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bioRxiv2021

A systems-level analysis of dynamic total-body PET data reveals complex skeletal energy metabolism networks in vivo

Bone is now regarded to be a key regulator of a number of metabolic processes, in addition to the regulation of mineral metabolism. However, our understanding of complex bone metabolic interactions at a systems level remains rudimentary, limiting our ability to assess systemic mechanisms underlying diseases and develop novel therapeutics. In vitro molecular biology and bioinformatics approaches have frequently been used to understand the mechanistic changes underlying disease at the cell level, however, these approaches lack the capability to interrogate dynamic multi-bone metabolic interactions in vivo. Here we present a novel and integrative approach to understand complex bone metabolic interactions in vivo using total-body positron emission tomography (PET) network analysis of murine 18F-FDG scans, as a biomarker of glucose metabolism signature in bones. In this report we show that different bones within the skeleton have a unique glucose metabolism and form a complex metabolic network. These data could have important therapeutic implications in the management of the metabolic syndrome and skeletal disease. The application of our approach to clinical and preclinical total-body PET studies promises to reveal further physiological and pathological tissue interactions, which simplistic PET standard uptake values analysis fail to interrogate, extending beyond skeletal metabolism, due to the diversity of PET radiotracers available and under development as well as the advent of clinical total-body PET systems. One Sentence SummaryBones form a complex metabolic network.

physiology↗

bioRxiv2021

Interpretable Drug Target Predictions using Self-Expressiveness

The identification of missing drug targets is critical for the development of treatments and for the molecular elucidation of drug side effects. Drug targets have been predicted by exploiting molecular, biological or pharmacological features of drugs and protein targets. Yet, developing integrative and interpretable machine learning models for predicting drug targets remains a challenging task. We present Inception, an integrative and interpretable matrix completion model for predicting drug targets. Inception is a self-expressive model that learns two similarity matrices: one for drugs and another for protein targets. These learned similarity matrices are key for our models interpretability: they can explain how a predicted drug-target interaction can be explain in terms of a linear combination of chemical, biological and pharmacological similarities. We develop a novel objective function with efficient closed-form solution. To demonstrate the ability of Inception at recovering missing drug-target interactions (DTIs), we perform cross-validation experiments with stringent controls of data imbalance, chemical similarities between drugs and sequence similarities between targets. We also assess the performance of our model using a simulated prospective approach. Having trained our model with DTIs from a snapshot 2011 of the DrugBank database, we test whether we could predict DTIs from a 2020 snapshot of DrugBank. Inception outperforms two state-of-the-art drug target prediction models in all the scenarios. This suggests that Inception could be useful for predicting missing drug target interactions while providing interpretable predictions.

bioinformatics↗

bioRxiv2021

Sexual antagonism in haplodiploids

Females and males may face different selection pressures, such that alleles conferring a benefit in one sex may be deleterious in the other. Such sexual antagonism has received a great deal of theoretical and empirical attention, almost all of which has focused on diploids. However, a sizeable minority of animals display an alternative haplodiploid mode of inheritance, encompassing both arrhenotoky, whereby males develop from unfertilized eggs, and paternal genome elimination (PGE), whereby males receive but do not transmit a paternal genome. Alongside unusual genetics, haplodiploids often exhibit social ecologies that modulate the relative value of females and males. Here we develop a series of evolutionary-genetic models of sexual antagonism for haplodiploids, incorporating details of their molecular biology and social ecology. We find that: 1) PGE promotes female-beneficial alleles more than arrhenotoky, and to an extent determined by the timing of elimination - and degree of silencing of - the paternal genome; 2) sib-mating relatively promotes female-beneficial alleles, as do other forms of inbreeding, including limited male-dispersal, oedipal-mating, and the pseudo-hermaphroditism of Icerya purchasi; 3) resource competition between related females relatively inhibits female-beneficial alleles; and 4) sexual antagonism foments conflicts between parents and offspring, endosymbionts and hosts, and maternal-origin and paternal-origin genes.

evolutionary biology↗

bioRxiv2021

Novel insights in the pathophysiology of α-synuclein dysregulation on D2 receptor activity contributing to the vulnerability of dopamine neurons

Pathophysiological damages and loss of function of dopamine neurons precedes their demise and contributes to the early phases of Parkinsons disease. The presence of aberrant intercellular pathological inclusions of the protein -synuclein within ventral midbrain dopaminergic neurons is one of the cardinal features of Parkinsons disease. We employed multiple complementary approaches in molecular biology, electrophysiology, and live-cell imaging to investigate how excessive -synuclein levels alters multiple characteristics of dopaminergic neuronal dynamics and dopamine transmission prior to neuronal demise. These studies demonstrate that -synuclein dysregulation of D2 receptor autoinhibition contributes to the vulnerability of dopaminergic neurons, and that modulation thereof can ameliorate the resulting pathophysiology. These novel findings provide mechanistic insights in the insidious loss of dopaminergic function and neurons that characterize Parkinsons disease progression with significant therapeutic implications.

neuroscience↗

bioRxiv2021

Towards community-driven metadata standards for light microscopy: tiered specifications extending the OME model

1 -Digital light microscopy provides powerful tools for quantitatively probing the real-time dynamics of subcellular structures. While the power of modern microscopy techniques is undeniable, rigorous record-keeping and quality control are required to ensure that imaging data may be properly interpreted (quality), reproduced (reproducibility), and used to extract reliable information and scientific knowledge which can be shared for further analysis (value). Keeping notes on microscopy experiments and quality control procedures ought to be straightforward, as the microscope is a machine whose components are defined and the performance measurable. Nevertheless, to this date, no universally adopted community-driven specifications exist that delineate the required information about the microscope hardware and acquisition settings (i.e., microscopy "data provenance" metadata) and the minimally accepted calibration metrics (i.e., microscopy quality control metadata) that should be automatically recorded by both commercial microscope manufacturers and customized microscope developers. In the absence of agreed guidelines, it is inherently difficult for scientists to create comprehensive records of imaging experiments and ensure the quality of resulting image data or for manufacturers to incorporate standardized reporting and performance metrics. To add to the confusion, microscopy experiments vary greatly in aim and complexity, ranging from purely descriptive work to complex, quantitative and even sub-resolution studies that require more detailed reporting and quality control measures. To solve this problem, the 4D Nucleome Initiative (4DN) (1, 2) Imaging Standards Working Group (IWG), working in conjunction with the BioImaging North America (BINA) Quality Control and Data Management Working Group (QC-DM-WG) (3), here propose light Microscopy Metadata specifications that scale with experimental intent and with the complexity of the instrumentation and analytical requirements. They consist of a revision of the Core of the Open Microscopy Environment (OME) Data Model, which forms the basis for the widely adopted Bio-Formats library (4-6), accompanied by a suite of three extensions, each with three tiers, allowing the classification of imaging experiments into levels of increasing imaging and analytical complexity (7, 8). Hence these specifications not only provide an OME-based comprehensive set of metadata elements that should be recorded, but they also specify which subset of the full list should be recorded for a given experimental tier. In order to evaluate the extent of community interest, an extensive outreach effort was conducted to present the proposed metadata specifications to members of several core-facilities and international bioimaging initiatives including the European Light Microscopy Initiative (ELMI), Global BioImaging (GBI), and European Molecular Biology Laboratory (EMBL) - European Bioinformatics Institute (EBI). Consequently, close ties were established between our endeavour and the undertakings of the recently established QUAlity Assessment and REProducibility for Instruments and Images in Light Microscopy global community initiative (9). As a result this flexible 4DN-BINA-OME (NBO namespace) framework (7, 8) represents a turning point towards achieving community-driven Microscopy Metadata standards that will increase data fidelity, improve repeatability and reproducibility, ease future analysis and facilitate the verifiable comparison of different datasets, experimental setups, and assays, and it demonstrates the method for future extensions. Such universally accepted microscopy standards would serve a similar purpose as the Encode guidelines successfully adopted by the genomic community (10, 11). The intention of this proposal is therefore to encourage participation, critiques and contributions from the entire imaging community and all stakeholders, including research and imaging scientists, facility personnel, instrument manufacturers, software developers, standards organizations, scientific publishers, and funders.

bioinformatics↗

bioRxiv2021

Dedifferentiation alters chondrocyte nuclear mechanics during in vitro culture and expansion

Dedifferentiation of chondrocytes during in vitro passaging before implantation, and post implantation in vivo, is a critical limitation in cartilage tissue engineering. Several biophysical features define the dedifferentiated state including a flattened cell morphology and increased stress fiber formation. However, how dedifferentiation influences nuclear mechanics, and the possible long-term implications of this state, are unknown. In this study, we investigated how chondrocyte dedifferentiation affects the mechanics of the chromatin architecture inside the cell nucleus and the gene expression of the structural proteins located at the nuclear envelope. Through an experimental model of cell stretching and a detailed spatial intranuclear strain quantification, we identified that strain is amplified and distribution of strain within the chromatin is altered under tensile loading in the dedifferentiated state. Further, using a confocal microscopy image-based finite element model and simulation of cell stretching, we found that the cell shape is the primary determinant of the strain amplification inside the chondrocyte nucleus in the dedifferentiated state. Additionally, we found that nuclear envelope proteins have lower gene expression in the dedifferentiated state suggesting a weaker nuclear envelope which can further intensify the intranuclear strain amplification. Our results indicate that dedifferentiation and altered nuclear strain could promote gene expression changes at the nuclear envelope, thus promoting further deviation from chondrocyte phenotype. This study highlights the role of cell shape on nuclear mechanics and lays the groundwork to design biophysical strategies for the maintenance and enhancement of the chondrocyte phenotype during expansion with a goal of successful cartilage tissue engineering. SIGNIFICANCEChondrocytes dedifferentiate into a fibroblast-like phenotype in a non-native biophysical environment. Using high resolution microscopy, intranuclear strain analysis, finite element method based computational modeling, and molecular biology techniques, we investigated how mechanical force causes abnormal intranuclear strain distribution in chondrocytes during the dedifferentiation process. Overall, our results suggest that the altered cell geometry aided by an altered or weakened nuclear envelope structure are responsible for abnormal intranuclear strain during chondrocyte dedifferentiation that can further deviate chondrocytes to a more dedifferentiated state.

biophysics↗

bioRxiv2021

A nanoscale reciprocating rotary mechanism with coordinated mobility control

Biological molecular motors transform chemical energy into mechanical work by coupling a cycle of catalytic reactions to large scale structural transitions. Mechanical deformation can be surprisingly efficient in realizing such coupling, as demonstrated by the celebrated example of F1Fo ATP synthase. Here, we describe a synthetic molecular mechanism that transforms a rotary motion of an asymmetric camshaft into reciprocating large-scale transitions in the structure of the surrounding stator orchestrated by mechanical deformation. We designed the mechanism using DNA origami, characterized the structure of the components and the entire mechanism using cryo-electron microscopy, and examined the mechanisms dynamic behavior using single-particle fluorescence microscopy and molecular dynamics simulations. The data indicates that, while the camshaft can rotate inside the stator by diffusion, mechanical deformation of the stator makes the camshaft pause at a set of preferred orientations. By changing the mechanical stiffness of the stator, we could accelerate or suppress the Brownian rotation within the mechanism, thereby demonstrating an allosteric coupling between the movement of the camshaft and of the stator, and the ability to tailor the free energy landscape that governs the rotary motion. Our mechanism provides a framework for the manufacture of artificial nanomachines that, just like the man-made machines in the macroscopic world, function because of coordinated movements of their components.

biophysics↗

bioRxiv2021

Sustained coevolution of phage Lambda and Escherichia coli involves inner as well as outer membrane defenses and counter-defenses

Bacteria often evolve resistance to phage through the loss or modification of cell-surface receptors. In Escherichia coli and phage {lambda}, such resistance can catalyze a coevolutionary arms race focused on host and phage structures that interact at the outer membrane. Here, we analyze another facet of this arms race involving interactions at the inner membrane, whereby E. coli evolves mutations in mannose permease-encoding genes manY and manZ that impair {lambda}s ability to eject its DNA into the cytoplasm. We show that these man mutants arose concurrently with the arms race at the outer membrane. We tested the hypothesis that {lambda} evolved an additional counter-defense that allowed them to infect bacteria with deleted man genes. The deletions severely impaired the ancestral {lambda}, but some evolved phage grew well on the deletion mutants, indicating they regained infectivity by evolving the ability to infect hosts independently of the mannose permease. This coevolutionary arms race fulfills the model of an inverse-gene-for-gene infection network. Taken together, the interactions at both the outer and inner membranes reveal that coevolutionary arms races can be richer and more complex than is often appreciated. IMPACT STATEMENTLaboratory studies of coevolution help us understand how host defenses and pathogen counter-defenses change over time, which is often essential for predicting the future dynamics of host-pathogen interactions. One particular model, termed "inverse-gene-for-gene" coevolution, predicts that coevolution proceeds through alternating steps, whereby hosts lose the features exploited by pathogens, and pathogens evolve to exploit alternative features. Using a classic model system in molecular biology, we describe the nature and timing of a previously overlooked step in the coevolution of E. coli and bacteriophage lambda. Our work demonstrates that this mode of coevolution can profoundly re-shape the interactions between bacteria and phage.

microbiology↗

bioRxiv2021

The Core Human Fecal Metabolome

Summary ParagraphAmong the biomolecules at the center of human health and molecular biology is a system of molecules that defines the human phenotype known as the metabolome. Through an untargeted metabolomic analysis of fecal samples from human individuals from Africa and the Americas--the birthplace and the last continental expansion of our species, respectively--we present the characterization of the core human fecal metabolome. The majority of detected metabolite features were ubiquitous across populations, despite any geographic, dietary, or behavioral differences. Such shared metabolite features included hyocholic acid and cholesterol. However, any characterization of the core human fecal metabolome is insufficient without exploring the influence of industrialization. Here, we show chemical differences along an industrialization gradient, where the degree of industrialization correlates with metabolomic changes. We identified differential metabolite features like leucyl-leucine dipeptides and urobilin as major metabolic correlates of these behavioral shifts. Our results indicate that industrialization significantly influences the human fecal metabolome, but diverse human lifestyles and behavior still maintain a core human fecal metabolome. This study represents the first characterization of the core human fecal metabolome through untargeted analyses of populations along an industrialization gradient.

biochemistry↗

bioRxiv2021

Rational Drug Design of Targeted and Enzyme Cleavable Vitamin E Analogs as Neoadjuvant to Chemotherapy: In Vitro and In Vivo Evaluation on Reduction of Cardiotoxicity of Doxorubicin.

Traditional drug design focus on specific target (s) expressed by cancer cells. However, cancer cells outsmart the interventions by activating survival pathways and/or downregulating cell death pathways. As the research in molecular biology of cancer grows exponentially, new methods of drug designs are needed to target multiple pathways/targets which are involved in survival of cancer cells. Vitamin E analogues including a-tocopheryl succinate (-TOS) is a well-known anti-tumoregenic agent which is well studied both in vitro and in vivo tumor models. However, lack of targeting cancer cells and unexpected toxicity along with the poor water solubility of -TOS compelled a rational drug design using both targeting and cleavable technologies incorporated in the new drug design. A plethora of Vitamin E derivatives (AMP-001, 002 and 003) were synthesized, characterized and studied for the improved efficacy and lowered toxicity in various cancer cells in vitro. Preliminary studies revealed AAAPT leading candidates reduced the invasive potential of brain tumor stem cells, synergized with different drugs and different treatments. AAAPT leading drug AMP-001 enhanced the therapeutic index of front-line drug Doxorubicin in triple negative breast cancer (TNBC) tumor rat model preserving the ventricular function when used as a neoadjuvant to Doxorubicin. These results may pave the way for reducing the cardiotoxicity of chemotherapy in clinical settings.

cancer biology↗

bioRxiv2021

Arousal-dependent auditory response in the brain of Bengalese finches as measured by gene expression

Songbirds use auditory feedback to maintain their own songs. Juveniles also memorize a tutor song and use memory as a template to make up their own songs through auditory feedback. A recent electrophysiological study revealed that HVC neurons respond to BOS playback only in low arousal, sleeping, or anesthetized conditions. One outstanding question is how does auditory suppression occur in the brain? Here, we determined how arousal affects auditory responses simultaneously in the whole brain and over the song neural circuit in Bengalese finches, using the immediate early gene egr-1 as a marker of neural activity. Our results showed that auditory responses in the low-arousal state were less susceptible to gating, which was also confirmed by gene expression, and that the suppression may be weaker than observed in previous zebra finch studies. This may be because the Bengalese finch is a domesticated species. In addition, our results suggest that information may flow from the MLd.I of the midbrain to higher auditory regions. Altogether, this study presents a new attempt to explore the auditory suppression network by simultaneously investigating the whole brain using molecular biology methods.

neuroscience↗

bioRxiv2021

Sequence analysis paradigm shift reveals unsuspected semantic properties of species proteomes

Alignment-based methods dominate molecular biology. However, by primarily allowing one-to-one comparisons, these methods are focused on a gene-centered viewpoint and lack the broad context essential to analyze how complex biological systems function and evolve. In actuality, a gene is part of genome where more than one sequence contributes to the functional network and evolutionary trajectory of the cell. The need for conservation of established interactions, is arguably more important to the evolutionary success of species than conservation of individual function. To test whether such contextual information exists, a distributional semantics method - Latent Semantic Analysis (LSA), was applied to thousands of species proteomes. Using natural language processing, Latent Taxonomic Signatures (LTSs) were identified that outperformed existing alignment-based BLAST methods when random protein sequences were being mapped to annotated taxonomy according to GenBank. LTSs are a novel proteome distributed feature, suggesting the existence of evolutionary constraints imposed on individual proteins by their proteome context. Even orphan proteins are exhibiting LTSs, which makes their uniqueness linked to a specific taxonomic level questionable. Unlike more simple bias, LTSs represent a self-similarity pattern, where random sets of species proteins show the same statistical properties of a complete proteome at many scales. Natural language processing and machine learning provide insights not easily discernable using alignment based methods suggestive there is more to species related differences than just translational optimization.

systems biology↗

bioRxiv2021

High-quality Arabidopsis thaliana genome assembly with Nanopore and HiFi long reads

Arabidopsis thaliana is an important and long-established model species for plant molecular biology, genetics, epigenetics, and genomics. However, the latest version of reference genome still contains significant number of missing segments. Here, we report a high-quality and almost complete Col-0 genome assembly with two gaps (Col-XJTU) using combination of Oxford Nanopore Technology ultra-long reads, PacBio high-fidelity long reads, and Hi-C data. The total genome assembly size is 133,725,193 bp, introducing 14.6 Mb of novel sequences compared to the TAIR10.1 reference genome. All five chromosomes of Col-XJTU assembly are highly accurate with consensus quality (QV) scores > 60 (ranging from 62 to 68), which are higher than those of TAIR10.1 reference (QV scores ranging from 45 to 52). We have completely resolved chromosome (Chr) 3 and Chr5 in a telomere-to-telomere manner. Chr4 has been completely resolved except the nucleolar organizing regions, which comprise long repetitive DNA fragments. The Chr1 centromere (CEN1), reportedly around 9 Mb in length, is particularly challenging to assemble due to the presence of tens of thousands of CEN180 satellite repeats. Using the cutting-edge sequencing data and novel computational approaches, we assembled about 4 Mb of sequence for CEN1 and a 3.5-Mb-long CEN2. We investigated the structure and epigenetics of centromeres. We detected four clusters of CEN180 monomers, and found that the centromere-specific histone H3-like protein (CENH3) exhibits a strong preference for CEN180 cluster 3. Moreover, we observed hypomethylation patterns in CENH3-enriched regions. We believe that this high-quality genome assembly, Col-XJTU, would serve as a valuable reference to better understand the global pattern of centromeric polymorphisms, as well as genetic and epigenetic features in plants.

genomics↗

bioRxiv2021

Spatial and temporal constraints on the composition of microbial communities in subsurface boreholes of the Edgar Experimental Mine

The deep biosphere hosts uniquely adapted microorganisms overcoming geochemical extremes at significant depths within the crust of the Earth. While numerous novel microbial members with unique physiological modifications remain to be identified, even greater attention is required to understand the near-subsurface and its continuity with surface systems. This raises key questions about networking of surface hydrology, geochemistry affecting near-subsurface microbial composition, and resiliency of subsurface ecosystems. Here, we apply molecular and geochemical approaches to determine temporal microbial composition and environmental conditions of filtered borehole fluid from the Edgar Experimental Mine ([~]150 meters below the surface) in Idaho Springs, CO. Samples were collected over a 4-year collection period from expandable packers deployed to accumulate fluid in previously drilled boreholes located centimeters to meters apart, revealing temporal evolution of borehole microbiology. Meteoric water feeding boreholes demonstrated variable recharge rates likely due to a complex and undefined fracture system within the host rock. 16S rRNA gene analysis determined unique microbial communities occupy the four boreholes examined. Two boreholes yielded sequences revealing the presence of Proteobacteria, Firmicutes, and Nanoarcheota associated with endemic subsurface communities. Two other boreholes presented sequences related to soil-originating microbiota, which likely indicate a direct link to surface infiltration. High concentrations of sulfate suggest sulfur-related metabolic strategies dominate within these near-subsurface boreholes. Overall, results indicate microbial community composition in the near-subsurface is highly dynamic at very fine spatial scales (<20cm) within fluid-rock equilibrated boreholes, which additionally supports the role of a relationship for surface geochemical processes infiltrating and influencing subsurface environments. ImportanceThe Edgar Experimental Mine, Idaho Springs, Colorado provides inexpensive and open access to borehole investigations for subsurface microbiology studies. Understanding how microbial processes in the near-subsurface are connected to surface hydrological influences like meteoric input is lacking. Investigating microbial communities of subsurface mine boreholes provides evidence of how geochemical processes are linked to biogeochemical processes within each borehole, and the geochemical connectedness and mobility of surface influences. This study details microbial community composition and fluid geochemistry over spatial and temporal scales from boreholes within the Edgar Mine. These findings are relevant to biogeochemistry of near-surface mines, caves and other voids across planetary terrestrial systems. In addition, this work can lead to understanding how microbial communities relating to both fluid-rock equilibration and geochemical influences may enhance our understanding of subsurface molecular biological tools that aid mining economic practices to reflect biological signals for lucrative veins in the near subsurface.

microbiology↗

bioRxiv2021

Unexpected gene activation following CRISPR-Cas9-mediated genome editing

The discovery of the Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR) and its development as a genome editing tool has revolutionized the field of molecular biology. In the DNA damage field, CRISPR has brought an alternative to induce endogenous double-strand breaks (DSB) at desired genomic locations and study the DNA damage response and its consequences. Many systems for sgRNA delivery have been reported in order to efficiency generate this DSB, including lentiviral vectors. However, some of the consequences of these systems are yet not well understood. Here we report that lentiviral-based sgRNA vectors can integrate into the endogenous genomic target location, leading to undesired activation of the target gene. By generating a DSB in the regulatory region of the ABCB1 gene using a lentiviral sgRNA vector, we can induce the formation of taxol-resistant colonies. We show that these colonies upregulated ABCB1 via integration of the EEF1A1 and the U6 promoters from the sgRNA vector. We believe that this is an unreported CRISPR/Cas9 artefact that researchers need to be aware of when using lentiviral vectors for genome editing.

genomics↗

bioRxiv2021

Structural analysis of 3'UTRs in insect flaviviruses reveals novel determinant of sfRNA biogenesis and provides new insights into flavivirus evolution

Insect-specific flaviviruses (ISFs) circulate in nature due to vertical transmission in mosquitoes and do not infect vertebrates. ISFs include two distinct lineages - classical ISFs (cISFs) that evolved independently and dual host associated ISFs (dISFs) that are proposed to diverge from mosquito-borne flaviviruses (MBFs). Compared to pathogenic flaviviruses, ISFs are relatively poorly studied, and their molecular biology remains largely unexplored. In this study we focused on the characterisation of ISF 3UTRs and their ability to produce subgenomic flaviviral RNAs - noncoding viral RNAs that are known as important determinants of transmission and replication of pathogenetic flaviviruses. We demonstrated that cISFs and dISFs produce sfRNAs by employing a highly conserved mechanism of resistance to degradation by the cellular 5-3 exoribonuclease XRN1. We determined the secondary structures of complete 3UTRs and experimentally identified structured RNA elements that resist degradation by XRN1 (xrRNAs) in divergent representatives of cISF and dISF clades. We discovered a novel class of xrRNAs in dISFs and identified structurally divergent xrRNA in Anopheles-associated cISFs. Phylogenetic analyses based on sequences and secondary structures of xrRNAs and complete 3UTRs reveal that xrRNAs of cISFs and MBFs/dISFs evolved from a common xrRNA ancestor similar to the xrRNA of Anopheles-associated cISFs. Additionally, we found that duplications of xrRNAs occurred independently in ISF and MBF clades. Using ISF mutants deficient in the production of sfRNAs, we found that individual sfRNAs of ISFs have redundant functions. We conclude that duplicated xrRNAs were selected in the evolution of flaviviruses to ensure that sfRNA is produced if one of the xrRNAs lose XRN1 resistance due to mutations or misfolding.

microbiology↗

bioRxiv2021

A Plasmid System with Tunable Copy Number

Plasmids are one of the most commonly used and time-tested molecular biology platforms for genetic engineering and recombinant gene expression in bacteria. Despite their ubiquity, little consideration is given to metabolic effects and fitness costs of plasmid copy numbers on engineered genetic systems. Here, we introduce two systems that allow for the finely-tuned control of plasmid copy number: a plasmid with an anhydrotetracycline-controlled copy number, and a massively parallel assay that is used to generate a continuous spectrum of ColE1-based copy number variants. Using these systems, we investigate the effects of plasmid copy number on cellular growth rates, gene expression, biosynthesis, and genetic circuit performance. We perform single-cell timelapse measurements to characterize plasmid loss, runaway plasmid replication, and quantify the impact of plasmid copy number on the variability of gene expression. Using our massively parallel assay, we find that each plasmid imposes a 0.063% linear metabolic burden on their hosts, hinting at a simple relationship between metabolic burdens and plasmid DNA synthesis. Our plasmid system with tunable copy number should allow for a precise control of gene expression and highlight the importance of tuning plasmid copy number as tool for the optimization of synthetic biological systems.

synthetic biology↗

bioRxiv2021

CRISPR-Cas9-mediated Large Cluster Deletion and Multiplex Genome Editing in Paenibacillus polymyxa

The use of molecular tools based on the clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) systems has rapidly advanced genetic engineering. These molecular biological tools have been applied for different genetic engineering purposes in multiple organisms, including the quite rarely explored Paenibacillus polymyxa. However, only limited studies on large cluster deletion and multiplex genome editing have been described for this highly interesting and versatile bacterium. Here, we demonstrate the utilization of a Cas9-based system to realize targeted deletions of four biosynthetic gene clusters in the range of 12 kb to 41 kb by the use of a single targeting sgRNA. Furthermore, we also harness the system for multiplex editing of genes and large genomic regions. Multiplex deletion was achieved with more than 80 % efficiency, while simultaneous integration at two distantly located sites was obtained with 58 % efficiency. The findings reported in this study are anticipated to accelerate future research in P. polymyxa and related species.

synthetic biology↗