Nanoarrays of Individual Liposomes and Bacterial Outer Membrane Vesicles by Liftoff Nanocontact Printing.

Analytical characterization of small biological particles, such as extracellular vesicles (EVs), is complicated by their extreme heterogeneity in size, lipid, membrane protein, and cargo composition. Analysis of individual particles is essential for illuminating particle property distributions that are obscured by ensemble measurements. To enable high-throughput analysis of individual particles, liftoff nanocontact printing (LNCP) is used to define hexagonal antibody and toxin arrays that have a 425 nm dot size, on average, and 700 nm periodicity. The LNCP process is rapid, simple, and does not require access to specialized nanofabrication tools. These densely packed, highly ordered arrays are used to capture liposomes and bacterial outer membrane vesicles on the basis of their surface biomarkers, with a maximum of one particle per array dot, resulting in densely packed arrays of particles. Despite the high particle density, the underlying antibody or toxin array ensured that neighboring individual particles are optically resolvable. Provided target particle biomarkers and suitable capture molecules are identified, this approach can be used to generate high density arrays of a wide variety of small biological particles, including other types of EVs like exosomes.

Rapid generation of CRISPR/dCas9-regulated, orthogonally repressible hybrid T7-lac promoters for modular, tuneable control of metabolic pathway fluxes in Escherichia coli.

Robust gene circuit construction requires use of promoters exhibiting low crosstalk. Orthogonal promoters have been engineered utilizing an assortment of natural and synthetic transcription factors, but design of large orthogonal promoter-repressor sets is complicated, labor-intensive, and often results in unanticipated crosstalk. The specificity and ease of targeting the RNA-guided DNA-binding protein dCas9 to any 20 bp user-defined DNA sequence makes it a promising candidate for orthogonal promoter regulation. Here, we rapidly construct orthogonal variants of the classic T7-lac promoter using site-directed mutagenesis, generating a panel of inducible hybrid promoters regulated by both LacI and dCas9. Remarkably, orthogonality is mediated by only two to three nucleotide mismatches in a narrow window of the RNA:DNA hybrid, neighboring the protospacer adjacent motif. We demonstrate that, contrary to many reports, one PAM-proximal mismatch is insufficient to abolish dCas9-mediated repression, and we show for the first time that mismatch tolerance is a function of target copy number. Finally, these promoters were incorporated into the branched violacein biosynthetic pathway as dCas9-dependent switches capable of throttling and selectively redirecting carbon flux in Escherichia coli We anticipate this strategy is relevant for any promoter and will be adopted for many applications at the interface of synthetic biology and metabolic engineering.

Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli.

Methanol is an attractive substrate for biological production of chemicals and fuels. Engineering methylotrophic Escherichia coli as a platform organism for converting methanol to metabolites is desirable. Prior efforts to engineer methylotrophic E. coli were limited by methanol dehydrogenases (Mdhs) with unfavorable enzyme kinetics. We engineered E. coli to utilize methanol using a superior NAD-dependent Mdh from Bacillus stearothermophilus and ribulose monophosphate (RuMP) pathway enzymes from B. methanolicus. Using 13C-labeling, we demonstrate this E. coli strain converts methanol into biomass components. For example, the key TCA cycle intermediates, succinate and malate, exhibit labeling up to 39%, while the lower glycolytic intermediate, 3-phosphoglycerate, up to 53%. Multiple carbons are labeled for each compound, demonstrating a cycling RuMP pathway for methanol assimilation to support growth. By incorporating the pathway to synthesize the flavanone naringenin, we demonstrate the first example of in vivo conversion of methanol into a specialty chemical in E. coli.

Experimental and computational optimization of an Escherichia coli co-culture for the efficient production of flavonoids.

Metabolic engineering and synthetic biology have enabled the use of microbial production platforms for the renewable production of many high-value natural products. Titers and yields, however, are often too low to result in commercially viable processes. Microbial co-cultures have the ability to distribute metabolic burden and allow for modular specific optimization in a way that is not possible through traditional monoculture fermentation methods. Here, we present an Escherichia coli co-culture for the efficient production of flavonoids in vivo, resulting in a 970-fold improvement in titer of flavan-3-ols over previously published monoculture production. To accomplish this improvement in titer, factors such as strain compatibility, carbon source, temperature, induction point, and inoculation ratio were initially optimized. The development of an empirical scaled-Gaussian model based on the initial optimization data was then implemented to predict the optimum point for the system. Experimental verification of the model predictions resulted in a 65% improvement in titer, to 40.7±0.1mg/L flavan-3-ols, over the previous optimum. Overall, this study demonstrates the first application of the co-culture production of flavonoids, the most in-depth co-culture optimization to date, and the first application of empirical systems modeling for improvement of titers from a co-culture system.

Heterogeneity of Size and Toxin Distribution in Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles.

Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium associated with localized aggressive periodontitis as well as some systemic diseases. The strains of A. actinomycetemcomitans most closely associated with disease produce more of a secreted leukotoxin (LtxA) than isolates from healthy carriers, suggesting a key role for this toxin in disease progression. LtxA is released into the bacterial cytosol in a free form as well as in association with the surface of outer membrane vesicles (OMVs). We previously observed that the highly leukotoxic A. actinomycetemcomitans strain JP2 produces two populations of OMVs: a highly abundant population of small (<100 nm) OMVs and a less abundant population of large (>300 nm) OMVs. Here, we have developed a protocol to isolate the OMVs produced during each specific phase of growth and used this to demonstrate that small OMVs are produced throughout growth and lack LtxA, while large OMVs are produced only during the exponential phase and are enriched with LtxA. Our results indicate that surface-associated DNA drives the selective sorting of LtxA into large OMVs. This study provides valuable insights into the observed heterogeneity of A. actinomycetemcomitans vesicles and emphasizes the importance of understanding these variations in the context of bacterial pathogenesis.

A globally synthesised and flagged bee occurrence dataset and cleaning workflow.

Species occurrence data are foundational for research, conservation, and science communication, but the limited availability and accessibility of reliable data represents a major obstacle, particularly for insects, which face mounting pressures. We present BeeBDC, a new R package, and a global bee occurrence dataset to address this issue. We combined >18.3 million bee occurrence records from multiple public repositories (GBIF, SCAN, iDigBio, USGS, ALA) and smaller datasets, then standardised, flagged, deduplicated, and cleaned the data using the reproducible BeeBDC R-workflow. Specifically, we harmonised species names (following established global taxonomy), country names, and collection dates and, we added record-level flags for a series of potential quality issues. These data are provided in two formats, "cleaned" and "flagged-but-uncleaned". The BeeBDC package with online documentation provides end users the ability to modify filtering parameters to address their research questions. By publishing reproducible R workflows and globally cleaned datasets, we can increase the accessibility and reliability of downstream analyses. This workflow can be implemented for other taxa to support research and conservation.

Sequential, low-temperature aqueous synthesis of Ag-In-S/Zn quantum dots via staged cation exchange under biomineralization conditions.

The development of high quality, non-toxic (i.e., heavy-metal-free), and functional quantum dots (QDs) via 'green' and scalable synthesis routes is critical for realizing truly sustainable QD-based solutions to diverse technological challenges. Herein, we demonstrate the low-temperature all-aqueous-phase synthesis of silver indium sulfide/zinc (AIS/Zn) QDs with a process initiated by the biomineralization of highly crystalline indium sulfide nanocrystals, and followed by the sequential staging of Ag+ cation exchange and Zn2+ addition directly within the biomineralization media without any intermediate product purification. Therein, we exploit solution phase cation concentration, the duration of incubation in the presence of In2S3 precursor nanocrystals, and the subsequent addition of Zn2+ as facile handles under biomineralization conditions for controlling QD composition, tuning optical properties, and improving the photoluminescence quantum yield of the AIS/Zn product. We demonstrate how engineering biomineralization for the synthesis of intrinsically hydrophilic and thus readily functionalizable AIS/Zn QDs with a quantum yield of 18% offers a 'green' and non-toxic materials platform for targeted bioimaging in sensitive cellular systems. Ultimately, the decoupling of synthetic steps helps unravel the complexities of ion exchange-based synthesis within the biomineralization platform, enabling its adaptation for the sustainable synthesis of 'green', compositionally diverse QDs.

Bacterial Outer Membrane Vesicles as Antibiotic Delivery Vehicles.

Bacterial outer membrane vesicles (OMVs) are nanometer-scale, spherical vehicles released by Gram-negative bacteria into their surroundings throughout growth. These OMVs have been demonstrated to play key roles in pathogenesis by delivering certain biomolecules to host cells, including toxins and other virulence factors. In addition, this biomolecular delivery function enables OMVs to facilitate intra-bacterial communication processes, such as quorum sensing and horizontal gene transfer. The unique ability of OMVs to deliver large biomolecules across the complex Gram-negative cell envelope has inspired the use of OMVs as antibiotic delivery vehicles to overcome transport limitations. In this review, we describe the advantages, applications, and biotechnological challenges of using OMVs as antibiotic delivery vehicles, studying both natural and engineered antibiotic applications of OMVs. We argue that OMVs hold great promise as antibiotic delivery vehicles, an urgently needed application to combat the growing threat of antibiotic resistance.

Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity.

The cell wall of Gram-negative bacteria consists of an inner (cytoplasmic) and outer membrane (OM), separated by a thin peptidoglycan layer. Throughout growth, the outer membrane can bleb to form spherical outer membrane vesicles (OMVs). These OMVs are involved in numerous cellular functions including cargo delivery to host cells and communication with bacterial cells. Recently, the therapeutic potential of OMVs has begun to be explored, including their use as vaccines and drug delivery vehicles. Although OMVs are derived from the OM, it has long been appreciated that the lipid and protein cargo of the OMV differs, often significantly, from that of the OM. More recently, evidence that bacteria can release multiple types of OMVs has been discovered, and evidence exists that size can impact the mechanism of their uptake by host cells. However, studies in this area are limited by difficulties in efficiently separating the heterogeneously sized OMVs. Density gradient centrifugation (DGC) has traditionally been used for this purpose; however, this technique is time-consuming and difficult to scale-up. Size exclusion chromatography (SEC), on the other hand, is less cumbersome and lends itself to the necessary future scale-up for therapeutic use of OMVs. Here, we describe a SEC approach that enables reproducible separation of heterogeneously sized vesicles, using as a test case, OMVs produced by Aggregatibacter actinomycetemcomitans, which range in diameter from less than 150 nm to greater than 350 nm. We demonstrate separation of "large" (350 nm) OMVs and "small" (<150 nm) OMVs, verified by dynamic light scattering (DLS). We recommend SEC-based techniques over DGC-based techniques for separation of heterogeneously sized vesicles due to its ease of use, reproducibility (including user-to-user), and possibility for scale-up.

Complete Biosynthesis of Anthocyanins Using E. coli Polycultures.

Fermentation-based chemical production strategies provide a feasible route for the rapid, safe, and sustainable production of a wide variety of important chemical products, ranging from fuels to pharmaceuticals. These strategies have yet to find wide industrial utilization due to their inability to economically compete with traditional extraction and chemical production methods. Here, we engineer for the first time the complex microbial biosynthesis of an anthocyanin plant natural product, starting from sugar. This was accomplished through the development of a synthetic, 4-strain Escherichia coli polyculture collectively expressing 15 exogenous or modified pathway enzymes from diverse plants and other microbes. This synthetic consortium-based approach enables the functional expression and connection of lengthy pathways while effectively managing the accompanying metabolic burden. The de novo production of specific anthocyanin molecules, such as calistephin, has been an elusive metabolic engineering target for over a decade. The utilization of our polyculture strategy affords milligram-per-liter production titers. This study also lays the groundwork for significant advances in strain and process design toward the development of cost-competitive biochemical production hosts through nontraditional methodologies.IMPORTANCE To efficiently express active extensive recombinant pathways with high flux in microbial hosts requires careful balance and allocation of metabolic resources such as ATP, reducing equivalents, and malonyl coenzyme A (malonyl-CoA), as well as various other pathway-dependent cofactors and precursors. To address this issue, we report the design, characterization, and implementation of the first synthetic 4-strain polyculture. Division of the overexpression of 15 enzymes and transcription factors over 4 independent strain modules allowed for the division of metabolic burden and for independent strain optimization for module-specific metabolite needs. This study represents the most complex synthetic consortia constructed to date for metabolic engineering applications and provides a new paradigm in metabolic engineering for the reconstitution of extensive metabolic pathways in nonnative hosts.

Optimization of naringenin and p-coumaric acid hydroxylation using the native E. coli hydroxylase complex, HpaBC.

Flavonoids are a growing class of bioactive natural products with distinct and interesting bioactivity both in vitro and in vivo. The extraction of flavonoids from plant sources is limited by their low natural abundance and commonly results in a mixture of products that are difficult to separate. However, due to recent advances, the microbial production of plant natural products has developed as a promising alternative for flavonoid production. Through optimization of media, induction temperature, induction point, and substrate delay time, we demonstrate the highest conversion of naringenin to eriodictyol (62.7 ± 2.7 mg/L) to date, using the native E. coli hydroxylase complex, HpaBC. We also show the first evidence of in vivo HpaBC activity towards the monohydroxylated flavan-3-ol afzelechin with catechin product titers of 34.7 ± 1.5 mg/L. This work confirms the wide applicability of HpaBC towards realizing efficient de novo production of various orthohydroxylated flavonoids and flavonoid derived products in E. coli.

Reliability of conventional and new pulse oximetry in neonatal patients.

OBJECTIVES: Pulse oximetry is widely used in the NICU, but clinicians often distrust the displayed values during patient motion, i.e., questionable oxygen saturation (SpO(2)) and pulse rate (PR) values. Masimo Corporation (Irvine, CA) has developed pulse oximetry with claims of resistance to sources of interference. To test this premise, we compared the performance of the Masimo SET pulse oximeter to a conventional device, Nellcor N-200, and then with three other new-generation pulse oximeters, Nellcor N-395, Novametrix MARS, and Philips Viridia 24C. STUDY DESIGN: We studied 26 nonsedated NICU infants who were on supplemental oxygen and/or mechanical ventilation. ECG heart rate (HR) from a bedside monitor and SpO(2) and PR from the two pulse oximeters were captured by a PC for a total of 156 hours. The ECG HR and pulse oximeter spectral waveform were analyzed at alarms for hypoxemia (SpO(2)< or = 85%) and/or bradycardia (HR< or = 80 bpm). We then compared the performance of the Masimo SET to three other new-generation pulse oximeters, Agilent Viridia 24C, Nellcor N-395, and Novametrix MARS, in a similar population of seven infants for a total of 28 hours. We added to the test criteria the ability of the various pulse oximeters to track acute changes in HR. RESULTS: Compared with Nellcor, Masimo SET had 86% fewer false alarms, which also were shorter in duration, resulting in 92% less total alarm time. Masimo SET also identified nearly all bradycardias versus 14% for the Nellcor. Compared with the new-generation pulse oximeters, false desaturations, data drop-outs, and false bradycardias were lowest for Masimo SET, as was the capture of true desaturations and bradycardias. Notably, the new-generation devices differed greatly in their ability to detect changes in HR (i.e., the frequency of frozen PR during times of ECG HR change was 0, 6, 11, and 46 for Masimo, Nellcor, Philips, and Novametrix, respectively). CONCLUSIONS: Masimo SET pulse oximetry recorded markedly fewer false SpO(2) and PR alarms and identified more true hypoxic and bradycardic events than either conventional or other new-generation pulse oximeters. Masimo SET also most closely reflected the ECG rate irrespective of accelerations or decelerations in HR. SPECULATION: Routine use of Masimo SET pulse oximetry in the NICU could improve clinician confidence in the parameter leading to more judicious titration of oxygen with possible reductions in hypoxic (e.g., pulmonary hypertension) and hyperoxic (e.g., retinopathy of prematurity) pathology. Additionally, a more trustworthy technology should equate with fewer confirmatory arterial blood gas analyses (less blood loss), and faster weaning from the mechanical ventilation (less chronic lung disease).