Biology subjects

Lin, X. N.

Publications and source records attributed to Lin, X. N..

5 recordsLinked to original sources

bioRxiv2026

BiDBiC: A novel ultra-high-throughput pipeline for Bead-in-Droplet Biofilm Cultivation and Characterization

Biofilms are a form of microbial growth consisting of cells, often attached to a surface, embedded in a structured 3D extracellular matrix that confers important emergent properties such as increased resistance to physical removal and antimicrobials. Despite the importance of biofilms to a variety of systems and despite increasing attention from both the public and private sectors, high-throughput approaches to study them are scarce, limiting investigations of complex mechanisms critical for the structure and function of biofilms, such as interactions in multispecies communities. We thus developed a novel workflow to grow and analyze bacterial cells adhered to plastic beads encapsulated within highly parallel nanoliter-scale water-in-oil microfluidic droplets. We term this pipeline for bead-in-droplet biofilm cultivation and characterization BiDBiC. To benchmark BiDBiC, we utilized a well-characterized biofilm former, Stenotrophomonas maltophilia, as well as a poorly studied drinking water biofilm isolate, Sphingopyxis sp. OPL5. Each bacterium exhibited strong adherent growth when co-encapsulated with polystyrene beads in droplets. Furthermore, we retrieved beads from the droplets and removed planktonic cells, enabling focused analysis of adhered cells. From bead-associated biomass, we extracted DNA and RNA for molecular analysis and recovered viable cells for subculturing. We conclude with a discussion of further development of the platform as well as suggestions for microbial biofilm systems that may benefit from ultra-high-throughput droplet-enabled cultivation and analysis. Insight BoxBiofilms are an important yet understudied form of microbial growth. In this study, we developed bead-in-droplet biofilm cultivation (BiDBiC), a novel ultra-high-throughput workflow to culture biofilms. The droplets act as massively parallelized miniature bioreactors, with co-encapsulated plastic beads providing a surface for cell attachment and growth. Using a well-characterized biofilm former, Stenotrophomonas maltophilia, as well as a drinking water biofilm isolate, Sphingopyxis sp. OPL5, we demonstrated robust adherent cell growth in droplets. We additionally efficiently separated beads from planktonic cells, enabling targeted molecular analysis and outgrowth of adherent cells. Adapting and extending BiDBiC could facilitate the study of numerous complex biofilm systems, such as diverse isolates or combinatorial subcommunities of microbiomes, to observe their phenotypes and probe underlying mechanisms.

microbiology↗

bioRxiv2025

Massively parallel metabarcoding of droplet co-cultures

A systems approach to microbial community ecology requires high-throughput tools for identifying key microbial interactions. While microfluidic droplets enable the high throughput generation, co-cultivation, and sorting of miniaturized co-cultures in sub-nanoliter, uniform water-in-oil emulsions, analyzing composition of individual droplet co-cultures at comparable throughputs has been challenging, particularly with environmentally isolated or less genetically tractable strains. To address this bottleneck, we present Cocoa-seq (combinatorial co-cultivation and amplicon sequencing), a droplet-based microfluidic workflow that enables 16S rRNA gene amplicon sequencing for thousands of droplet co-cultures in parallel. In summary, after co-cultivation of microbial co-cultures in agarose droplets, which are then set and recovered as discrete gel beads, Cocoa-seq pairs individual gel beads with barcoded primer beads in new droplets to produce multiplexed amplicon libraries via droplet PCR for sequencing. To benchmark Cocoa-seq, we used a model two-species co-culture and four-member mock communities with three different compositions. The community profiles derived from Cocoa-seq were qualitatively consistent with fluorescence microscopy-based estimates for the two-species co-culture and correlated well with mock community expectations, even for low-abundance representatives. Attempts to incorporate spike-in 16S standards for quantification of absolute abundance were hindered by PCR stochasticity at the single-molecule level, and we provide a simulation-based explanation of this bias and recommend against relying on spike-ins for quantitative inference. ImportanceResearchers in microbial ecology are increasingly utilizing sub-nanoliter microfluidic droplets to construct, grow, sort, and analyze miniaturized microbial co-cultures to identify and characterize critical microbial interactions. However, while the generation, incubation, and sorting of droplet co-cultures is scalable, analyzing the composition of these droplet co-cultures in large number is much more difficult. We demonstrate and benchmark a workflow for multiplexing 16S rRNA amplicon libraries derived from thousands of individual droplet co-cultures in a single sequencing run. This addition in the arsenal of growing microfluidic capabilities will support future study of critical interactions within complex microbial systems. Study fundingNational Science Foundation (Awards 2120909, 2426415) (awarded to XNL)

microbiology↗

bioRxiv2023

Microdroplet-enabled high-throughput cultivation of vaginal bacteria using cervicovaginal fluids

The human vaginal microbiome (HVM) is closely associated with the health of the host. In particular, bacterial vaginosis, a condition where vaginal lactobacilli are reduced dramatically by an overgrowth of various other bacteria, has been linked to increased risk of sexually transmitted infectious diseases, including HIV, and preterm birth. Recent culture-independent studies leveraging next-generation sequencing technology have revealed that the HVM composition differs between women and changes over time. However, questions remain as to the underlying mechanisms and culture-dependent studies are needed for further elucidation of the HVMs genotype-phenotype relationships and system-level properties in vivo. In this work, we have adapted a previously developed microdroplet-based high-throughput cultivation platform for the investigation of vaginal bacteria using the cervicovaginal fluid (CVF) as cultivation medium. Using undiluted CVF collected with Softdiscs discs, we observed the growth of L. iners in microdroplets containing CVF pooled from samples with a high prevalence of L. crispatus. Although demonstrated with L. iners, this work establishes a new framework for culturing microorganisms under clinically-relevant conditions ex vivo using minute volumes of host fluids; it can be further extended and adapted for addressing numerous questions about the HVM and other complex microbiomes.

microbiology↗

bioRxiv2020

Temperature regulation as a tool to program synthetic microbial community composition

Engineering of synthetic microbial communities is emerging as a powerful new paradigm for performing various industrially, medically, and environmentally important processes. To reach the fullest potential, however, this approach requires further development in many aspects, a key one being regulating the community composition. Here we leverage well established mechanisms in ecology which govern the relative abundance of multi-species ecosystems and develop a new tool for programming the composition of synthetic microbial communities. Using a simple model system consisting of two microorganisms Escherichia coli and Pseudomonas putida, which occupy different but partially overlapping thermal niches, we demonstrate that temperature regulation can be used to enable coexistence and program the community composition. We first investigate a constant temperature regime and show that different temperatures lead to different community compositions. Next, we invent a new cycling temperature regime and show that it can dynamically tune the microbial community, achieving a wide range of compositions depending on parameters that are readily manipulatable. Our work provides conclusive proof of concept that temperature regulation is a versatile and powerful tool capable of programming compositions of synthetic microbial communities.

systems biology↗

bioRxiv2019

Syntrophic co-culture amplification of production phenotype for high-throughput screening of microbial strain libraries

Microbes can be engineered to synthesize a wide array of bioproducts, yet production phenotype evaluation remains a frequent bottleneck in the design-build-test cycle where strain development requires iterative rounds of library construction and testing. Here, we present Syntrophic Co-culture Amplification of Production phenotype (SnoCAP). Through a metabolic cross-feeding circuit, the production level of a target molecule is translated into highly distinguishable co-culture growth characteristics, which amplifies differences in production into highly distinguishable growth phenotypes. We demonstrate SnoCAP with the screening of Escherichia coli strains for production of two target molecules: 2-ketoisovalerate, a precursor of the drop-in biofuel isobutanol, and L-tryptophan. The dynamic range of the screening can be tuned by employing an inhibitory analog of the target molecule. Screening based on this framework requires compartmentalization of individual producers with the sensor strain. We explore three formats of implementation with increasing throughput capability: confinement in microtiter plates (102-104 assays/experiment), spatial separation on agar plates (104-105 assays/experiment), and encapsulation in microdroplets (105-107 assays/experiment). Using SnoCAP, we identified an efficient isobutanol production strain from a random mutagenesis library, reaching a final titer that is 5-fold higher than that of the parent strain. The framework can also be extended to screening for secondary metabolite production using a push-pull strategy. We expect that SnoCAP can be readily adapted to the screening of various microbial species, to improve production of a wide range of target molecules.

synthetic biology↗