High-throughput analysis and protein engineering using microcapillary arrays
Abstract/Contents
- Abstract
- Over the past decade, high-throughput technologies have allowed researchers to gain unprecedented insights into intrinsically complex and interconnected biological systems. As examples, whole-genome sequencing has enabled the identification of crucial genes and mutations underlying disease pathophysiology, DNA microarrays have been used to elucidate transcription patterns involved in healthy and diseased states, and large-scale proteomics methods have helped map the connectivity of cell signaling networks that orchestrate responses to growth factors and other external stimuli. In contrast, analogously powerful approaches for rapidly and deeply interrogating the sequence-structure-activity relationship of proteins, with functional read-outs that span a range of biophysical and biochemical measurements, have lagged because of technical challenges. In this dissertation, I describe the development of a new technology platform, termed µSCALE (microcapillary single-cell analysis and laser extraction), that aims to improve high-throughput protein characterization and engineering. The µSCALE platform is built around a microcapillary array, automated fluorescence imaging, and a novel laser-based sample recovery method. The platform spatially segregates millions of cells or their protein products in a microcapillary array and interrogates the dense array within minutes. µSCALE also enables recovery of target sample from the microcapillary array using a precise laser-based extraction technique. To showcase capabilities and breadth of µSCALE, I applied it to three distinct protein-analysis and engineering applications, using libraries expressed in yeast or bacteria. In each case, I demonstrated feasibility and then engineered a novel protein variant, identifying new binders against a clinical target, creating a novel fluorescent protein biosensor, or reducing inhibition of an enzyme. Compared to conventional screening techniques, such as FACS and plate-based screens, the µSCALE affords several distinctions advantages, including a five orders of magnitude improvement in throughput, direct cell imaging, and high sorting purity and yield. Collectively, these benefits present µSCALE as an exciting, novel technology to the field of protein engineering and beyond.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Copyright date | 2016 |
| Publication date | 2015, c2016; 2015 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Chen, Bob |
|---|---|
| Associated with | Stanford University, Department of Bioengineering. |
| Primary advisor | Cochran, Jennifer R |
| Thesis advisor | Cochran, Jennifer R |
| Thesis advisor | Baer, Thomas, 1945- |
| Thesis advisor | Endy, Andrew D |
| Advisor | Baer, Thomas, 1945- |
| Advisor | Endy, Andrew D |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Bob Chen. |
|---|---|
| Note | Submitted to the Department of Bioengineering. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2016. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2016 by Bob Chen
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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