering multicellular behaviours in yeast by developing modular toolkits for two key mechanisms seen in multicellularity, contact-dependent signalling and specific cell-to-cell adhesion. MARS (Mating-peptide Anchored Response System) is a toolkit based on surface-displayed fungal mating peptides and G protein-coupled receptor (GPCR) signalling which can mimic juxtacrine signalling between yeasts. SATURN (Saccharomyces Adhesion Toolkit for multicellUlar patteRNing) surface displays adhesion-proteins pairs on yeasts and facilitates the creation of cell aggregation patterns. Together they can be used to create multicellular logic circuits, equivalent to developmental programs that lead to cell differentiation based on the local population. Using MARS and SATURN, we further developed JUPITER (JUxtacrine sensor for Protein-protein InTERaction), a genetic sensor for assaying protein-protein interactions in culture, demonstrating this as a tool to select for high affinity binders among a population of mutated nanobodies. Collectively, MARS, SATURN, and JUPITER present valuable tools that facilitate the engineering of complex multicellularity with yeast and expand the scope of its biotechnological applications.

synthetic biology↗

bioRxiv2024

Combinatorial Design Testing in Genomes with POLAR-seq

Synthetic biology projects increasingly use modular DNA assembly or synthetic in vivo recombination to generate diverse combinatorial libraries of genetic constructs for testing. But as these designs expand to multigene systems it becomes challenging to sequence these in a cost-effective way that reveals the genotype to phenotype relationships in the libraries. Here, we introduce a new quick, low-cost method designed for assessing combinational designs of genome-integrated multigene constructs that we call Pool of Long Amplified Reads (POLAR) sequencing. POLAR-seq takes genomic DNA isolated from library pools and uses long range PCR to amplify target genomic regions up to 35 kb long containing combinatorial designs. The pool of long amplicons is then directly read by nanopore sequencing with full length reads then used to identify the gene content and structural variation of individual genotypes in the library and read count indicating how abundant a genotype is within the pool. Using yeast cells with loxP-containing synthetic gene clusters that rearrange in vivo in the presence of Cre recombinase, we demonstrate how POLAR-seq can be used to identify global patterns from combinatorial experiments, find the most abundant genotypes in a pool and also be adapted to sequence-verify gene clusters from isolated strains.

synthetic biology↗

bioRxiv2024

Synthetic genome modules designed for programmable silencing of functions and chromosomes

Unlike in bacteria, eukaryotes rarely cluster sets of genes in their genomes according to function, instead having most genes spread randomly across different chromosomes and loci. However, with the advent of genome engineering, synthetic co-location of genes that together encode a cell function has now become possible. Here, using Saccharomyces cerevisiae we demonstrate the feasibility of reorganising a set of yeast genes encoding a cell function, tryptophan biosynthesis, into a synthetic genome module by deleting these genes and their regulatory elements from their native genomic loci while in parallel reconstructing them into gene cluster format by synthetic DNA assembly. As part of synthetic module design, loxPsym sequences recognised by Cre recombinase are placed between all module genes, and we leverage these for a novel master regulation system we call dCreSIR. Using dCreSIR we externally control silencing of synthetic modules by targeted binding of chromatin recruiters to loxPsym sites and this leads to inhibition of local transcription. We further show that dCreSIR can go beyond modules and be used to specifically downregulate expression across an entire synthetic yeast chromosome containing loxPsym sites. Together, our work offers insights into yeast genome organisation and establishes new principles and tools for the future design and construction of modular synthetic yeast genomes.

synthetic biology↗

bioRxiv2024

Randomizing the human genome by engineering recombination between repeat elements

While protein-coding genes are characterized increasingly well, 99% of the human genome is non-coding and poorly understood. This gap is due to a lack of tools for engineering variants that affect sequence to the necessary extent. To bridge this gap, we have developed a toolbox to create deletions, inversions, translocations, and extrachromosomal circular DNA at scale by highly multiplexed insertion of recombinase recognition sites into repetitive sequences with CRISPR prime editing. Using this strategy, we derived stable human cell lines with several thousand clonal insertions, the highest number of novel sequences inserted into single human genomes. Subsequent recombinase induction generated an average of more than one hundred megabase-sized rearrangements per cell, and thousands across the whole population. The ability to detect rearrangements as they are generated and to track their abundance over time allowed us to measure the selection pressures acting on different types of structural changes. We observed a consolidation towards shorter variants that preferentially delete growth-inhibiting genes and a depletion of translocations. We isolated and characterized 21 clones with multiple recombinase-induced rearrangements. These included viable haploid clones with deletions that span hundreds of kilobases as well as triploid HEK293T clones with aneuploidies and fold back chromosomes. We mapped the impact of these genetic changes on gene expression to decipher how structural variants affect gene regulation. The genome scrambling strategy developed here makes it possible to delete megabases of sequence, move sequences between and within chromosomes, and implant regulatory elements into new contexts which will shed light on the genome organization principles of humans and other species.

genomics↗

bioRxiv2023

Self-dyeing textiles grown from cellulose-producing bacteria with engineered tyrosinase expression

Environmental concerns are driving interests in post-petroleum synthetic textiles produced from microbial and fungal sources. Bacterial cellulose is a promising sustainable leather alternative, on account of its material properties, low infrastructure needs and biodegradability. However, for alternative textiles like bacterial cellulose to be fully sustainable, alternative ways to dye textiles need to be developed alongside alternative production methods. To address this, we here use genetic engineering of Komagataeibacter rhaeticus to create a bacterial strain that grows self-dyeing bacterial cellulose. Dark black pigmentation robust to material use is achieved through melanin biosynthesis in the bacteria from recombinant tyrosinase expression. Melanated bacterial cellulose production can be scaled up for the construction of prototype fashion products, and we illustrate the potential of combining engineered self-dyeing with tools from synthetic biology, via the optogenetic patterning of gene expression in cellulose-producing bacteria. With this work, we demonstrate that combining genetic engineering with current and future methods of textile biofabrication has the potential to create a new class of textiles.

synthetic biology↗