Biology subjects

Bernstein, H. C.

Publications and source records attributed to Bernstein, H. C..

10 recordsLinked to original sources

bioRxiv2024

Fine Tuning Genetic Circuits via Host Context and RBS Modulation

The choice of organism to host a genetic circuit - the chassis - is often defaulted to model organisms due to their amenability. The chassis-design space has therefore remained underexplored as an engineering variable. In this work, we explored the design space of a genetic toggle switch through variations in nine ribosome binding sites compositions and three host contexts, creating 27 circuit variants. Characterization of performance metrics in terms of toggle switch output and host growth dynamics unveils a spectrum of performance profiles from our circuit library. We find that changes in host-context causes large shifts in overall performance, while modulating ribosome binding sites leads to more incremental changes. We find that a combined ribosome binding site and host-context modulation approach can be used to fine tune the properties of a toggle switch according to user-defined specifications, such as towards greater signaling strength, inducer sensitivity or both. Other auxiliary properties, such as inducer tolerance, are also exclusively accessed through changes in host-context. We demonstrate here that exploration of the chassis-design space can offer significant value, reconceptualizing the chassis-organism as an important part in the synthetic biologists toolbox with important implications for the field of synthetic biology. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/604438v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1ff45d0org.highwire.dtl.DTLVardef@4060d7org.highwire.dtl.DTLVardef@95d5d8org.highwire.dtl.DTLVardef@10a98d5_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Pangenomic landscapes shape the genetic circuit performance in a Stutzerimonas biodesign toolkit

Engineering identical genetic circuits into different species typically results in large differences in performance due to the unique cellular environmental context of each host, a phenomenon known as the "chassis-effect". A better understanding of how genomic and physiological contexts underpin the chassis-effect will greatly improve biodesign strategies across diverse microorganisms. Here, we combined a pangenomics-based gene expression analysis with quantitative measurements of performance from an engineered genetic inverter device to uncover how genome structure and function relates to the observed chassis-effect across six closely related Stutzerimonas hosts. Our results reveal that genome architecture underpins divergent responses between our chosen non-model bacterial hosts to engineered genetic circuits. Specifically, differential expression of the core genome, gene clusters shared between all hosts, were found to be the main source of significant concordance to the observed genetic device performance, whereas specialty genes from respective accessory genomes were not significant. A data-driven investigation revealed that genes involved in denitrification and components of trans-membrane transporter proteins were among the most differentially expressed gene clusters in response to the genetic device. Our results show the chassis-effect can be traced along differences among genome-encoded functions that are mostly conserved and that these differences create a unique biodesign space among closely related species. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/580380v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@173161dorg.highwire.dtl.DTLVardef@b0fdf3org.highwire.dtl.DTLVardef@1f09130org.highwire.dtl.DTLVardef@cc1eb3_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

The bioreactor microbiome of mass cultivated marine diatoms for industrial carbon capture and utilization

Marine microalgae are a promising innovation platform for carbon capture and utilization (CCU) biotechnologies to mitigate industrial greenhouse gas emissions. However, industrial-scale cultivation of algal mono-cultures is challenging and often unscalable. Non-axenic microalgae in large semi-open photobioreactors lead to the co-cultivation of diverse microbial communities. There is limited knowledge about the "bioreactor ecology" involving microalgae interacting with the microbiome and its subsequent impact on process stability and productivity. In this study, we describe the semi-continuous industrial mass cultivation of the cold-adapted marine diatom, Porosira glacialis UiT201, by investigating the prokaryotic and microeukaryotic (phytoplankton and heterotrophic protist) communities. Data were collected in two consecutive time series experiments, representing the initiation and operation of an preindustrial scale CCU photobioreactor (300,000 liters). The first experiment experienced a culture "crash" of the focal strain after 39 days, while the second culture remained stable and "healthy" for 60 days. The results highlight that this mass cultivation system represents a unique industrial marine microbial ecosystem. The succession of the prokaryotic community was primarily driven by species replacement, indicating turnover due to selective bioreactor conditions and/or biological interactions. Nonetheless, the bioreactor consistently harbors a recurring and abundant core microbiome, suggesting that the closely associated bacterial community is influenced by microalgae-specific properties and can endure a dynamic and variable environment. The observed culture collapse of P. glacialis coincided with changes in the core microbiome structure and different environmental growth conditions compared to the stable and "healthy" experiment. These findings imply that cohabiting microbial taxa within industrial microalgae cultivation likely play a critical role in stabilizing the conversion of industrial CO2 into marine biomass, and changes in community structure serve as an indicator of process stability.

microbiology↗

bioRxiv2023

Revealing the chassis-effect on a broad-host-range genetic switch and its concordance with interspecies bacterial physiologies

Broad-host-range synthetic biology is an emerging frontier that aims to expand our current engineerable domain of microbial hosts for biodesign applications. As more novel species are brought to "model status", synthetic biologists are discovering that identically engineered genetic circuits can exhibit different performances depending on the organism it operates within, an observation referred to as the "chassis-effect". It remains a major challenge to uncover which genome encoded and physiological biological determinants will underpin chassis effects that govern the performance of engineered genetic devices. In this study, we compared model and novel bacterial hosts to ask whether phylogenomic relatedness or similarity in host physiology is a better predictor of toggle switch performance. This was accomplished using comparative framework based on multivariate statistical approaches to systematically demonstrate the chassis-effect and characterize the performance dynamics of a genetic toggle switch operating within six Gammaproteobacteria. Our results solidify the notion that genetic devices are significantly impacted by host-context. Furthermore, we formally determined that hosts exhibiting more similar metrics of growth and molecular physiology also exhibit more similar toggle switch performance, indicating that specific bacterial physiology underpins measurable chassis effects. The result of this study contributes to the field of broad-host-range synthetic biology by lending increased predictive power to the implementation of genetic devices in less-established microbial hosts.

synthetic biology↗

bioRxiv2022

Microbial community dynamics during a harmful Chrysochromulina leadbeateri bloom

A harmful algae bloom occurred in late spring 2019 across multiple, interconnected fjords and bays in northern Norway. The event was caused by the haptophyte Chrysochromulina leadbeateri and led to severe fish mortality at several salmon aquaculture facilities. This study reports on the spatial and temporal succession dynamics of the holistic marine microbiome associated with this bloom by relating all detectable 18S and 16S rRNA gene ASVs to the relative abundance of the C. leadbeateri focal taxon. A k-medoids clustering enabled inferences on how the causative focal taxon co-bloomed with diverse groups of bacteria and microeukaryotes. These co-blooming patterns showed high temporal variability and were distinct between two geographically separated time series stations during the regional harmful algae bloom. The distinct blooming patterns observed with respect to each station were poorly connected to environmental conditions suggesting that other factors, such as biological interactions, may be at least as important in shaping the dynamics of this type of harmful algae bloom. A deeper understanding of microbiome succession patterns during these rare but destructive events will help guide future efforts to forecast deviations from the natural bloom cycles of the northern Norwegian coastal marine ecosystems that are home to intensive aquaculture activities.

microbiology↗

bioRxiv2022

Environment constrains fitness advantages of division of labor in microbial consortia engineered for metabolite push or pull interactions

Fitness benefits from division of labor in microbial consortia are well documented, but the dependency of the benefits on environmental context is poorly understood. Two synthetic Escherichia coli consortia were built to test the relationships between exchanged organic acid, local environment, and opportunity costs of different metabolic strategies. Opportunity costs quantify benefits not realized due to selecting one phenotype over another. The consortia catabolized glucose and exchanged either acetic or lactic acid to create producer-consumer food webs. The organic acids had different inhibitory properties and different opportunity costs associated with their positions in central metabolism. The exchanged metabolites modulated different consortial dynamics. The acetic acid-exchanging (AAE) consortium had a push interaction motif where acetic acid was secreted faster by the producer than the consumer imported it, while the lactic acid-exchanging (LAE) consortium had a pull interaction motif where the consumer imported lactic acid at a comparable rate to its production. The LAE consortium outperformed wild type (WT) batch cultures under the environmental context of weakly buffered conditions, achieving a 55% increase in biomass titer, a 51% increase in biomass per proton yield, an 86% increase in substrate conversion, and the complete elimination of byproduct accumulation all relative to the WT. However, the LAE consortium had the tradeoff of a 42% lower specific growth rate. The AAE consortium did not outperform the WT in any considered fitness metric. Fitness advantages of the LAE consortium were sensitive to environment; increasing the medium buffering capacity negated the fitness advantages compared to WT. IMPORTANCEMost naturally occurring microorganisms persist in consortia where metabolic interactions are common and often essential to ecosystem function. This study uses synthetic ecology to test how different cellular interaction motifs influence fitness properties of consortia. Environmental context ultimately controlled the division of labor fitness as shifts from weakly buffered to highly buffered conditions negated the benefits of the strategy. Understanding the limits of division of labor advances our understanding of natural community functioning which is central to nutrient cycling and provides design rules for assembling consortia used in applied bioprocessing.

synthetic biology↗

bioRxiv2019

Selection, succession and stabilization of soil microbial consortia

Soil microorganisms play fundamental roles in cycling of soil carbon, nitrogen and other nutrients, yet we have a poor understanding of how soil microbiomes are shaped by their nutritional and physical environment. Here we investigated the successional dynamics of a soil microbiome during 21-weeks of enrichment on chitin and its monomer, N-acetylglucosamine. We examined succession of the soil communities in a physically heterogeneous soil matrix as well as a highly mixed liquid medium. The guiding hypothesis was that the initial species richness would influence the tendency for the selected consortia to stabilize and maintain relatively constant community structure over time. We also hypothesized that long term, substrate-driven growth would result in consortia with reduced species richness as compared to the parent microbiome and that this process would be deterministic with relatively little variation between replicates. We found that the initial species richness does influence the long-term community stability in both liquid media and soil and that lower initial richness results in a more rapid convergence to stability. Despite use of the same soil inoculum and access to the same major substrate, the resulting community composition differed greatly in soil compared to liquid medium. Hence, distinct selective pressures in soils relative to homogenous liquid media exist and can control community succession dynamics. This difference is likely related to the fact that soil microbiomes are more likely to thrive, with fewer compositional changes, in a soil matrix compared to liquid environments. IMPORTANCEThe soil microbiome carries out important ecosystem functions, but interactions between soil microbial communities have been difficult to study due to the high microbial diversity and complexity of the soil habitat. Here we successfully obtained stable consortia with reduced complexity that contained species found in the original source soil. These consortia and the methods used to obtain them can be a valuable resource for exploration of specific mechanisms underlying soil microbial community ecology. The results of this study also provide new experimental context to better inform how soil microbial communities are shaped by new environments and how a combination of initial taxonomic structure and physical environment influences stability.

microbiology↗

bioRxiv2018

A broad-host-range event detector: expanding and quantifying performance across bacterial species

Modern microbial biodesign relies on the principle that well-characterized genetic parts can be reused and reconfigured for different functions. However, this paradigm has only been successful in a limited set of hosts, mostly comprised from common lab strains of Escherichia coli. It is clear that new applications - such as chemical sensing and event logging in complex environments - will benefit from new host chassis. This study quantitatively compared how a chemical event logger performed across multiple microbial species. An integrase-based sensor and memory device was operated by two representative soil Pseudomonads - Pseudomonas fluorescens SBW25 and Pseudomonas putida DSM 291. Quantitative comparisons were made between these two non-traditional hosts and two bench-mark Escherichia coli chassis including the probiotic Nissle 1917 and common cloning strain DH5. The performance of sensor and memory components changed according to each host, such that a clear chassis effect was observed and quantified. These results were obtained via fluorescence from reporter proteins that were transcriptionally fused to the integrase and down-stream recombinant region and via data-driven kinetic models. The Pseudomonads proved to be acceptable chassis for the operation of this event logger, which outperformed the common E. coli DH5 in many ways. This study advances an emerging frontier in synthetic biology that aims to build broad-host-range devices and understand the context by which different species can execute programmable genetic operations.

synthetic biology↗

bioRxiv2018

Lactobacillus acidophilus disrupts collaborative multispecies bile acid metabolism

Bile acids are metabolic links between hosts and their gut microbiomes, yet little is known about the roles they play in microbe-to-microbe interactions. Here we present a study designed to investigate the effect that a common probiotic, Lactobacillus acidophilus, has on microbial interactions that lead to formation of secondary bile acids. A model microbial consortium was built from three human gut isolates, Clostridium scindens, Collinsella aerofaciens, and Blautia obeum, and cultured under different bile acid and probiotic treatments. A multi-omics platform that included mass spectrometry-based metabolomics and activity-based proteomic probes was used to produce two major results. The first, was that an uncommon secondary bile acid - ursocholate - was produced by a multi-species chemical synthesis pathway. This result highlights a new microbe-to-microbe interaction mediated by bile acids. The second finding was that the probiotic strain, L. acidophilus, quenched the observed interactions and effectively halted consortial synthesis of ursocholate. Little is known about the role that ursocholate plays in human health and development. However, we did discover that a decrease in ursocholate abundance corresponded with successful weight loss in patients after gastric bypass surgery versus those who did not lose weight after surgery. Hence, this study uncovered basic knowledge that may aid future designs of custom probiotic therapies to combat obesity.

microbiology↗

bioRxiv2018

Forfeiting the founder effect: turnover defines biofilm community succession

Microbial community succession is a fundamental process that effects underlying functions of almost all ecosystems; yet the roles and fates of the most abundant colonizers are poorly understood. Does early abundance spur long term persistence? How do deterministic and stochastic processes influence the roles of founder species? We performed a succession experiment within a hypersaline microbial mat ecosystem to investigate how ecological processes contributed to the turnover of founder species. Bacterial and micro-eukaryotic founder species were identified from primary succession and tracked through a defined maturation period using 16S and 18S rRNA gene amplicon sequencing in combination with high resolution imaging that utilized stable isotope tracers to evaluate basic functional capabilities. The majority of the founder species did not maintain high relative abundances in later stages of succession. Turnover (versus nestedness) was the dominant process shaping the final community structure. We also asked if different ecological processes acted on bacteria versus eukaryotes during successional stages and found that deterministic and stochastic forces corresponded more with eukaryote and bacterial colonization, respectively. Our results show that taxa from different kingdoms, that share habitat in the tight spatial confines of a biofilm, were influenced by different ecological forces and time scales of succession.

ecology↗