자료유형  

 학위논문

Control Number  

0016933696

International Standard Book Number  

9798379566593

Dewey Decimal Classification Number  

576

Main Entry-Personal Name  

Tan, James Yi.

Publication, Distribution, etc. (Imprint  

[S.l.] : University of Michigan., 2023

Publication, Distribution, etc. (Imprint  

Ann Arbor : ProQuest Dissertations & Theses, 2023

Physical Description  

1 online resource(189 p.)

General Note  

Source: Dissertations Abstracts International, Volume: 84-12, Section: B.

General Note  

Advisor: Lin, Nina.

Dissertation Note  

Thesis (Ph.D.)--University of Michigan, 2023.

Restrictions on Access Note  

This item must not be sold to any third party vendors.

Restrictions on Access Note  

This item must not be added to any third party search indexes.

Summary, Etc.  

요약Microbiomes, the collective communities of microorganisms in a particular habitat, play critical roles in host-associated, natural, and built environments. The cell-cell interactions in microbiomes form expansive and complex networks, driving community structure and behavior. However, they are difficult to characterize, which prevents the rational design of interventions. As an emerging technology for elucidating interactions in microbiomes, microfluidic droplets (microdroplets), which are nanoliter-scale, monodisperse water-in-oil emulsions, show tremendous promise. Microdroplets enable reductionist, high-throughput study of interactions by encapsulating subsets of communities for co-growth and analysis. Yet, major barriers to realizing this potential include: (1) deficiencies in our understanding of how microdroplet parameters affect microbial co-growth, and (2) the lack of generalizable approaches for high-throughput and high-resolution characterization of co-cultivated sub-communities in microdroplets.One critical parameter in droplet-enabled co-cultivation that has evaded evaluation is the droplet size. Given the same number of initial cells, a larger droplet increases the length scale secreted metabolites must diffuse and dilutes the initial concentration of cells, impacting community dynamics. To evaluate the effect of droplet size on a spectrum of syntrophic interactions, we cultivated a synthetic model system consisting of two E. coli auxotrophs, whose interactions could be modulated through supplementation of related amino acids in the medium. Our results demonstrate that the droplet size impacts numerous aspects of the growth of a cross-feeding bi-culture, particularly the growth capacity, growth rate, and lag time, depending on the degree of the interaction. This work suggests that the droplet size should be more carefully evaluated based on the system of study or research objectives.The first approach to address the lack of technical capabilities is the utilization of metagenomic shot-gun sequencing for individual droplets. We demonstrated this approach with the encapsulation and co-cultivation of droplet sub-communities from a human fecal sample. From a selection of 22 droplets, we observed this approach provides accessibility to previously uncharacterized gut commensals. We applied metagenomic sequencing for the de novo reconstruction of genomes from one droplet sub-community and demonstrated the capability to dissect sub-communities with high genomic resolution. Genomic characterization of one novel member of the family Neisseriaceae revealed novel pathways such as the production of atherogenic intermediates. Future adaptation and application of this approach would enable the inference of specific interactions based on genomic complementarity.The second approach is droplet-resolved, quantitative 16S amplicon sequencing to profile the absolute abundance of thousands of droplet subcommunities. The eventual application of this methodology would be to infer interactions in co-cultivated droplet sub-communities based on membership and the degree of co-growth in each droplet. We developed novel microfluidic and molecular biology workflows for high-throughput, droplet-based barcoding of 16S sequences and the incorporation of molecular standards. We benchmarked the workflow throughput and accuracy with constructed mock communities. The benchmarking data showed good accuracy in terms of relative community composition, but further work is needed to assess absolute quantification and improve even amplification and sequencing depth across droplets. Additionally, for wide applicability across biological systems, the degree of throughput and generalizability need to be improved.In summary, we have expanded the understanding and technical capabilities regarding the utilization of microdroplets in deciphering interactions in microbiomes, enabling the technology to further advance fundamental discoveries in the field of microbiome science.

Subject Added Entry-Topical Term  

Microbiology.

Subject Added Entry-Topical Term  

Chemical engineering.

Index Term-Uncontrolled  

Microbiome

Index Term-Uncontrolled  

Microfluidics

Index Term-Uncontrolled  

Microbial interactions

Index Term-Uncontrolled  

Microdroplets

Index Term-Uncontrolled  

Metagenomics

Added Entry-Corporate Name  

University of Michigan Chemical Engineering

Host Item Entry  

Dissertations Abstracts International. 84-12B.

Host Item Entry  

Dissertation Abstract International

Electronic Location and Access  

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Control Number  

joongbu:643720