Anaerobic single-cell dispensing facilitates the cultivation of human gut bacteria.

Cultivation via classical agar plate (CAP) approaches is widely used to study microbial communities, but they are time-consuming. An alternative approach is the application of single-cell dispensing (SCD), which allows high-throughput, label-free sorting of microscopic particles. We aimed to develop a new anaerobic SCD workflow to cultivate human gut bacteria and compared it with CAP using faecal communities on three rich culture media. We found that the SCD approach significantly decreased the experimental time to obtain pure cultures from 17 ± 4 to 5 ± 0 days, while the isolate diversity and relative abundance coverage were comparable for both approaches. We further tested the total captured fraction by sequencing the sorted bacteria directly after growth as bulk biomass from 2400 dispensed single cells without downstream identification of individual strains. In this approach, the cultured fraction increased from 35.2% to 52.2% for SCD, highlighting the potential for deeper cultivation projects from single samples. SCD-based cultivation also captured species not detected by sequencing (16 ± 5 per sample, including seven novel taxa). From this work, 82 human gut bacterial species across five phyla (Actinobacteriota, Bacteroidota, Desulfobacterota, Firmicutes and Proteobacteria) and 24 families were obtained, including the first cultured member of 11 novel genera and 10 novel species that were fully characterized taxonomically.

Communities of Niche-optimized Strains (CoNoS) - Design and creation of stable, genome-reduced co-cultures.

Current bioprocesses for production of value-added compounds are mainly based on pure cultures that are composed of rationally engineered strains of model organisms with versatile metabolic capacities. However, in the comparably well-defined environment of a bioreactor, metabolic flexibility provided by various highly abundant biosynthetic enzymes is much less required and results in suboptimal use of carbon and energy sources for compound production. In nature, non-model organisms have frequently evolved in communities where genome-reduced, auxotrophic strains cross-feed each other, suggesting that there must be a significant advantage compared to growth without cooperation. To prove this, we started to create and study synthetic communities of niche-optimized strains (CoNoS) that consists of two strains of the same species Corynebacterium glutamicum that are mutually dependent on one amino acid. We used both the wild-type and the genome-reduced C1* chassis for introducing selected amino acid auxotrophies, each based on complete deletion of all required biosynthetic genes. The best candidate strains were used to establish several stably growing CoNoS that were further characterized and optimized by metabolic modelling, microfluidic experiments and rational metabolic engineering to improve amino acid production and exchange. Finally, the engineered CoNoS consisting of an l-leucine and l-arginine auxotroph showed a specific growth rate equivalent to 83% of the wild type in monoculture, making it the fastest co-culture of two auxotrophic C. glutamicum strains to date. Overall, our results are a first promising step towards establishing improved biobased production of value-added compounds using the CoNoS approach.

N-dependent dynamics of root growth and nitrate and ammonium uptake are altered by the bacterium Herbaspirillum seropedicae in the cereal model Brachypodium distachyon.

Nitrogen (N) fixation in cereals by root-associated bacteria is a promising solution for reducing use of chemical N fertilizers in agriculture. However, plant and bacterial responses are unpredictable across environments. We hypothesized that cereal responses to N-fixing bacteria are dynamic, depending on N supply and time. To quantify the dynamics, a gnotobiotic, fabricated ecosystem (EcoFAB) was adapted to analyse N mass balance, to image shoot and root growth, and to measure gene expression of Brachypodium distachyon inoculated with the N-fixing bacterium Herbaspirillum seropedicae. Phenotyping throughput of EcoFAB-N was 25-30 plants h-1 with open software and imaging systems. Herbaspirillum seropedicae inoculation of B. distachyon shifted root and shoot growth, nitrate versus ammonium uptake, and gene expression with time; directions and magnitude depended on N availability. Primary roots were longer and root hairs shorter regardless of N, with stronger changes at low N. At higher N, H. seropedicae provided 11% of the total plant N that came from sources other than the seed or the nutrient solution. The time-resolved phenotypic and molecular data point to distinct modes of action: at 5 mM NH4NO3 the benefit appears through N fixation, while at 0.5 mM NH4NO3 the mechanism appears to be plant physiological, with H. seropedicae promoting uptake of N from the root medium.Future work could fine-tune plant and root-associated microorganisms to growth and nutrient dynamics.

The iSplit GFP assay detects intracellular recombinant proteins in Bacillus subtilis.

BACKGROUND: Bacillus subtilis is one of the most important microorganisms for recombinant protein production. It possesses the GRAS (generally recognized as safe) status and a potent protein secretion capacity. Secretory protein production greatly facilitates downstream processing and thus significantly reduces costs. However, not all heterologous proteins are secreted and intracellular production poses difficulties for quantification. To tackle this problem, we have established a so-called intracellular split GFP (iSplit GFP) assay in B. subtilis as a tool for the in vivo protein detection during expression in batch cultures and at a single-cell level. For the iSplit GFP assay, the eleventh ß-sheet of sfGFP is fused to a target protein and can complement a detector protein consisting of the respective truncated sfGFP (GFP1-10) to form fluorescent holo-GFP. RESULTS: As proof of concept, the GFP11-tag was fused C-terminally to the E. coli ß-glucuronidase GUS, resulting in fusion protein GUS11. Variable GUS and GUS11 production levels in B. subtilis were achieved by varying the ribosome binding site via spacers of increasing lengths (4-12 nucleotides) for the GUS-encoding gene. Differences in intracellular enzyme accumulation were determined by measuring the GUS11 enzymatic activity and subsequently by adding the detector protein to respective cell extracts. Moreover, the detector protein was co-produced with the GUS11 using a two-plasmid system, which enabled the in vivo detection and online monitoring of glucuronidase production. Using this system in combination with flow cytometry and microfluidics, we were able to monitor protein production at a single-cell level thus yielding information about intracellular protein distribution and culture heterogeneity. CONCLUSION: Our results demonstrate that the iSplit GFP assay is suitable for the detection, quantification and online monitoring of recombinant protein production in B. subtilis during cultivation as well as for analyzing production heterogeneity and intracellular localization at a single-cell level.

Importance of Pyruvate Sensing and Transport for the Resuscitation of Viable but Nonculturable Escherichia coli K-12.

Escherichia coli and many other bacterial species can enter into a viable but nonculturable (VBNC) state, which is a survival strategy adopted by cells exposed to adverse environmental conditions. Pyruvate is known to be one factor that promotes resuscitation of VBNC cells. Here we studied the role of a pyruvate-sensing network, composed of the histidine kinase-response regulator systems BtsS/BtsR and YpdA/YpdB and the target gene btsT, encoding the high-affinity pyruvate/H+ symporter BtsT, in the resuscitation of VBNC E. coli K-12 cells after exposure to cold for 120 days. Analysis of the proteome of VBNC cells revealed upregulation, relative to exponentially growing cells, of BtsT and other proteins involved in pyruvate metabolism. Provision of pyruvate stimulated protein and DNA biosynthesis, and thus resuscitation, in wild-type but not btsSR ypdAB mutant VBNC cells. This result was corroborated by time-dependent tracking of the resuscitation of individual VBNC E. coli cells observed in a microfluidic system. Finally, transport assays revealed that 14C-labeled pyruvate was rapidly taken up into VBNC cells by BtsT. These results provide the first evidence that pyruvate is taken up as a carbon source for the resuscitation of VBNC E. coli cells.IMPORTANCE Viable but nonculturable (VBNC) bacteria do not form colonies in standard medium but otherwise retain their metabolic activity and can express toxic proteins. Many bacterial genera, including Escherichia, Vibrio, and Listeria, have been shown to enter the VBNC state upon exposure to adverse conditions, such as low temperature, radiation, and starvation. Ultimately, these organisms pose a public health risk with potential implications for the pharmaceutical and food industries, as dormant organisms are especially difficult to selectively eliminate and VBNC bacteria can be resuscitated if placed in an environment with appropriate nutrition and temperature. Here we used a microfluidic system to monitor the resuscitation of single VBNC cells over time. We provide new molecular insights into the initiation of resuscitation by demonstrating that VBNC E. coli cells rapidly take up pyruvate with an inducible high-affinity transporter, whose expression is triggered by the BtsSR-YpdAB sensing network.

A Single-Cell View of the BtsSR/YpdAB Pyruvate Sensing Network in Escherichia coli and Its Biological Relevance.

Fluctuating environments and individual physiological diversity force bacteria to constantly adapt and optimize the uptake of substrates. We focus here on two very similar two-component systems (TCSs) of Escherichia coli belonging to the LytS/LytTR family: BtsS/BtsR (formerly YehU/YehT) and YpdA/YpdB. Both TCSs respond to extracellular pyruvate, albeit with different affinities, typically during postexponential growth, and each system regulates expression of a single transporter gene, yjiY and yhjX, respectively. To obtain insights into the biological significance of these TCSs, we analyzed the activation of the target promoters at the single-cell level. We found unimodal cell-to-cell variability; however, the degree of variance was strongly influenced by the available nutrients and differed between the two TCSs. We hypothesized that activation of either of the TCSs helps individual cells to replenish carbon resources. To test this hypothesis, we compared wild-type cells with the btsSR ypdAB mutant under two metabolically modulated conditions: protein overproduction and persister formation. Although all wild-type cells were able to overproduce green fluorescent protein (GFP), about half of the btsSR ypdAB population was unable to overexpress GFP. Moreover, the percentage of persister cells, which tolerate antibiotic stress, was significantly lower in the wild-type cells than in the btsSR ypdAB population. Hence, we suggest that the BtsS/BtsR and YpdA/YpdB network contributes to a balancing of the physiological state of all cells within a population.IMPORTANCE Histidine kinase/response regulator (HK/RR) systems enable bacteria to respond to environmental and physiological fluctuations. Escherichia coli and other members of the Enterobacteriaceae possess two similar LytS/LytTR-type HK/RRs, BtsS/BtsR (formerly YehU/YehT) and YpdA/YpdB, which form a functional network. Both systems are activated in response to external pyruvate, typically when cells face overflow metabolism during post-exponential growth. Single-cell analysis of the activation of their respective target genes yjiY and yhjX revealed cell-to-cell variability, and the range of variation was strongly influenced by externally available nutrients. Based on the phenotypic characterization of a btsSR ypdAB mutant compared to the parental strain, we suggest that this TCS network supports an optimization of the physiological state of the individuals within the population.

Silencing of cryptic prophages in Corynebacterium glutamicum.

DNA of viral origin represents a ubiquitous element of bacterial genomes. Its integration into host regulatory circuits is a pivotal driver of microbial evolution but requires the stringent regulation of phage gene activity. In this study, we describe the nucleoid-associated protein CgpS, which represents an essential protein functioning as a xenogeneic silencer in the Gram-positive Corynebacterium glutamicum CgpS is encoded by the cryptic prophage CGP3 of the C. glutamicum strain ATCC 13032 and was first identified by DNA affinity chromatography using an early phage promoter of CGP3. Genome-wide profiling of CgpS binding using chromatin affinity purification and sequencing (ChAP-Seq) revealed its association with AT-rich DNA elements, including the entire CGP3 prophage region (187 kbp), as well as several other elements acquired by horizontal gene transfer. Countersilencing of CgpS resulted in a significantly increased induction frequency of the CGP3 prophage. In contrast, a strain lacking the CGP3 prophage was not affected and displayed stable growth. In a bioinformatics approach, cgpS orthologs were identified primarily in actinobacterial genomes as well as several phage and prophage genomes. Sequence analysis of 618 orthologous proteins revealed a strong conservation of the secondary structure, supporting an ancient function of these xenogeneic silencers in phage-host interaction.

Homogenizing bacterial cell factories: Analysis and engineering of phenotypic heterogeneity.

In natural habitats, microbes form multispecies communities that commonly face rapidly changing and highly competitive environments. Thus, phenotypic heterogeneity has evolved as an innate and important survival strategy to gain an overall fitness advantage over cohabiting competitors. However, in defined artificial environments such as monocultures in small- to large-scale bioreactors, cell-to-cell variations are presumed to cause reduced production yields as well as process instability. Hence, engineering microbial production toward phenotypic homogeneity is a highly promising approach for synthetic biology and bioprocess optimization. In this review, we discuss recent studies that have unraveled the cell-to-cell heterogeneity observed during bacterial gene expression and metabolite production as well as the molecular mechanisms involved. In addition, current single-cell technologies are briefly reviewed with respect to their applicability in exploring cell-to-cell variations. We highlight emerging strategies and tools to reduce phenotypic heterogeneity in biotechnological expression setups. Here, strain or inducer modifications are combined with cell physiology manipulations to achieve the ultimate goal of equalizing bacterial populations. In this way, the majority of cells can be forced into high productivity, thus reducing less productive subpopulations that tend to consume valuable resources during production. Modifications in uptake systems, inducer molecules or nutrients represent valuable tools for diminishing heterogeneity. Finally, we address the challenge of transferring homogeneously responding cells into large-scale bioprocesses. Environmental heterogeneity originating from extrinsic factors such as stirring speed and pH, oxygen, temperature or nutrient distribution can significantly influence cellular physiology. We conclude that engineering microbial populations toward phenotypic homogeneity is an increasingly important task to take biotechnological productions to the next level of control.

Live cell imaging of SOS and prophage dynamics in isogenic bacterial populations.

Almost all bacterial genomes contain DNA of viral origin, including functional prophages or degenerated phage elements. A frequent but often unnoted phenomenon is the spontaneous induction of prophage elements (SPI) even in the absence of an external stimulus. In this study, we have analyzed SPI of the large, degenerated prophage CGP3 (187 kbp), which is integrated into the genome of the Gram-positive Corynebacterium glutamicum ATCC 13032. Time-lapse fluorescence microscopy of fluorescent reporter strains grown in microfluidic chips revealed the sporadic induction of the SOS response as a prominent trigger of CGP3 SPI but also displayed a considerable fraction (∼30%) of RecA-independent SPI. Whereas approx. 20% of SOS-induced cells recovered from this stress and resumed growth, the spontaneous induction of CGP3 always led to a stop of growth and likely cell death. A carbon source starvation experiment clearly emphasized that SPI only occurs in actively proliferating cells, whereas sporadic SOS induction was still observed in resting cells. These data highlight the impact of sporadic DNA damage on the activity of prophage elements and provide a time-resolved, quantitative description of SPI as general phenomenon of bacterial populations.

Photocaged Arabinose: A Novel Optogenetic Switch for Rapid and Gradual Control of Microbial Gene Expression.

Controlling cellular functions by light allows simple triggering of biological processes in a non-invasive fashion with high spatiotemporal resolution. In this context, light-regulated gene expression has enormous potential for achieving optogenetic control over almost any cellular process. Here, we report on two novel one-step cleavable photocaged arabinose compounds, which were applied as light-sensitive inducers of transcription in bacteria. Exposure of caged arabinose to UV-A light resulted in rapid activation of protein production, as demonstrated for GFP and the complete violacein biosynthetic pathway. Moreover, single-cell analysis revealed that intrinsic heterogeneity of arabinose-mediated induction of gene expression was overcome when using photocaged arabinose. We have thus established a novel phototrigger for synthetic bio(techno)logy applications that enables precise and homogeneous control of bacterial target gene expression.

Vizardous: interactive analysis of microbial populations with single cell resolution.

MOTIVATION: Single cell time-lapse microscopy is a powerful method for investigating heterogeneous cell behavior. Advances in microfluidic lab-on-a-chip technologies and live-cell imaging render the parallel observation of the development of individual cells in hundreds of populations possible. While image analysis tools are available for cell detection and tracking, biologists are still confronted with the challenge of exploring and evaluating this data. RESULTS: We present the software tool Vizardous that assists scientists with explorative analysis and interpretation tasks of single cell data in an interactive, configurable and visual way. With Vizardous, lineage tree drawings can be augmented with various, time-resolved cellular characteristics. Associated statistical moments bridge the gap between single cell and the population-average level. AVAILABILITY AND IMPLEMENTATION: The software, including documentation and examples, is available as executable Java archive as well as in source form at https://github.com/modsim/vizardous. CONTACT: k.noeh@fz-juelich.de. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Light-Controlled Cell Factories: Employing Photocaged Isopropyl-ß-d-Thiogalactopyranoside for Light-Mediated Optimization of lac Promoter-Based Gene Expression and (+)-Valencene Biosynthesis in Corynebacterium glutamicum.

Precise control of microbial gene expression resulting in a defined, fast, and homogeneous response is of utmost importance for synthetic bio(techno)logical applications. However, even broadly applied biotechnological workhorses, such as Corynebacterium glutamicum, for which induction of recombinant gene expression commonly relies on the addition of appropriate inducer molecules, perform moderately in this respect. Light offers an alternative to accurately control gene expression, as it allows for simple triggering in a noninvasive fashion with unprecedented spatiotemporal resolution. Thus, optogenetic switches are promising tools to improve the controllability of existing gene expression systems. In this regard, photocaged inducers, whose activities are initially inhibited by light-removable protection groups, represent one of the most valuable photoswitches for microbial gene expression. Here, we report on the evaluation of photocaged isopropyl-ß-d-thiogalactopyranoside (IPTG) as a light-responsive control element for the frequently applied tac-based expression module in C. glutamicum In contrast to conventional IPTG, the photocaged inducer mediates a tightly controlled, strong, and homogeneous expression response upon short exposure to UV-A light. To further demonstrate the unique potential of photocaged IPTG for the optimization of production processes in C. glutamicum, the optogenetic switch was finally used to improve biosynthesis of the growth-inhibiting sesquiterpene (+)-valencene, a flavoring agent and aroma compound precursor in food industry. The variation in light intensity as well as the time point of light induction proved crucial for efficient production of this toxic compound. IMPORTANCE: Optogenetic tools are light-responsive modules that allow for a simple triggering of cellular functions with unprecedented spatiotemporal resolution and in a noninvasive fashion. Specifically, light-controlled gene expression exhibits an enormous potential for various synthetic bio(techno)logical purposes. Before our study, poor inducibility, together with phenotypic heterogeneity, was reported for the IPTG-mediated induction of lac-based gene expression in Corynebacterium glutamicum By applying photocaged IPTG as a synthetic inducer, however, these drawbacks could be almost completely abolished. Especially for increasing numbers of parallelized expression cultures, noninvasive and spatiotemporal light induction qualifies for a precise, homogeneous, and thus higher-order control to fully automatize or optimize future biotechnological applications.

Spatiotemporal microbial single-cell analysis using a high-throughput microfluidics cultivation platform.

Cell-to-cell heterogeneity typically evolves due to a manifold of biological and environmental factors and special phenotypes are often relevant for the fate of the whole population but challenging to detect during conventional analysis. We demonstrate a microfluidic single-cell cultivation platform that incorporates several hundred growth chambers, in which isogenic bacteria microcolonies growing in cell monolayers are tracked by automated time-lapse microscopy with spatiotemporal resolution. The device was not explicitly developed for a specific organism, but has a very generic configuration suitable for various different microbial organisms. In the present study, we analyzed Corynebacterium glutamicum microcolonies, thereby generating complete lineage trees and detailed single-cell data on division behavior and morphology in order to demonstrate the platform's overall capabilities. Furthermore, the occurrence of spontaneously induced stress in individual C. glutamicum cells was investigated by analyzing strains with genetically encoded reporter systems and optically visualizing SOS response. The experiments revealed spontaneous SOS induction in the absence of any external trigger comparable to results obtained by flow cytometry (FC) analyzing cell samples from conventional shake flask cultivation. Our microfluidic setup delivers detailed single-cell data with spatial and temporal resolution; complementary information to conventional FC results.

Analysis of SOS-induced spontaneous prophage induction in Corynebacterium glutamicum at the single-cell level.

The genome of the Gram-positive soil bacterium Corynebacterium glutamicum ATCC 13032 contains three integrated prophage elements (CGP1 to -3). Recently, it was shown that the large lysogenic prophage CGP3 (∼187 kbp) is excised spontaneously in a small number of cells. In this study, we provide evidence that a spontaneously induced SOS response is partly responsible for the observed spontaneous CGP3 induction. Whereas previous studies focused mainly on the induction of prophages at the population level, we analyzed the spontaneous CGP3 induction at the single-cell level using promoters of phage genes (Pint2 and Plysin) fused to reporter genes encoding fluorescent proteins. Flow-cytometric analysis revealed a spontaneous CGP3 activity in about 0.01 to 0.08% of the cells grown in standard minimal medium, which displayed a significantly reduced viability. A PrecA-eyfp promoter fusion revealed that a small fraction of C. glutamicum cells (∼0.2%) exhibited a spontaneous induction of the SOS response. Correlation of PrecA to the activity of downstream SOS genes (PdivS and PrecN) confirmed a bona fide induction of this stress response rather than stochastic gene expression. Interestingly, the reporter output of PrecA and CGP3 promoter fusions displayed a positive correlation at the single-cell level (ρ = 0.44 to 0.77). Furthermore, analysis of the PrecA-eyfp/Pint2-e2-crimson strain during growth revealed the highest percentage of spontaneous PrecA and Pint2 activity in the early exponential phase, when fast replication occurs. Based on these studies, we postulate that spontaneously occurring DNA damage induces the SOS response, which in turn triggers the induction of lysogenic prophages.

Beyond growth rate 0.6: What drives Corynebacterium glutamicum to higher growth rates in defined medium.

In a former study we showed that Corynebacterium glutamicum grows much faster in defined CGXII glucose medium when growth was initiated in highly diluted environments [Grünberger et al. (2013b) Biotechnol Bioeng]. Here we studied the batch growth of C. glutamicum in CGXII at a comparable low starting biomass concentration of OD ≈ 0.005 in more detail. During bioreactor cultivations a bi-phasic growth behavior with changing growth rates was observed. Initially the culture grew with µË†=0.61±0.02 h-1 before the growth rate dropped to µË†=0.46±0.02 h-1. We were able to confirm the elevated growth rate for C. glutamicum in CGXII and showed for the first time a growth rate beyond 0.6 in lab-scale bioreactor cultivations on defined medium. Advanced growth studies combining well-designed bioreactor and microfluidic single-cell cultivations (MSCC) with quantitative transcriptomics, metabolomics and integrative in silico analysis revealed protocatechuic acid as a hidden co-substrate for accelerated growth within CGXII. The presented approach proves the general applicability of MSCC to investigate and validate the effect of single medium components on microorganism growth during cultivation in liquid media, and therefore might be of interest for any kind of basic growth study.

Beyond growth rate 0.6: Corynebacterium glutamicum cultivated in highly diluted environments.

Fast growth of industrial microorganisms, such as Corynebacterium glutamicum, is a direct amplifier for the productivity of any growth coupled or decoupled production process. Recently, it has been shown that C. glutamicum when grown in a novel picoliter bioreactor (PLBR) exhibits a 50% higher growth rate compared to a 1 L batch cultivation [Grünberger et al. (2012) Lab Chip]. We here compare growth of C. glutamicum with glucose as substrate at different scales covering batch cultivations in the liter range down to single cell cultivations in the picoliter range. The maximum growth rate of standard batch cultures as estimated from different biomass quantification methods is mu = 0.42 ± 0.03 h(-1) even for microtiter scale cultivations. In contrast, growth in a microfluidic perfusion system enabling analysis of single cells reproducibly reveals a higher growth rate of mu = 0.62 ± 0.02 h(-1). When in the same perfusion system cell-free supernatant from exponentially grown shake flask cultures is used the growth rate of single cells is reduced to mu = 0.47 ± 0.02 h(-1). Likewise, when fresh medium is additionally supplied with 5 mM acetate, a growth rate of mu = 0.51 ± 0.01 h(-1) is determined. These results prove that higher growth rates of C. glutamicum than known from typical batch cultivations are possible, and that growth is definitely impaired by very low concentrations of byproducts such as acetate.

Assessing the growth kinetics and stoichiometry of Escherichia coli at the single-cell level.

Microfluidic cultivation and single-cell analysis are inherent parts of modern microbial biotechnology and microbiology. However, implementing biochemical engineering principles based on the kinetics and stoichiometry of growth in microscopic spaces remained unattained. We here present a novel integrated framework that utilizes distinct microfluidic cultivation technologies and single-cell analytics to make the fundamental math of process-oriented biochemical engineering applicable at the single-cell level. A combination of non-invasive optical cell mass determination with sub-pg sensitivity, microfluidic perfusion cultivations for establishing physiological steady-states, and picoliter batch reactors, enabled the quantification of all physiological parameters relevant to approximate a material balance in microfluidic reaction environments. We determined state variables (biomass concentration based on single-cell dry weight and mass density), biomass synthesis rates, and substrate affinities of cells grown in microfluidic environments. Based on this data, we mathematically derived the specific kinetics of substrate uptake and growth stoichiometry in glucose-grown Escherichia coli with single-cell resolution. This framework may initiate microscale material balancing beyond the averaged values obtained from populations as a basis for integrating heterogeneous kinetic and stoichiometric single-cell data into generalized bioprocess models and descriptions.

Reliable cell retention of mammalian suspension cells in microfluidic cultivation chambers.

Microfluidic cultivation, with its high level of environmental control and spatio-temporal resolution of cellular behavior, is a well-established tool in today's microfluidics. Yet, reliable retention of (randomly) motile cells inside designated cultivation compartments still represents a limitation, which prohibits systematic single-cell growth studies. To overcome this obstacle, current approaches rely on complex multilayer chips or on-chip valves, which makes their application for a broad community of users infeasible. Here, we present an easy-to-implement cell retention concept to withhold cells inside microfluidic cultivation chambers. By introducing a blocking structure into a cultivation chamber's entrance and nearly closing it, cells can be manually pushed into the chamber during loading procedures but are unable to leave it autonomously in subsequent long-term cultivation. CFD simulations as well as trace substance experiments confirm sufficient nutrient supply within the chamber. Through preventing recurring cell loss, growth data obtained from Chinese hamster ovary cultivation on colony level perfectly match data determined from single-cell data, which eventually allows reliable high throughput studies of single-cell growth. Due to its transferability to other chamber-based approaches, we strongly believe that our concept is also applicable for a broad range of cellular taxis studies or analyses of directed migration in basic or biomedical research.

Enabling oxygen-controlled microfluidic cultures for spatiotemporal microbial single-cell analysis.

Microfluidic cultivation devices that facilitate O2 control enable unique studies of the complex interplay between environmental O2 availability and microbial physiology at the single-cell level. Therefore, microbial single-cell analysis based on time-lapse microscopy is typically used to resolve microbial behavior at the single-cell level with spatiotemporal resolution. Time-lapse imaging then provides large image-data stacks that can be efficiently analyzed by deep learning analysis techniques, providing new insights into microbiology. This knowledge gain justifies the additional and often laborious microfluidic experiments. Obviously, the integration of on-chip O2 measurement and control during the already complex microfluidic cultivation, and the development of image analysis tools, can be a challenging endeavor. A comprehensive experimental approach to allow spatiotemporal single-cell analysis of living microorganisms under controlled O2 availability is presented here. To this end, a gas-permeable polydimethylsiloxane microfluidic cultivation chip and a low-cost 3D-printed mini-incubator were successfully used to control O2 availability inside microfluidic growth chambers during time-lapse microscopy. Dissolved O2 was monitored by imaging the fluorescence lifetime of the O2-sensitive dye RTDP using FLIM microscopy. The acquired image-data stacks from biological experiments containing phase contrast and fluorescence intensity data were analyzed using in-house developed and open-source image-analysis tools. The resulting oxygen concentration could be dynamically controlled between 0% and 100%. The system was experimentally tested by culturing and analyzing an E. coli strain expressing green fluorescent protein as an indirect intracellular oxygen indicator. The presented system allows for innovative microbiological research on microorganisms and microbial ecology with single-cell resolution.

The development and application of a single-cell biosensor for the detection of l-methionine and branched-chain amino acids.

The detection and quantification of specific metabolites in single bacterial cells is a major goal for industrial biotechnology. We have developed a biosensor based on the transcriptional regulator Lrp that detects intracellular l-methionine and branched-chain amino acids in Corynebacterium glutamicum. In assays, fluorescence output showed a linear relationship with cytoplasmic concentrations of the effector amino acids. In increasing order, the affinity of Lrp for the amino acids is l-valine, l-isoleucine, l-leucine and l-methionine. The sensor was applied for online monitoring and analysis of cell-to-cell variability of l-valine production by the pyruvate dehydrogenase-deficient C. glutamicum strain ΔaceE. Finally, the sensor system was successfully used in a high-throughput (HT) FACS screen for the isolation of amino acid-producing mutants after random mutagenesis of a non-producing wild type strain. These applications illustrate how one of nature's sensor devices - transcriptional regulators - can be used for the analysis, directed evolution and HT screening for microbial strain development.