Reliable functional composition of a recombinase device family - Subsoontorn, Pakpoom; Endy, Andrew D; Calos, Michele P; Covert, Markus; Horowitz, Mark (Mark Alan); Stanford University, Department of Bioengineering (2014)
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.
Submitted to the Department of Bioengineering.
Thesis (Ph.D.)--Stanford University, 2014.
Rights: (c) Copyright 2014 by Pakpoom Subsoontorn
Rights: This work is licensed under a Creative Commons Attribution 3.0 Unported license (CC BY).
Keywords: theses