Silberg, Jonathan J.St-Pierre , Francois2022-09-262022-09-262022-052022-04-21May 2022Lee, Jihwan. "Accelerating bioengineering using computer vision, automated microscopy, and synthetic biology." (2022) Diss., Rice University. <a href="https://hdl.handle.net/1911/113360">https://hdl.handle.net/1911/113360</a>.https://hdl.handle.net/1911/113360Engineering biological entities and systems can provide innovative solutions to healthcare, agriculture, environmental, and manufacturing problems. Nucleic acids, proteins, and cells can be repurposed to function as biosensors, reporters, biochemical foundries and genetic circuits. However, engineering biology is slow and challenging, especially when spatial and temporal properties need to be optimized. This is because traditional methods to optimize spatial and temporal properties have extremely low throughput. Also, transient plasmid transfections in mammalian cells produce high expression heterogeneity making biological systems less predictable and challenging to engineer. We overcame these challenges by leveraging recent developments in computer vision, automated microscopy, and synthetic biology. We developed SPOTlight, a versatile high-throughput bioengineering platform that can screen pooled single-cell variants using a microscope. SPOTlight relies on imaging visual phenotypes by automated microscopy, precise optical tagging of single target cells, and retrieval of tagged cells by fluorescence-activated cell sorting. We used the platform to screen for 3 million cells expressing mutagenesis libraries and identified a bright variant, mGold, the most photostable yellow fluorescent protein reported to date. Separately, to address the problem of large gene expression variability in mammalian cells, we developed a series of plasmid-based circuits called Equalizers. Equalizers can robustly buffer gene expression heterogeneity caused by plasmid dosage variation between individual mammalian cells. We also show that Equalizers ported on a self-replicating episomal plasmid enable the rapid generation of extrachromosomal cell lines with stable and uniform gene expression. Equalizers are a plasmid-based gene expression system that can facilitate bioengineering in mammalian cells. We believe SPOTlight and Equalizers will enable the rapid engineering of beneficial biological entities and systems that can be deployed to solve pressing problems in many different fields.application/pdfengCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.High-throughput screeningSingle-cell pooled screeningHigh-throughput screeningFluorescent proteinPhotostabilitySynthetic biologyComputer visionThesis2022-09-26