Ultra high-throughput evolution towards the brightest monomeric green fluorescent protein
Abstract/Contents
- Abstract
- Since the first demonstration of exogenous Green Fluorescent Protein (GFP) expression in E.coli and C. elegans, fluorescent proteins have served as revolutionary molecular tools in the life sciences. The brightest fluorescent proteins enable super resolution imaging, a microscopic technique with resolution at the nanometer scale. Here, we engineered a green fluorescent protein for enhanced brightness using the ultra-high throughput μSCALE engineering platform. The μSCALE platform utilizes a glass array comprised of millions of densely packed 10um capillaries, enabling the screening and isolation of the brightest clones from libraries with nearly 106 variants. Starting from a single domain of a recently isolated four-domain Anthoathecata protein, a bright dimer with brightness exceeding five times the parent was evolved in only four rounds of evolution. The dimer was rationally monomerized through disruption of a hydrophobic interface to produce a monomer, the desired oligomeric status for a molecular imaging tool. The resulting monomer was again evolved for improved brightness, surpassing that of the parent while retaining monomeric character.
Description
| Type of resource | text |
|---|---|
| Date created | June 2015 - May 2016 |
Creators/Contributors
| Author | Filsinger Interrante, Maria |
|---|---|
| Advisor | Cochran, Jennifer |
| Advisor | Altman, Russ B. |
| Advisor | Deisseroth, Karl |
| Degree granting institution | Stanford University. Department of Bioengineering. |
Subjects
| Subject | fluorescent protein |
|---|---|
| Subject | evolution |
| Genre | Thesis |
Bibliographic information
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- Use and reproduction
- User agrees that, where applicable, content will not be used to identify or to otherwise infringe the privacy or confidentiality rights of individuals. Content distributed via the Stanford Digital Repository may be subject to additional license and use restrictions applied by the depositor.
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
Preferred citation
- Preferred Citation
- Filsinger Interrante, Maria. Ultra high-throughput evolution towards the brightest monomeric green fluorescent protein. Stanford Digital Repository. Available at: http://purl.stanford.edu/ct365bc0153
Collection
Undergraduate Theses, School of Engineering
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- Contact
- mariav@stanford.edu
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