Robotic Antimicrobial Susceptibility Platform (RASP): a next-generation approach to One Health surveillance of antimicrobial resistance.

BACKGROUND: Surveillance of antimicrobial resistance (AMR) is critical to reducing its wide-reaching impact. Its reliance on sample size invites solutions to longstanding constraints regarding scalability. A robotic platform (RASP) was developed for high-throughput AMR surveillance in accordance with internationally recognized standards (CLSI and ISO 20776-1:2019) and validated through a series of experiments. METHODS: Experiment A compared RASP's ability to achieve consistent MICs with that of a human technician across eight replicates for four Escherichia coli isolates. Experiment B assessed RASP's agreement with human-performed MICs across 91 E. coli isolates with a diverse range of AMR profiles. Additionally, to demonstrate its real-world applicability, the RASP workflow was then applied to five faecal samples where a minimum of 47 E. coli per animal (239 total) were evaluated using an AMR indexing framework. RESULTS: For each drug-rater-isolate combination in Experiment A, there was a clear consensus of the MIC and deviation from the consensus remained within one doubling dilution (the exception being gentamicin at two dilutions). Experiment B revealed a concordance correlation coefficient of 0.9670 (95% CI: 0.9670-0.9670) between the robot- and human-performed MICs. RASP's application to the five faecal samples highlighted the intra-animal diversity of gut commensal E. coli, identifying between five and nine unique isolate AMR phenotypes per sample. CONCLUSIONS: While adhering to internationally accepted guidelines, RASP was superior in throughput, cost and data resolution when compared with an experienced human technician. Integration of robotics platforms in the microbiology laboratory is a necessary advancement for future One Health AMR endeavours.

Direct on-the-spot detection of SARS-CoV-2 in patients.

IMPACT STATEMENT: Humanity is currently experiencing a global pandemic with devastating implications on human health and the economy. Most countries are gradually exiting their lockdown state. We are currently lacking rapid and simple viral detections, especially methods that can be performed in the household. Here, we applied RT-LAMP directly on human clinical swabs and self-collected saliva samples. We adjusted the method to allow simple and rapid viral detection, with no RNA purification steps. By testing our method on over 180 human samples, we determined its sensitivity, and by applying it to other viruses, we determined its specificity. We believe this method has a promising potential to be applied world-wide as a simple and cheap surveillance test for SARS-CoV-2.

A Protocol for Simple, Rapid, and Direct Detection of SARS-CoV-2 from clinical samples, using Reverse Transcribed Loop-Mediated Isothermal Amplification (RT-LAMP).

SARS-CoV-2 has quickly spread all around the globe causing illness and wide damages. Most countries were unprepared for such a rapid spread and crisis. This led to various strategies for effective control of the new pandemic. A key aspect in all countries was to effectively test the population for the virus. Most countries chose a lockdown strategy in which many workplaces and activities are completely closed, leading to substantial economy costs. Here, we present a protocol we recently developed that allows rapid and simple detection of SARS-CoV-2 for the large population, eliminating costs and involvement of professional teams and laboratories. This protocol is based on Reverse Transcribed Loop-Mediated Isothermal Amplification (RT-LAMP). We tested this protocol directly on patient samples, both nasal and throat clinical swabs as well as saliva. Notably, this protocol is simple, cheap and can be easily applied to other pathogens as well.

Quantifying outdoor water consumption of urban land use/land cover: sensitivity to drought.

Outdoor water use is a key component in arid city water systems for achieving sustainable water use and ensuring water security. Using evapotranspiration (ET) calculations as a proxy for outdoor water consumption, the objectives of this research are to quantify outdoor water consumption of different land use and land cover types, and compare the spatio-temporal variation in water consumption between drought and wet years. An energy balance model was applied to Landsat 5 TM time series images to estimate daily and seasonal ET for the Central Arizona Phoenix Long-Term Ecological Research region (CAP-LTER). Modeled ET estimations were correlated with water use data in 49 parks within CAP-LTER and showed good agreement (r² = 0.77), indicating model effectiveness to capture the variations across park water consumption. Seasonally, active agriculture shows high ET (>500 mm) for both wet and dry conditions, while the desert and urban land cover types experienced lower ET during drought (<300 mm). Within urban locales of CAP-LTER, xeric neighborhoods show significant differences from year to year, while mesic neighborhoods retain their ET values (400-500 mm) during drought, implying considerable use of irrigation to sustain their greenness. Considering the potentially limiting water availability of this region in the future due to large population increases and the threat of a warming and drying climate, maintaining large water-consuming, irrigated landscapes challenges sustainable practices of water conservation and the need to provide amenities of this desert area for enhancing quality of life.

Recursive construction and error correction of DNA molecules and libraries from synthetic and natural DNA.

Making error-free, custom DNA assemblies from potentially faulty building blocks is a fundamental challenge in synthetic biology. Here, we show how recursion can be used to address this challenge using a recursive procedure that constructs error-free DNA molecules and their libraries from error-prone synthetic oligonucleotides and naturally existing DNA. Specifically, we describe how divide and conquer (D&C), the quintessential recursive problem-solving technique, is applied in silico to divide target DNA sequences into overlapping, albeit error prone, oligonucleotides, and how recursive construction is applied in vitro to combine them to form error-prone DNA molecules. To correct DNA sequence errors, error-free fragments of these molecules are then identified, extracted, and used as new, typically longer and more accurate, inputs to another iteration of the recursive construction procedure; the entire process repeats until an error-free target molecule is formed. The method allows combining synthetic and natural DNA fragments into error-free designer DNA libraries, thus providing a foundation for the design and construction of complex synthetic DNA assemblies.

Processing DNA molecules as text.

UNLABELLED: Polymerase Chain Reaction (PCR) is the DNA-equivalent of Gutenberg's movable type printing, both allowing large-scale replication of a piece of text. De novo DNA synthesis is the DNA-equivalent of mechanical typesetting, both ease the setting of text for replication. What is the DNA-equivalent of the word processor? Biology labs engage daily in DNA processing-the creation of variations and combinations of existing DNA-using a plethora of manual labor-intensive methods such as site-directed mutagenesis, error-prone PCR, assembly PCR, overlap extension PCR, cleavage and ligation, homologous recombination, and others. So far no universal method for DNA processing has been proposed and, consequently, no engineering discipline that could eliminate this manual labor has emerged. Here we present a novel operation on DNA molecules, called Y, which joins two DNA fragments into one, and show that it provides a foundation for DNA processing as it can implement all basic text processing operations on DNA molecules including insert, delete, replace, cut and paste and copy and paste. In addition, complicated DNA processing tasks such as the creation of libraries of DNA variants, chimeras and extensions can be accomplished with DNA processing plans consisting of multiple Y operations, which can be executed automatically under computer control. The resulting DNA processing system, which incorporates our earlier work on recursive DNA composition and error correction, is the first demonstration of a unified approach to DNA synthesis, editing, and library construction. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11693-010-9059-y) contains supplementary material, which is available to authorized users.

Invariant distribution of promoter activities in Escherichia coli.

Cells need to allocate their limited resources to express a wide range of genes. To understand how Escherichia coli partitions its transcriptional resources between its different promoters, we employ a robotic assay using a comprehensive reporter strain library for E. coli to measure promoter activity on a genomic scale at high-temporal resolution and accuracy. This allows continuous tracking of promoter activity as cells change their growth rate from exponential to stationary phase in different media. We find a heavy-tailed distribution of promoter activities, with promoter activities spanning several orders of magnitude. While the shape of the distribution is almost completely independent of the growth conditions, the identity of the promoters expressed at different levels does depend on them. Translation machinery genes, however, keep the same relative expression levels in the distribution across conditions, and their fractional promoter activity tracks growth rate tightly. We present a simple optimization model for resource allocation which suggests that the observed invariant distributions might maximize growth rate. These invariant features of the distribution of promoter activities may suggest design constraints that shape the allocation of transcriptional resources.

Molecular implementation of simple logic programs.

Autonomous programmable computing devices made of biomolecules could interact with a biological environment and be used in future biological and medical applications. Biomolecular implementations of finite automata and logic gates have already been developed. Here, we report an autonomous programmable molecular system based on the manipulation of DNA strands that is capable of performing simple logical deductions. Using molecular representations of facts such as Man(Socrates) and rules such as Mortal(X) <-- Man(X) (Every Man is Mortal), the system can answer molecular queries such as Mortal(Socrates)? (Is Socrates Mortal?) and Mortal(X)? (Who is Mortal?). This biomolecular computing system compares favourably with previous approaches in terms of expressive power, performance and precision. A compiler translates facts, rules and queries into their molecular representations and subsequently operates a robotic system that assembles the logical deductions and delivers the result. This prototype is the first simple programming language with a molecular-scale implementation.

De novo DNA synthesis using single molecule PCR.

The throughput of DNA reading (sequencing) has dramatically increased recently due to the incorporation of in vitro clonal amplification. The throughput of DNA writing (synthesis) is trailing behind, with cloning and sequencing constituting the main bottleneck. To overcome this bottleneck, an in vitro alternative for in vivo DNA cloning must be integrated into DNA synthesis methods. Here we show how a new single molecule PCR (smPCR)-based procedure can be employed as a general substitute to in vivo cloning thereby allowing for the first time in vitro DNA synthesis. We integrated this rapid and high fidelity in vitro procedure into our earlier recursive DNA synthesis and error correction procedure and used it to efficiently construct and error-correct a 1.8-kb DNA molecule from synthetic unpurified oligos completely in vitro. Although we demonstrate incorporating smPCR in a particular method, the approach is general and can be used in principle in conjunction with other DNA synthesis methods as well.

The incoherent feed-forward loop can generate non-monotonic input functions for genes.

Gene regulation networks contain recurring circuit patterns called network motifs. One of the most common network motif is the incoherent type 1 feed-forward loop (I1-FFL), in which an activator controls both gene and repressor of that gene. This motif was shown to act as a pulse generator and response accelerator of gene expression. Here we consider an additional function of this motif: the I1-FFL can generate a non-monotonic dependence of gene expression on the input signal. Here, we study this experimentally in the galactose system of Escherichia coli, which is regulated by an I1-FFL. The promoter activity of two of the gal operons, galETK and galP, peaks at intermediate levels of the signal cAMP. We find that mutants in which the I1-FFL is disrupted lose this non-monotonic behavior, and instead display monotonic input functions. Theoretical analysis suggests that non-monotonic input functions can be achieved for a wide range of parameters by the I1-FFL. The models also suggest regimes where a monotonic input-function can occur, as observed in the mglBAC operon regulated by the same I1-FFL. The present study thus experimentally demonstrates how upstream circuitry can affect gene input functions and how an I1-FFL functions within its natural context in the cell.

Recursive construction of perfect DNA molecules from imperfect oligonucleotides.

Making faultless complex objects from potentially faulty building blocks is a fundamental challenge in computer engineering, nanotechnology and synthetic biology. Here, we show for the first time how recursion can be used to address this challenge and demonstrate a recursive procedure that constructs error-free DNA molecules and their libraries from error-prone oligonucleotides. Divide and Conquer (D&C), the quintessential recursive problem-solving technique, is applied in silico to divide the target DNA sequence into overlapping oligonucleotides short enough to be synthesized directly, albeit with errors; error-prone oligonucleotides are recursively combined in vitro, forming error-prone DNA molecules; error-free fragments of these molecules are then identified, extracted and used as new, typically longer and more accurate, inputs to another iteration of the recursive construction procedure; the entire process repeats until an error-free target molecule is formed. Our recursive construction procedure surpasses existing methods for de novo DNA synthesis in speed, precision, amenability to automation, ease of combining synthetic and natural DNA fragments, and ability to construct designer DNA libraries. It thus provides a novel and robust foundation for the design and construction of synthetic biological molecules and organisms.

Diverse two-dimensional input functions control bacterial sugar genes.

Cells respond to signals by regulating gene expression. The relation between the level of input signals and the transcription rate of the gene is called the gene's input function. Because most genes are regulated by more than one signal, the input functions are usually multidimensional. To understand cellular responses, it is essential to know the shapes of these functions. Here, we map the two-dimensional input functions of 19 sugar-utilization genes at high resolution in living E. coli cells. We find diverse, intricately shaped input functions, despite the similarity in the regulatory circuitry of these genes. Surprisingly, some of the input functions are nonmonotonic, peaking at intermediate signal levels. Furthermore, most of the input functions show separation of variables, in the sense that they can be described as the product of simple functions that depend on a single input. This first broad survey of two-dimensional input functions can be extended to map the logic of gene regulation in other systems.

Reconstruction of cell lineage trees in mice.

The cell lineage tree of a multicellular organism represents its history of cell divisions from the very first cell, the zygote. A new method for high-resolution reconstruction of parts of such cell lineage trees was recently developed based on phylogenetic analysis of somatic mutations accumulated during normal development of an organism. In this study we apply this method in mice to reconstruct the lineage trees of distinct cell types. We address for the first time basic questions in developmental biology of higher organisms, namely what is the correlation between the lineage relation among cells and their (1) function, (2) physical proximity and (3) anatomical proximity. We analyzed B-cells, kidney-, mesenchymal- and hematopoietic-stem cells, as well as satellite cells, which are adult skeletal muscle stem cells isolated from their niche on the muscle fibers (myofibers) from various skeletal muscles. Our results demonstrate that all analyzed cell types are intermingled in the lineage tree, indicating that none of these cell types are single exclusive clones. We also show a significant correlation between the physical proximity of satellite cells within muscles and their lineage. Furthermore, we show that satellite cells obtained from a single myofiber are significantly clustered in the lineage tree, reflecting their common developmental origin. Lineage analysis based on somatic mutations enables performing high resolution reconstruction of lineage trees in mice and humans, which can provide fundamental insights to many aspects of their development and tissue maintenance.

Estimating cell depth from somatic mutations.

The depth of a cell of a multicellular organism is the number of cell divisions it underwent since the zygote, and knowing this basic cell property would help address fundamental problems in several areas of biology. At present, the depths of the vast majority of human and mouse cell types are unknown. Here, we show a method for estimating the depth of a cell by analyzing somatic mutations in its microsatellites, and provide to our knowledge for the first time reliable depth estimates for several cells types in mice. According to our estimates, the average depth of oocytes is 29, consistent with previous estimates. The average depth of B cells ranges from 34 to 79, linearly related to the mouse age, suggesting a rate of one cell division per day. In contrast, various types of adult stem cells underwent on average fewer cell divisions, supporting the notion that adult stem cells are relatively quiescent. Our method for depth estimation opens a window for revealing tissue turnover rates in animals, including humans, which has important implications for our knowledge of the body under physiological and pathological conditions.

A universal mechanism ties genotype to phenotype in trinucleotide diseases.

Trinucleotide hereditary diseases such as Huntington disease and Friedreich ataxia are cureless diseases associated with inheriting an abnormally large number of DNA trinucleotide repeats in a gene. The genes associated with different diseases are unrelated and harbor a trinucleotide repeat in different functional regions; therefore, it is striking that many of these diseases have similar correlations between their genotype, namely the number of inherited repeats and age of onset and progression phenotype. These correlations remain unexplained despite more than a decade of research. Although mechanisms have been proposed for several trinucleotide diseases, none of the proposals, being disease-specific, can account for the commonalities among these diseases. Here, we propose a universal mechanism in which length-dependent somatic repeat expansion occurs during the patient's lifetime toward a pathological threshold. Our mechanism uniformly explains for the first time to our knowledge the genotype-phenotype correlations common to trinucleotide disease and is well-supported by both experimental and clinical data. In addition, mathematical analysis of the mechanism provides simple explanations to a wide range of phenomena such as the exponential decrease of the age-of-onset curve, similar onset but faster progression in patients with Huntington disease with homozygous versus heterozygous mutation, and correlation of age of onset with length of the short allele but not with the long allele in Friedreich ataxia. If our proposed universal mechanism proves to be the core component of the actual mechanisms of specific trinucleotide diseases, it would open the search for a uniform treatment for all these diseases, possibly by delaying the somatic expansion process.

Genomic variability within an organism exposes its cell lineage tree.

What is the lineage relation among the cells of an organism? The answer is sought by developmental biology, immunology, stem cell research, brain research, and cancer research, yet complete cell lineage trees have been reconstructed only for simple organisms such as Caenorhabditis elegans. We discovered that somatic mutations accumulated during normal development of a higher organism implicitly encode its entire cell lineage tree with very high precision. Our mathematical analysis of known mutation rates in microsatellites (MSs) shows that the entire cell lineage tree of a human embryo, or a mouse, in which no cell is a descendent of more than 40 divisions, can be reconstructed from information on somatic MS mutations alone with no errors, with probability greater than 99.95%. Analyzing all approximately 1.5 million MSs of each cell of an organism may not be practical at present, but we also show that in a genetically unstable organism, analyzing only a few hundred MSs may suffice to reconstruct portions of its cell lineage tree. We demonstrate the utility of the approach by reconstructing cell lineage trees from DNA samples of a human cell line displaying MS instability. Our discovery and its associated procedure, which we have automated, may point the way to a future "Human Cell Lineage Project" that would aim to resolve fundamental open questions in biology and medicine by reconstructing ever larger portions of the human cell lineage tree.

Extraction of transcription regulatory signals from genome-wide DNA-protein interaction data.

Deciphering gene regulatory network architecture amounts to the identification of the regulators, conditions in which they act, genes they regulate, cis-acting motifs they bind, expression profiles they dictate and more complex relationships between alternative regulatory partnerships and alternative regulatory motifs that give rise to sub-modalities of expression profiles. The 'location data' in yeast is a comprehensive resource that provides transcription factor-DNA interaction information in vivo. Here, we provide two contributions: first, we developed means to assess the extent of noise in the location data, and consequently for extracting signals from it. Second, we couple signal extraction with better characterization of the genetic network architecture. We apply two methods for the detection of combinatorial associations between transcription factors (TFs), the integration of which provides a global map of combinatorial regulatory interactions. We discover the capacity of regulatory motifs and TF partnerships to dictate fine-tuned expression patterns of subsets of genes, which are clearly distinct from those displayed by most genes assigned to the same TF. Our findings provide carefully prioritized, high-quality assignments between regulators and regulated genes and as such should prove useful for experimental and computational biologists alike.