Strategies for the genetic characterization of the oral streptococci

Wyllie, Ryan M

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https://hdl.handle.net/2142/115874 Copy

Description

Title

Strategies for the genetic characterization of the oral streptococci

Author(s)

Wyllie, Ryan M

Issue Date

2022-07-12

Director of Research (if dissertation) or Advisor (if thesis)

Jensen, Paul A

Doctoral Committee Chair(s)

Jensen, Paul A

Committee Member(s)

• Perez-Pinera, Pablo
• Sirk, Shannon
• Miller, Michael

Department of Study

Bioengineering

Discipline

Bioengineering

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• gene expression
• oral streptococci
• synthetic biology
• transcriptomics
• promoter
• ribosome binding site

Abstract

The oral streptococci make up a diverse community of Gram-positive bacteria that inhabit the human oral cavity. Members of the oral streptococci include dental pathogens, like Streptococcus mutans and Streptococcus sobrinus, as well as commensal bacteria, like Streptococcus salivarius, which produce a broad range of antimicrobial compounds effective against human pathogens. However, the complex genetic circuits of these microbes, which govern metabolism, virulence, community-level communication, and stress responses, remain poorly understood. Central to this knowledge deficit is the lack of tools available for the genetic characterization of the oral streptococci. In this dissertation, I develop synthetic biology tools and experimental methods that can be used to characterize genetic systems in the oral streptococci by modulating genetic inputs and measuring transcriptomic outputs. Using a novel, Golden Gate-compatible gene expression chassis designed specifically for the oral streptococci, I assembled thousands of combinatorial gene expression cassettes derived from an initial collection of 6,646 promoters, 22 ribosome binding sites (RBS), and 6 open reading frames (ORFs); and characterized their transcriptional and translational strengths. From these data, I provide a set of 12 characterized promoters for controlling transcription and 15 characterized promoter-RBS combinations for fine-tuned control of protein expression in S. mutans. In the interest of addressing the high labor and monetary costs of current prokaryotic transcriptomic library preps, I describe Splintlock-seq, a novel library prep technology based on the activity of SplintR ligase on padlock probes targeting 95% of the entire annotated transcriptome for S. mutans UA159. I demonstrate how top-down, automated padlock probe design can be combined with next-generation sequencing assays to achieve a characterized probe pool capable of identifying differentially-expressed genes in S. mutans. Splintlock-seq is shown to produce comparable transcriptomic profiling data to RNA-seq, but for a fraction of the overall cost.

Graduation Semester

2022-08

Type of Resource

Thesis

Copyright and License Information

Copyright 2022 Ryan M. Wyllie, please embargo for the maximum amount of time

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