Reliable functional composition of a recombinase device family
Abstract/Contents
- Abstract
- This dissertation describes design principles and demonstrates applications of a recombinase device family to provide examples for how to compose reliable synthetic gene systems. I begin by introducing the functional composition challenges in building a cell cycle counter, a theoretical example of complex synthetic gene systems requiring hundreds of genetic parts. I develop novel designs for combinatorial counters that could store exponentially more states than existing examples. Using numerical simulation, I demonstrate the feasibility and limits of each design; the lack of reliably rewritable binary state devices for use in genetic counting systems is also highlighted. I then focus on experimental proof-of-concepts, failure analyses and design principles for a Recombinase Addressable Data (RAD) device built from bacteriophage integrases and excisionases. RAD devices based on Bxb1 integrase-excisionase are capable of storing state over one hundred cell generations and can be switched repeatedly without performance degradation. The tedious process of engineering a first RAD device raises the question of whether it is practical to scale such devices beyond a single bit, to implement other logical operations beyond set-reset, and to couple such devices to application-specific control signals. To address these questions, I reconfigure RAD devices to operate entirely from genomic DNA, generalize rewritable RAD principles to three other integrase-excisionase pairs, and implement single-use two input logics, buffer gates and integrase-excisionase cascades. I also demonstrate the feasibility of using competitive binding to multiplex the control of different recombination sites via the same recombinase. Finally, I show that the outcomes of composing recombinase devices with novel transcription sources can be predicted, leading to an implementation of autonomous switches driven by growth-phase dependent promoters. Taken together, the work developed here comprises an initial framework for composing complex yet reliable genetic systems using recombinase enzymes.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2014 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Subsoontorn, Pakpoom | |
|---|---|---|
| Associated with | Stanford University, Department of Bioengineering. | |
| Primary advisor | Endy, Andrew D | |
| Thesis advisor | Endy, Andrew D | |
| Thesis advisor | Calos, Michele P | |
| Thesis advisor | Covert, Markus | |
| Thesis advisor | Horowitz, Mark (Mark Alan) | |
| Advisor | Calos, Michele P | |
| Advisor | Covert, Markus | |
| Advisor | Horowitz, Mark (Mark Alan) |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Pakpoom Subsoontorn. |
|---|---|
| Note | Submitted to the Department of Bioengineering. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2014. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2014 by Pakpoom Subsoontorn
- License
- This work is licensed under a Creative Commons Attribution 3.0 Unported license (CC BY).
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