Investigating routes for in vitro and in vivo data storage

Abstract

Computing Science, Synthetic Biology and Nanotechnology are converging. Synthetic Biology and Nanotechnology compose the “hardware” platform, whilst Computing Science formulates the logic, data storage and processing pipelines in order to create complex yet controlled behaviour at the nanoscale. Although much work has been done on information processing at the nanoscale via in vivo constructs, e.g. logic gates in various organisms, relatively little has been done on implementing data structure, a fundamental building block for computation. This dissertation proposes and investigates methods to implement data structures by employing biological molecules via both a Synthetic Biology and a Nanotechnological approach. A data structure implemented at the nanoscale could help to substantially increase the complexity of behaviours that could be programmed and embedded in living cells or at the interface between living cells and other nano-substrates, with potential applications in intelligent drug factories and delivery nanosystems, biosensors, and environmental cleaning bionanotechnologies. This work explores the possibility of implementing via DNA constructs, both in vitro and in vivo, "list-like" data structure that can potentially hold an unlimited number of items. This has not been achieved before. Thus, the text describes designs and test prototypes. Firstly, this thesis focuses on an in vitro approach. This is achieved through a DNA-based machinery implementing a signal recorder based on DNA strand displacement reactions. Such DNA architecture can in principle implement a stack machine, capable of storing data providing a dynamic temporary memory capable of pushing and popping data-items encoded in DNA nanostructures (called DNA "bricks”). The "list-like" data is thus represented by a growing (or shrinking) chain of DNA bricks. iv Secondly, I introduce a potential design and initial experiments for an in vivo approach presenting, a synthetic genetic circuit designed to record and accumulate extracellular signals digitally within a "tape" DNA molecule inside a living cell. The core is based on the engineering of the self-splicing group II retrotransposon Ll.LtrB of Lactococcus lactis. Together, these two in vitro and in vivo routes expand our knowledge in the context of molecular memory devices and the biological operations we can compute.