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bioRxiv2025

The E. coli PeptideAtlas Build: Characterizing the observed Escherichia coli pan-proteome and its post-translational modifications

Escherichia coli is a widely used model organism in molecular biology. Despite its pivotal role, a comprehensive proteome resource covering the E. coli pan-proteome and its post-translational modifications (PTMs) has been lacking. Here we present the E. coli PeptideAtlas build, the first comprehensive pan-proteome analysis of E. coli, generated from 40 high-quality public and in-house datasets spanning a broad diversity of strains, sample types, and experimental conditions, and comprising over 73 million MS/MS spectra. All datasets were reprocessed using both a closed search (Trans-Proteomic Pipeline using MSFragger) and an open search (ionbot). The E. coli PeptideAtlas build provides evidence for 4,755 proteins, including 1,410 previously lacking protein-level support in UniProt. The resource offers protein coverage, modification sites, raw spectra with matched peptides, and manually annotated metadata for the E. coli pan proteome. PTM profiling identified over 10,000 modification sites, including phosphorylation (3,806), acetylation (754), methylation (730), glutathionylation (352) and phosphoribosylation (226). Analysis of the glutathionylation sites revealed potential links to metal binding regulation. We also detected proteins likely associated with phages, underscoring the value of pan-proteomic approaches for studying host-phage interactions. All identifications are publicly accessible and traceable through the PeptideAtlas interface. We expect that the E. coli PeptideAtlas build will provide a useful resource for the community, which supports, for example, targeted MS experiment design, PTM enrichment method development, and strain typing. It allows straightforward lookups of protein and peptide identifications and facilitates comparative proteomic analyses by enabling the assessment of protein presence and variability across different E. coli strains. The build is available at https://db.systemsbiology.net/sbeams/cgi/PeptideAtlas/buildDetails?atlas_build_id=585.

bioinformatics↗

bioRxiv2025

Reducing the environmental footprint of laboratory research using visual communications: A pilot study in Spain

Addressing the environmental impact of laboratory research provides a meaningful opportunity for workplace-level mitigation. However, in Spain, the importance of sustainability in research is only starting to be recognised at a national level. We developed a scalable, visual-oriented communications strategy that was designed with input from scientific staff and included a live seminar and a coherent set of graphic materials. Five pillars were targeted: three key areas of consumption (energy, water, and plastic use), procurement, and colleague engagement. The scheme was piloted in two of the largest biomedical research centres in Spain: the Severo Ochoa Molecular Biology Centre (CBM) and the Spanish National Centre for Biotechnology (CNB). The pilot engaged 50 laboratories and services across both centres, of which 34 were followed up for 5 months. The results indicate engagement with sustainability measures and shifts toward best practice across metrics, including equipment switch-off, water use, plastic item reuse, and purchasing decisions. During the follow-up period, 23% of participants adopted new measures related to freezer maintenance, decluttering, or sample organization, 55% removed old data from storage to save energy, 100% reduced their use of lifts, and 94% discussed research sustainability with their laboratory/group. The findings also revealed key areas for improvement, including the management of shared resources and resistance to change protocols. These results offer a wide range of quantitative and qualitative insights to guide further efforts and scaling at regional and national levels. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/675379v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@b50baeorg.highwire.dtl.DTLVardef@11ccdeorg.highwire.dtl.DTLVardef@a13b46org.highwire.dtl.DTLVardef@13a9b0b_HPS_FORMAT_FIGEXP M_FIG C_FIG Sustainability spotlightThis paper describes a scalable, visual-oriented communications strategy that can be used to promote sustainable behaviours in research laboratories. We present pilot results indicating engagement in sustainability and shifts toward best practice across metrics. Our work contributes to UN Sustainable Development Goal (SDG) 9 (industry and infrastructure) and SDG 12 (responsible consumption and production).

scientific communication and education↗

bioRxiv2025

Characterisation of the Tilapia Lake Virus proteome and identification of an 11th protein, S9-F3

Tilapia Lake Virus (TiLV) is an emerging negative-sense, single-stranded RNA virus that poses a significant threat to global tilapia aquaculture. Of the proteins encoded by its ten genomic segments, the first four, which encode a viral polymerase (segments 1-3) and a nucleoprotein (segment 4) have been characterised. However, the functions of the polypeptides encoded by the remaining segments remain largely unknown. Here, we systematically investigated the expression and subcellular localisation of all ten predicted TiLV-encoded proteins using in vitro translation and expression in mammalian and fish cells as well as mass spectrometry of virus infected cells. These approaches confirmed the synthesis of major polypeptides from each segment and identified an additional 11th protein, S9-F3, translated from an alternative reading frame of segment 9. Microscopy of individually expressed green fluorescent protein (GFP)-tagged constructs revealed that S2 and S10 polypeptides were predominantly nuclear, while S1, S3, S5, S8 and S9-F3 were mostly cytoplasmic with S5 and S6 displaying perinuclear foci. Bioinformatic analysis suggested a potential nuclear export signal in S9-F3 and use of the inhibitor leptomycin B confirmed its CRM1-dependent nuclear export. Evolutionary analyses indicated that both S9 and S9-F3 are under selective pressure, as well as the presence of an S9-F3 homologue in a TiLV-like guppy virus. These findings uncover an alternative translation product and a regulated nuclear export mechanism in TiLV, providing new insights into the molecular biology of this virus and its interaction with host cellular pathways.

microbiology↗

bioRxiv2025

Investigating polarity effects in DNA base stacking

Nucleic acid structures are stabilized by both base pairing and base stacking. While energetics of base pairing interactions are relatively well established, our understanding of the energetic contributions of base stacking remain incomplete. Here, we use a combination of single-molecule and computational biophysics approaches to investigate the effect of strand polarity on base-stacking energetics. We designed pairs of DNA constructs with reversed stacking polarities at nick sites, along with corresponding no-stack controls to isolate stacking contributions. Performing single-molecule force-clamp assays with a Centrifuge Force Microscope (CFM), we observed polarity-dependent differences in stacking energetics. These differences were most pronounced in purine-purine and certain purine-pyrimidine interactions. Notably, a 5' purine stacked on a 3' pyrimidine was generally more stable than the reverse polarity. We employed molecular dynamics (MD) simulations to observe stacking interfaces in the DNA constructs. The simulations were qualitatively consistent with our experiments, and showed positional differences between opposite polarity stacking pairs, giving some insight into the origin of these polarity differences. Overall, these results demonstrate that base polarity can modulate stacking stability and should be considered when designing short duplex regions such as overhangs in molecular biology and biotechnology applications.

biophysics↗

bioRxiv2025

Metabolic Trans-Omic Analysis Reveals Key Regulatory Disruption of Energy Metabolism in Alzheimer's Disease

Alzheimers disease (AD), a leading cause of dementia, has been recognized as a disease with profound metabolic dysregulation. However, a systems-level view of metabolic regulation across multiple omic modalities in AD remains elusive. Here, we integrated public multi-omic datasets (transcriptome, proteome, and metabolome) from the dorsolateral prefrontal cortex of AD patients and controls. By leveraging existing molecular biological knowledge, we reconstructed a multi-layered metabolic regulatory network to systematically map the potential interplay among mRNAs, proteins, and metabolites in AD. Our analysis revealed a putative coordinated downregulation of energy producing pathways, including the TCA cycle, oxidative phosphorylation, and ketone body metabolism, driven by reduced enzyme abundance and inhibitory allosteric effects. In contrast, the glycolysis pathway appeared to be influenced by opposing enzymatic and allosteric regulations. These findings highlight key metabolic dysregulations that may contribute to the bioenergetic deficits in AD.

systems biology↗

bioRxiv2025

BindPred: A Framework for Predicting Protein-Protein Binding Affinity from Language Model Embeddings

MotivationReliable predictions of protein-protein binding affinities are essential for molecular biology and therapeutic discovery. However, most computational methods rely on three-dimensional structural models, which are often unavailable for many complexes. ResultsWe introduce BindPred, a structure-agnostic input framework that predicts affinities directly from amino acid sequences by combining embeddings from large protein language models with gradient boosting trees. On the PPB-Affinity benchmark, which comprises 11,919 diverse complexes, BindPred achieves a Pearson correlation coefficient of 0.86 in random split five-fold cross-validation, where the training and test sets share <30% global sequence identity. Ablation analysis indicates that evolutionary embeddings alone capture most of the predictive signals, while augmenting with physics-based energy terms from PyRosetta and BindCraft increases the correlation by only 0.01. A more stringent protein-level split that places entire protein families (wild-type and all mutants) exclusively in either training or testing sets, results in only a modest decline in performance, demonstrating robust generalization to novel interaction pairs. Because BindPred operates exclusively on sequence input, it enables rapid inference (approximately 3 million complexes per GPU (T4) hour), making proteome-scale screening computationally feasible. AvailabilityThe pretrained model and inference pipeline are available in a Google Colab notebook: BindPred Colab notebook. The training dataset, code, and model weights are available on the Hugging Face: BindPred Contactcostas@psu.edu Supplementary informationSupplementary data are available at Bioinformatics online.

bioinformatics↗

bioRxiv2025

Fluorescence in situ hybridization (FISH) and detection of Bacteria and Archaea cells in fecal samples

Oligonucleotide probes have been used to detect bacteria and archaea that colonize the cattle and pig digestive system. The Fluorescent in situ hybridization (FISH) is a molecular biology technique that uses oligonucleotides of 15 to 25 nucleotides of length associated with a fluorescent molecule. In microbiology, the FISH technique utilizes probes targeting ribosomal RNA (rRNA). It is one amongst others, a staining technique that allows the identification, detection and quantification of microorganisms without prior cultivation by means of epifluorescence and confocal laser scanning microscopy (CLSM). In this study, we describe the usage of the confocal laser scanning microscopy coupled with fluorescence in situ hybridization (FISH), in order to detect and quantify of bacteria and archaea in fecal samples from cattles manure and swine slurry.

microbiology↗

bioRxiv2025

Extracellular electron transfer in cable bacteria enables growth rates comparable to aerobic respiration

Cable bacteria are filamentous sulphide-oxidisers performing long-distance electron transfer in redox-stratified sediments by transporting electrons over centimetre-scale distances to reduce oxygen. Here, we show that the freshwater cable bacterium Electronema aureum GS can respire insoluble electron acceptors under anoxic conditions via a versatile extracellular electron transfer (EET) system, supporting growth rates comparable to those under aerobic conditions. Using electrochemical and molecular biology analyses, we demonstrate that E. aureum GS engages in both direct and mediated electron transfer to electrodes, including at +600 mV vs. Ag/AgCl--an unusually high redox potential typically not accessed by electroactive bacteria. Two distinct cell-surface redox components were identified, which are metal-dependent, pH-sensitive, and heat-labile, consistent with outer-membrane-localised cytochromes. Moreover, the redox shuttle riboflavin accumulated extracellularly and enhanced current production in bioelectrochemical systems, indicating a role for soluble mediators in cable bacteria. Together, these findings reveal a previously unrecognised respiratory flexibility in E. aureum GS and highlight EET as a key alternative strategy for energy conservation in fluctuating redox environments.

microbiology↗

bioRxiv2025

Tagging and harvesting of developing rice spikelets to demarcate grain developmental stages

PremiseIn rice, grain is the major sink for nutrient storage and is a rich source of carbohydrates, seed storage proteins, lipids, minerals, and vitamins, making it a principal food source for over half of the worlds population. Grain development in rice generally spans over a month and has been divided into five stages, S1-S5, depending upon various morphological and physiological changes occurring in the developing seed. To completely understand the process, rice grain development needs to be examined at each stage. However, there are no detailed protocols available to demarcate grain developmental stages. Methods and ResultsThis protocol describes how to tag and harvest the developing rice spikelets. The process starts with anthesis and continues till seed maturation, demarcating the five stages of rice grain development. Seeds are collected, labelled, and stored for use in molecular experiments. Using this protocol, we were able to identify and distinguish different seed developmental stages as observed by histochemical screening. ConclusionThis is an easy, efficient, and cost-effective method. This protocol ensures the collection of the entire panel of rice seed developmental stages. The stored tissue can later be used for various molecular biology based experiments.

plant biology↗

bioRxiv2025

Sulbactam-mediated inhibition of TEM-1 β-lactamase increases specific yields of ampicillin-resistant plasmids

Ampicillin resistance is the most widely used selection marker in molecular biology. Most ampicillin-resistant plasmids originate from highly active TEM-1 {beta}-lactamase cassette which causes very fast antibiotic decay and negatively affects long-term plasmid retention. The present work proposes a method of controlling the {beta}-lactamase activity by the addition of an irreversible {beta}-lactamase inhibitor. The positive effect of a {beta}-lactamase inhibitor, specifically sulbactam, on plasmid retention is confirmed by much higher specific plasmid yields: 4-8 times higher titres in shake-flask and almost 3 times higher in bioreactor scales were observed. Among all commercially available {beta}-lactamase inhibitors sulbactam provides the best combination of affordability and activity. The sulbactam addition is simple, fast and economic solution allowing to screen thousands of legacy pBR322 or pUC-derived plasmids harbouring TEM-1 {beta}-lactamase cassette without engineering their sequence.

microbiology↗

bioRxiv2025

Phospho-eIF2B-epsilon links stress response-driven translation to amyloidogenesis in Alzheimer's disease

A central etiopathogenic event in Alzheimers disease (AD) is the accumulation of amyloid {beta}-peptide (A{beta}) derived from the amyloidogenic processing of the amyloid precursor protein, a pathway initiated by BACE1. Chronic activation of the Integrated Stress Response is linked to AD, typically through eIF2 phosphorylation, which selectively enhances translation of key stress-responsive mRNAs like ATF4 and BACE1. We investigated a novel regulatory mechanism mediated by Glycogen Synthase Kinase-3{beta} (GSK-3{beta}) a hyperactive kinase in AD, on the translational factor eIF2B, the guanine nucleotide exchange factor (GEF) for eIF2. The methodology combined cellular and molecular biology approaches (western blot, ELISA, immunofluorescence, and luciferase assays) using pharmacological inhibitors and plasmid transfection in established cell models, along with computational structural modeling (AlphaFold3). Findings were validated in post-mortem human brain tissue, with statistical analyses (t-tests and ANOVA) applied throughout. We treated cells (SH-SY5Y) with a GSK-3{beta} inhibitor (NP031112) or overexpressed a constitutively active GSK-3{beta} mutant (GSK-3{beta}-S9A) to modulate GSK-3{beta} activity. GSK-3{beta} inhibition significantly reduced ATF4 and BACE1 protein levels in SH-SY5Y cells in a dose-dependent manner as we tested by western blot (P<0.001), independent of eIF2 phosphorylation. This effect was translational, as the inhibitor still reduced BACE1 levels when transcription was blocked. Crucially, NP031112 reversed stress-induced eIF2B{varepsilon} phosphorylation at Ser540 (P<0.001). Furthermore, H2O2-induced A{beta}1-42 secretion by SH-SY5Y cells was significantly reversed by GSK-3{beta} inhibition (P<0.001). Luciferase reporter assays using the BACE1 5 untranslated region (5 UTR) confirmed that both eIF2B{varepsilon} silencing and a phosphomimetic mutant eIF2B{varepsilon}-S540E increased BACE1 5 UTR-driven translation (P<0.001), demonstrating that reduced functional eIF2B enhances BACE1 translation. Finally, immunohistofluorescence and western blot analysis of human AD hippocampi showed a significant increase in pS540-eIF2B{varepsilon}, ATF4, and BACE1 levels in AD patients compared to non-demented controls (P<0.001 for all three). Our findings establish a novel GSK-3{beta}-eIF2B{varepsilon}-BACE1 axis that links stress response dysregulation to amyloidogenesis, independent of canonical eIF2 phosphorylation. The phosphorylation of eIF2B{varepsilon} functionally mimics the effects of eIF2 phosphorylation by reducing the available functional eIF2B pool, thereby reprogramming translation to favour the production of stress proteins like ATF4 and BACE1. We proposed eIF2B{varepsilon} phosphorylation is a key etiological mechanism in AD.

neuroscience↗

bioRxiv2025

How do selective serotonin reuptake inhibitors effect metabolism? A lesson for teaching non-majors about enzymes and metabolic pathways

Understanding foundational concepts in molecular biology and biochemistry can be challenging for many college students. For non-science majors, an introductory biology course may be their sole exposure to college-level science and only opportunity to learn about enzymes and metabolism. Framing these topics within issues that are personally relevant can make the material more meaningful and impactful for non-majors. There is a need to develop curricula that instructors of large-enrollment introductory biology courses can use to teach non-biology majors about enzymes and metabolism. Here, we designed and implemented a lesson that integrates enzyme function and metabolism within the context of selective serotonin reuptake inhibitors (SSRIs). SSRIs are a widely prescribed class of antidepressants whose efficacy can be influenced by enzymes. Genetic polymorphisms in the genes encoding these enzymes can result in individual differences in metabolic responses and lead to medication-induced weight gain. Framing the content in this way allows non-major students to connect enzyme function to genetics, energy balance, and metabolic processes through a health-related, societally relevant issue. In our lesson, students engaged in collaborative learning activities that emphasized interpreting biological data, graphing, and scientific reasoning. Pre- and post-assessment data from a conceptual inventory showed learning gains that exceeded baseline data and were greater than those observed with the traditional curriculum. Students also reported a more coherent understanding of enzyme function and a greater appreciation of the contents relevance to their own lives. This lesson provides an example of embedding biochemical concepts in real-world contexts for students who are non-science majors.

scientific communication and education↗

bioRxiv2025

Identifying Phage Host Receptors Using TraDIS

The growing antimicrobial resistance crisis has sparked renewed interest in using bacteriophage (phage) as a treatment for antibiotic-resistant infections. Phage are viruses that infect and often kill bacteria, and have served as key models for many molecular biology studies. There are many complex phage-bacterial interactions that dictate the success or failure of the phage life cycle, but many of these are not well understood or characterised. These include the specific interactions to recognize a bacterium during the infection process, bacterial phage defence mechanisms and metabolic pathways that are key to phage replication. In this study a novel Escherichia coli phage, Arnold, is characterised and the genome sequence is reported. We then use transposon-directed insertion sequencing (TraDIS) to screen a library of E. coli mutants to identify over 200 genes involved in Arnold phage infection or in phage resistance. Using this approach, we identified both an outer membrane protein (BtuB, vitamin B12 transporter) and an inner membrane protein (SdaC, serine/proton symporter) involved in phage infection. Deletions in these genes were shown to provide immunity to infection by this phage. Understanding the genetic basis of phage infection and phage resistance should allow us to select or design phage to target specific bacterial pathogens in the future.

microbiology↗

bioRxiv2017

A toolbox of anti-mouse and rabbit IgG secondary nanobodies

Polyclonal anti-IgG secondary antibodies are essential tools for many molecular biology techniques and diagnostic tests. Their animal-based production is, however, a major ethical problem. Here, we introduce a sustainable alternative, namely nanobodies against all mouse IgG subclasses and rabbit IgG. They can be produced at large scale in E. coli and could thus make secondary antibody-production in animals obsolete. Their recombinant nature allows fusion with affinity tags or reporter enzymes as well as efficient maleimide chemistry for fluorophore-coupling. We demonstrate their superior performance in Western Blotting, both in peroxidase- and fluorophore-linked form. Their site-specific labeling with multiple fluorophores creates bright imaging reagents for confocal and super-resolution microscopy with much smaller label displacement than traditional secondary antibodies. They also enable simpler and faster immunostaining protocols and even allow multi-target localization with primary IgGs from the same species and of the same class.

biochemistry↗

bioRxiv2017

MAPLE: a Modular Automated Platform for Large-scale Experiments, a low-cost robot for integrated animal-handling and phenotyping

Genetic model system animals have significant scientific value in part because of large-scale experiments like screens, but performing such experiments over long time periods by hand is arduous and risks errors. Thus the field is poised to benefit from automation, just as molecular biology did from liquid-handling robots. We developed a Modular Automated Platform for Large-scale Experiments (MAPLE), a Drosophila-handling robot capable of conducting lab tasks and experiments. We demonstrate MAPLEs ability to accelerate the collection of virgin female flies (a pervasive experimental chore in fly genetics) and assist high-throughput phenotyping assays. Using MAPLE to autonomously run a novel social interaction experiment, we found that 1) pairs of flies exhibit persistent idiosyncrasies in affiliative behavior, 2) these dyad-specific interactions require olfactory and visual cues, and 3) social interaction network structure is topologically stable over time. These diverse examples demonstrate MAPLEs versatility as a general platform for conducting fly science automatically.

bioengineering↗

bioRxiv2018

Time- and cost-efficient high-throughput transcriptomics enabled by Bulk RNA Barcoding and sequencing

Genome-wide gene expression analyses by RNA sequencing (RNA-seq) have quickly become a standard in molecular biology because of the widespread availability of high throughput sequencing technologies. While powerful, RNA-seq still has several limitations, including the time and cost of library preparation, which makes it difficult to profile many samples simultaneously. To deal with these constraints, the single-cell transcriptomics field has implemented the early multiplexing principle, making the library preparation of hundreds of samples (cells) markedly more affordable. However, the current standard methods for bulk transcriptomics (such as TruSeq Stranded mRNA) remain expensive, and relatively little effort has been invested to develop cheaper, but equally robust methods. Here, we present a novel approach, Bulk RNA Barcoding and sequencing (BRB-seq), that combines the multiplexing-driven cost-effectiveness of a single-cell RNA-seq workflow with the performance of a bulk RNA-seq procedure. BRB-seq produces 3 enriched cDNA libraries that exhibit similar gene expression quantification to TruSeq and that maintain this quality, also in terms of number of detected differentially expressed genes, even with low quality RNA samples. We show that BRB-seq is about 25 times less expensive than TruSeq, enabling the generation of ready to sequence libraries for up to 192 samples in a day with only 2 hours of hands-on time. We conclude that BRB-seq constitutes a powerful alternative to TruSeq as a standard bulk RNA-seq approach. Moreover, we anticipate that this novel method will eventually replace RT-qPCR-based gene expression screens given its capacity to generate genome-wide transcriptomic data at a cost that is comparable to profiling 4 genes using RT-qPCR.\n\n SoftwareWe developed a suite of open source tools (BRB-seqTools) to aid with processing BRB-seq data and generating count matrices that are used for further analyses. This suite can perform demultiplexing, generate count/UMI matrices and trim BRB-seq constructs and is freely available at http://github.com/DeplanckeLab/BRB-seqTools\n\nHighlightsO_LIRapid (~2h hands on time) and low-cost approach to perform transcriptomics on hundreds of RNA samples\nC_LIO_LIStrand specificity preserved\nC_LIO_LIPerformance: number of detected genes is equal to Illumina TruSeq Stranded mRNA at same sequencing depth\nC_LIO_LIHigh capacity: low cost allows increasing the number of biological replicates\nC_LIO_LIProduces reliable data even with low quality RNA samples (down to RIN value = 2)\nC_LIO_LIComplete user-friendly sequencing data pre-processing and analysis pipeline allowing result acquisition in a day\nC_LI

genomics↗

bioRxiv2018

Engineering of new-to-nature halogenated indigo precursors in plants

Plants are versatile chemists producing a tremendous variety of specialized compounds. Here, we describe the engineering of entirely novel metabolic pathways in planta enabling generation of halogenated indigo precursors as non-natural plant products.\n\nIndican (indolyl-{beta}-D-glucopyranoside) is a secondary metabolite characteristic of a number of dyers plants. Its deglucosylation and subsequent oxidative dimerization leads to the blue dye, indigo. Halogenated indican derivatives are commonly used as detection reagents in histochemical and molecular biology applications; their production, however, relies largely on chemical synthesis. To attain the de novo biosynthesis in a plant-based system devoid of indican, we employed a sequence of enzymes from diverse sources, including three microbial tryptophan halogenases substituting the amino acid at either C5, C6, or C7 of the indole moiety. Subsequent processing of the halotryptophan by bacterial tryptophanase TnaA in concert with a mutant of the human cytochrome P450 monooxygenase 2A6 and glycosylation of the resulting indoxyl derivatives by an endogenous tobacco glucosyltransferase yielded corresponding haloindican variants in transiently transformed Nicotiana benthamiana plants. Accumulation levels were highest when the 5-halogenase PyrH was utilized, reaching 0.93 {+/-}0.089 mg/g dry weight of 5-chloroindican. The identity of the latter was unambiguously confirmed by NMR analysis. Moreover, our combinatorial approach, facilitated by the modular assembly capabilities of the GoldenBraid cloning system and inspired by the unique compartmentation of plant cells, afforded testing a number of alternative subcellular localizations for pathway design. In consequence, chloroplasts were validated as functional biosynthetic venues for haloindican, with the requisite reducing augmentation of the halogenases as well as the cytochrome P450 monooxygenase fulfilled by catalytic systems native to the organelle.\n\nThus, our study puts forward a viable alternative production platform for halogenated fine chemicals, eschewing reliance on fossil fuel resources and toxic chemicals. We further contend that in planta generation of halogenated indigoid precursors previously unknown to nature offers an extended view on and, indeed, pushes forward the established frontiers of biosynthetic capacity of plants.\n\nGraphical abstract\n\nHighlightsO_LIEntirely novel combinatorial pathway yielded new-to-nature halogenated indigoids.\nC_LIO_LIAn array of specific chloro- and bromoindican regioisomers was generated.\nC_LIO_LISignificant retrieval rates afforded unambiguous metabolite identification.\nC_LIO_LIEngineered plants emerge as alternative manufacture chassis for fine chemicals.\nC_LI

synthetic biology↗

bioRxiv2018

CONSTRUCTION OF WHOLE GENOMES FROM SCAFFOLDS USING SINGLE CELL STRAND-SEQ DATA

Accurate reference genome sequences provide the foundation for modern molecular biology and genomics as the interpretation of sequence data to study evolution, gene expression and epigenetics depends heavily on the quality of the genome assembly used for its alignment. Correctly organising sequenced fragments such as contigs and scaffolds in relation to each other is a critical and often challenging step in the construction of robust genome references. We previously identified misoriented regions in the mouse and human reference assemblies using Strand-seq, a single cell sequencing technique that preserves DNA directionality1, 2. Here we demonstrate the ability of Strand-seq to build and correct full-length chromosomes, by identifying which scaffolds belong to the same chromosome and determining their correct order and orientation, without the need for overlapping sequences. We demonstrate that Strand-seq exquisitely maps assembly fragments into large related groups and chromosome-sized clusters without using new assembly data. Using template strand inheritance as a bi-allelic marker, we employ genetic mapping principles to cluster scaffolds that are derived from the same chromosome and order them within the chromosome based solely on directionality of DNA strand inheritance. We prove the utility of our approach by generating improved genome assemblies for several model organisms including the ferret, pig, Xenopus, zebrafish, Tasmanian devil and the Guinea pig.

genomics↗