Expanding and evaluating sense codon reassignment for genetic code expansion
| dc.contributor.author | Biddle, C. William, author | |
| dc.contributor.author | Fisk, John D., advisor | |
| dc.contributor.author | Ackerson, Chris, committee member | |
| dc.contributor.author | Henry, Charles, committee member | |
| dc.contributor.author | Stasevich, Tim, committee member | |
| dc.date.accessioned | 2017-09-14T16:04:26Z | |
| dc.date.available | 2018-09-12T16:04:38Z | |
| dc.date.issued | 2017 | |
| dc.description.abstract | Genetic code expansion is a field of synthetic biology that aims to incorporate non-canonical amino acids (ncAAs) into proteins as though they were one of the 20 "natural" amino acids. The amino acids which naturally make up proteins are chemical limited, and ncAAs can carry new chemical functionality into proteins. Proteins are of interest because they are simple to produce with good consistency and have immense potential due to the diversity of structure and function. Incorporating ncAA into proteins expands the scope of function of proteins even further. Two methods have been widely used for genetic code expansion, global amino acid replacement and amber stop codon suppression. Global amino acid replacement exchanges one of the natural amino acids for a ncAA, producing an altered 20 amino acid genetic code. Amber stop codon suppression incorporates ncAA in response to the UAG stop codon making a 21 amino acid genetic code, but is limited in incorporation efficiency and producing proteins with multiple instances of a ncAA is challenging. We wanted to use a third genetic code expansion system called sense codon reassignment which has not been widely employed at all but should enable multisite incorporation of ncAAs. When the work presented in this dissertation was started, a single report of sense codon reassignment existed in the literature. We set out to improve and expand sense codon reassignment for the incorporation of multiple copies of ncAAs into proteins. We quickly discovered disparities in what was known regarding the variables that could be used to manipulate genetic code expansion, and the focus of our work shifted to systems for improving sense codon reassignment using quantitative measurements. The first chapter of this dissertation is an introduction to genetic code expansion and the processes of translation and gene expression that are likely involved or could be involved in genetic code expansion. The three following chapters will build upon the fundamentals described in Chapter 1. The second chapter is a complete story about how a screen to quantify sense codon reassignment was developed. The fluorescence based screen was used in a high throughput fashion to screen a directed evolution library of variants for increased sense codon reassignment efficiency at the Lys AAG sense codon. While evaluating various sense codons for potential reassignment efficiency, the AUG anticodon was found to be incapable of discriminating between the CAU and CAC codons. This was anomalous relative to the other anticodons we tested. Chapter 3 describes how unintended modifications to an engineered tRNA were identified and then how the fluorescence based screen was used to engineer the tRNA further for increased sense codon reassignment efficiency and to avoid the unintentional modification. Most applications of genetic code expansion rely on modifications to tRNAs but few reports actually consider them, The final chapter of this dissertation is a manuscript in preparation describing the reassignment of a rare sense codon to incorporate ncAAs. The chapter focuses on how improvements made in a system specific for an amino acid can be transferred to systems specific for other ncAAs. Over 150 different ncAAs have been incorporated into proteins using genetic code expansion technologies, but the extent to which the various systems are combinable has barely been evaluated. This dissertation is a story about developing sense codon reassignment to functional levels and quantifying the effects of different variables along the way. | |
| dc.format.medium | born digital | |
| dc.format.medium | doctoral dissertations | |
| dc.identifier | Biddle_colostate_0053A_14265.pdf | |
| dc.identifier.uri | https://hdl.handle.net/10217/183902 | |
| dc.language | English | |
| dc.language.iso | eng | |
| dc.publisher | Colorado State University. Libraries | |
| dc.relation.ispartof | 2000-2019 | |
| dc.rights | Copyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright. | |
| dc.subject | green fluorescent protein | |
| dc.subject | sense codon reassignment | |
| dc.subject | genetic code expansion | |
| dc.subject | unnatural amino acid | |
| dc.subject | non-canonical amino acid | |
| dc.title | Expanding and evaluating sense codon reassignment for genetic code expansion | |
| dc.type | Text | |
| dcterms.embargo.expires | 2018-09-12 | |
| dcterms.embargo.terms | 2018-09-12 | |
| dcterms.rights.dpla | This Item is protected by copyright and/or related rights (https://rightsstatements.org/vocab/InC/1.0/). You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s). | |
| thesis.degree.discipline | Chemistry | |
| thesis.degree.grantor | Colorado State University | |
| thesis.degree.level | Doctoral | |
| thesis.degree.name | Doctor of Philosophy (Ph.D.) |
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