Engineering biological organisms is a complex process and challenging that could benefit from a combination of standardization and automation. This Commentary discusses the advantages and challenges ...of achieving high levels of autonomy in synthetic biology.
Engineering biological organisms is a complex, challenging, and often slow process. Other engineering domains have addressed such challenges with a combination of standardization and automation, enabling a divide‐and‐conquer approach to complexity and greatly increasing productivity. For example, standardization and automation allow rapid and predictable translation of prototypes into fielded applications (e.g., “design for manufacturability”), simplify sharing and reuse of work between groups, and enable reliable outsourcing and integration of specialized subsystems. Although this approach has also been part of the vision of synthetic biology, almost since its very inception (Knight & Sussman, 1998), this vision still remains largely unrealized (Carbonell et al, 2019). Despite significant progress over the last two decades, which have for example allowed obtaining and editing DNA sequences in easier and cheaper ways, the full process of organism engineering is still typically rather slow, manual, and artisanal.
As synthetic biology becomes increasingly capable and accessible, it is likewise increasingly critical to be able to make accurate biosecurity determinations regarding the pathogenicity or toxicity ...of particular nucleic acid or amino acid sequences. At present, this is typically done using the BLAST algorithm to determine the best match with sequences in the NCBI nucleic acid and protein databases. Neither BLAST nor any of the NCBI databases, however, are actually designed for biosafety determination. Critically, taxonomic errors or ambiguities in the NCBI nucleic acid and protein databases can also cause errors in BLAST-based taxonomic categorization. With heavily studied taxa and frequently used biotechnology tools, even low frequency taxonomic categorization issues can lead to high rates of errors in biosecurity decision-making. Here we focus on the implications for false positives, finding that BLAST against NCBI's protein database will now incorrectly categorize a number of commonly used biotechnology tool sequences as the pathogens or toxins with which they have been used. Paradoxically, this implies that problems are expected to be most acute for the pathogens and toxins of highest interest and for the most widely used biotechnology tools. We thus conclude that biosecurity tools should shift away from BLAST against general purpose databases and towards new methods that are specifically tailored for biosafety purposes.
Self-stabilizing distance estimation algorithms are an important building block of many distributed systems, such as seen in the emerging field of aggregate computing. Their safe use in feedback ...systems or under persistent perturbations has not previously been formally analyzed. Self-stabilization only involves eventual convergence, and is not endowed with robustness properties associated with global uniform asymptotic stability and thus does not guarantee stability under perturbations or feedback. We formulate a Lyapunov function to analyze the Adaptive Bellman-Ford distance estimation algorithm and use it to prove global uniform asymptotic stability, a property which the classical Bellman-Ford algorithm lacks. Global uniform asymptotic stability assures a measure of robustness to structural perturbations, empirically observed by us in a previous work. We also show that the algorithm is ultimately bounded under bounded measurement error and device mobility and provide a tight bound on the ultimate bound and the time to attain it.
Since the COVID-19 pandemic onset, the antibody response to SARS-CoV-2 has been extensively characterized. Antibodies to the receptor binding domain (RBD) on the spike protein are frequently encoded ...by IGHV3-53/3-66 with a short complementarity-determining region (CDR) H3. Germline-encoded sequence motifs in heavy chain CDRs H1 and H2 have a major function, but whether any common motifs are present in CDR H3, which is often critical for binding specificity, is not clear. Here, we identify two public clonotypes of IGHV3-53/3-66 RBD antibodies with a 9-residue CDR H3 that pair with different light chains. Distinct sequence motifs on CDR H3 are present in the two public clonotypes that seem to be related to differential light chain pairing. Additionally, we show that Y58F is a common somatic hypermutation that results in increased binding affinity of IGHV3-53/3-66 RBD antibodies with a short CDR H3. These results advance understanding of the antibody response to SARS-CoV-2.
In humans, the uterus undergoes a dramatic transformation to form an endometrial stroma-derived secretory tissue, termed decidua, during early pregnancy. The decidua secretes various factors that act ...in an autocrine/paracrine manner to promote stromal differentiation, facilitate maternal angiogenesis, and influence trophoblast differentiation and development, which are critical for the formation of a functional placenta. Here, we investigated the mechanisms by which decidual cells communicate with each other and with other cell types within the uterine milieu. We discovered that primary human endometrial stromal cells (HESCs) secrete extracellular vesicles (EVs) during decidualization and that this process is controlled by a conserved HIF2α-RAB27B pathway. Mass spectrometry revealed that the decidual EVs harbor a variety of protein cargo, including cell signaling molecules, growth modulators, metabolic regulators, and factors controlling endothelial cell expansion and remodeling. We tested the hypothesis that EVs secreted by the decidual cells mediate functional communications between various cell types within the uterus. We demonstrated that the internalization of EVs, specifically those carrying the glucose transporter 1 (GLUT1), promotes glucose uptake in recipient HESCs, supporting and advancing the decidualization program. Additionally, delivery of HESC-derived EVs into human endothelial cells stimulated their proliferation and led to enhanced vascular network formation. Strikingly, stromal EVs also promoted the differentiation of trophoblast stem cells into the extravillous trophoblast lineage. Collectively, these findings provide a deeper understanding of the pleiotropic roles played by EVs secreted by the decidual cells to ensure coordination of endometrial differentiation and angiogenesis with trophoblast function during the progressive phases of decidualization and placentation.
Variation in the DNA sequence upstream of bacterial promoters is known to affect the expression levels of the products they regulate, sometimes dramatically. While neutral synthetic insulator ...sequences have been found to buffer promoters from upstream DNA context, there are no established methods for designing effective insulator sequences with predictable effects on expression levels. We address this problem with Degenerate Insulation Screening (DIS), a novel method based on a randomized 36-nucleotide insulator library and a simple, high-throughput, flow-cytometry-based screen that randomly samples from a library of 436 potential insulated promoters. The results of this screen can then be compared against a reference uninsulated device to select a set of insulated promoters providing a precise level of expression. We verify this method by insulating the constitutive, inducible, and repressible promotors of a four transcriptional-unit inverter (NOT-gate) circuit, finding both that order dependence is largely eliminated by insulation and that circuit performance is also significantly improved, with a 5.8-fold mean improvement in on/off ratio.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The field of synthetic biology promises to revolutionize our ability to engineer biological systems, providing important benefits for a variety of applications. Recent advances in DNA synthesis and ...automated DNA assembly technologies suggest that it is now possible to construct synthetic systems of significant complexity. However, while a variety of novel genetic devices and small engineered gene networks have been successfully demonstrated, the regulatory complexity of synthetic systems that have been reported recently has somewhat plateaued due to a variety of factors, including the complexity of biology itself and the lag in our ability to design and optimize sophisticated biological circuitry.
To address the gap between DNA synthesis and circuit design capabilities, we present a platform that enables synthetic biologists to express desired behavior using a convenient high-level biologically-oriented programming language, Proto. The high level specification is compiled, using a regulatory motif based mechanism, to a gene network, optimized, and then converted to a computational simulation for numerical verification. Through several example programs we illustrate the automated process of biological system design with our platform, and show that our compiler optimizations can yield significant reductions in the number of genes (~ 50%) and latency of the optimized engineered gene networks.
Our platform provides a convenient and accessible tool for the automated design of sophisticated synthetic biological systems, bridging an important gap between DNA synthesis and circuit design capabilities. Our platform is user-friendly and features biologically relevant compiler optimizations, providing an important foundation for the development of sophisticated biological systems.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
The mouse decidua secretes many factors that act in a paracrine/autocrine manner to critically control uterine decidualization, neovascularization, and tissue remodeling that ensure proper ...establishment of pregnancy. The precise mechanisms that dictate intercellular communications among the uterine cells during early pregnancy remain unknown. We recently reported that conditional deletion of the gene encoding the hypoxia-inducible transcription factor 2 alpha (Hif2α) in mouse uterus led to infertility. Here, we report that HIF2α in mouse endometrial stromal cells (MESCs) acts via the cellular trafficking regulator RAB27b to control the secretion of extracellular vesicles (EVs) during decidualization. We also found that Hif2α-regulated pathways influence the biogenesis of EVs. Proteomic analysis of EVs secreted by decidualizing MESCs revealed that they harbor a wide variety of protein cargoes whose composition changed as the decidualization process progressed. The EVs enhanced the differentiation capacity of MESCs and the production of angiogenic factors by these cells. We also established that matrix metalloproteinase-2, a prominent EV cargo protein, modulates uterine remodeling during decidualization. Collectively, our results support the concept that EVs are central to the mechanisms by which the decidual cells communicate with each other and other cell types within the uterus to facilitate successful establishment of pregnancy.
For synthetic biology to mature, composition of devices into functional systems must become routine. This requires widespread adoption of comparable and replicable units of measurement. ...Interlaboratory studies organized through the International Genetically Engineered Machine (iGEM) competition show that fluorescence can be calibrated with simple, low-cost protocols, so fluorescence should no longer be published without units.
Fluorescent reporters are commonly used to quantify activities or properties of both natural and engineered cells. Fluorescence is still typically reported only in arbitrary or normalized units, ...however, rather than in units defined using an independent calibrant, which is problematic for scientific reproducibility and even more so when it comes to effective engineering. In this paper, we report an interlaboratory study showing that simple, low-cost unit calibration protocols can remedy this situation, producing comparable units and dramatic improvements in precision over both arbitrary and normalized units. Participants at 92 institutions around the world measured fluorescence from E. coli transformed with three engineered test plasmids, plus positive and negative controls, using simple, low-cost unit calibration protocols designed for use with a plate reader and/or flow cytometer. In addition to providing comparable units, use of an independent calibrant allows quantitative use of positive and negative controls to identify likely instances of protocol failure. The use of independent calibrants thus allows order of magnitude improvements in precision, narrowing the 95% confidence interval of measurements in our study up to 600-fold compared to normalized units.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
PDF
