Synthetic Auxin Engineering: Building a Biofoundry Platform

Genetic regulatory circuits control metabolism, development, and environmental response across all kingdoms of life. Genetic circuit engineering facilitates sustainable and efficient production of biopharmaceutical, chemical, fiber, and food products that keep humans healthy, nourished, and clothed. However, the complexity of most genetic regulatory circuits, particularly in the context of multicellular eukaryotes, often prevents them from being leveraged as tools or applied technologies with bioeconomic relevance. However, synthetic biology enables the transfer of genes, circuits, networks, and even whole chromosomes between organisms. This approach can be leveraged to port genetic circuits into simple model organisms to control existing and engineer new cellular functions. Still, porting genes to non-native contexts can affect circuit function due to unknown factors. For this reason, iterative design-build-test-learn (DBTL) cycles are necessary for optimizing circuits in new contexts. To facilitate the DBTL cycle, automation approaches can be deployed for streamlining synthetic genetic circuits optimization. Here, I provide a case study for how using synthetic biology and automation – a biofoundry approach – has facilitated engineering of the auxin signaling pathway in a synthetic yeast system. Auxin is a phytohormone involved in nearly every aspect of plant growth and development, and this striking versatility designates it as a target for biotechnology development and a candidate for engineering. First, I provide a literature review of the history of synthetic auxin engineering in yeast, a survey of tools available for expanding yeast synthetic biology, and a summary of applicable automation tools and platforms. Next, I describe and validate a platform called AssemblyTron, which deploys liquid handling robotics for DNA assembly and can serve as the foundation of a biofoundry platform. I then introduce TidyTron, which is a protocol library for automated wash and reuse of single use lab plastics to promote biofoundry sustainability. Next, I expand the AssemblyTron package by providing protocols for mutant and modular indexed plasmid library assembly. Finally, I describe a modular indexed plasmid library (toolkit) for rapid assembly of auxin circuit variants and validate it by building and optimizing an auxin circuit.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Biological Systems Engineering

Department dc:contributor.department

Biological Systems Engineering

Grantor dc:publisher

Virginia Tech

Year dc:date.issued

2024

Author dc:creator

• Bryant, John Alexander Jr.

Chairs dc:contributor.committeechair

• Wright, Robert Clay
• Bargmann, Bastiaan

Committee members dc:contributor.committeemember

• Senger, Ryan S.
• Sherif, Sherif Mohamed
• Strader, Lucia

• synthetic biology
• liquid handling robotics
• lab automation
• auxin signaling
• modular cloning
• sustainability

Statement dc:rights

• Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International

Licence dc:rights.uri

• http://creativecommons.org/licenses/by-nc-sa/4.0/

Language dc:language.iso

en

Dc Identifier Other

vt_gsexam:40262

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/119231