Rewritable digital data storage in live cells via engineered control of recombination directionality

Abstract

The use of synthetic biological systems in research, healthcare, and manufacturing often requires autonomous history-dependent behavior and therefore some form of engineered biological memory. For example, the study or reprogramming of aging, cancer, or development would benefit from genetically encoded counters capable of recording up to several hundred cell division or differentiation events. Although genetic material itself provides a natural data storage medium, tools that allow researchers to reliably and reversibly write information to DNA in vivo are lacking. Here, we demonstrate a rewriteable recombinase addressable data (RAD) module that reliably stores digital information within a chromosome. RAD modules use serine integrase and excisionase functions adapted from bacteriophage to invert and restore specific DNA sequences. Our core RAD memory element is capable of passive information storage in the absence of heterologous gene expression for over 100 cell divisions and can be switched repeatedly without performance degradation, as is required to support combinatorial data storage. We also demonstrate how programmed stochasticity in RAD system performance arising from bidirectional recombination can be achieved and tuned by varying the synthesis and degradation rates of recombinase proteins. The serine recombinase functions used here do not require cell-specific cofactors and should be useful in extending computing and control methods to the study and engineering of many biological systems.DNA inversion | synthetic biology | genetic engineering | standard biological parts M ost engineered genetic data storage systems use auto-or cross-regulating bistable systems of transcription repressors or activators to define and hold state via continuous gene expression (1-4). Such epigenetic storage systems can be subject to evolutionary counter selection due to resource burdens placed on the host cell or spontaneous switching due to putatively stochastic fluctuations in cellular processes, including gene expression. Moreover, heterologous expression-based systems are difficult to redeploy given differences in gene regulatory mechanisms across organisms.Another approach for storing data inside organisms is to code extrinsic information within genetic material (5). Nucleic acids have undergone natural selection to serve as heritable data storage material in organismal lineages. Moreover, DNA provides attractive features in terms of data storage robustness, scalability, and stability (6). In addition, engineered transmission of DNA molecules could support data exchange between organisms as needed to implement higher-order multicellular behaviors within programmed consortia (6, 7).Practically, researchers have begun to use enzymes that modify DNA, typically site-specific recombinases, to study and control engineered genetic systems. For example, recombinases can catalyze strand exchange between specific DNA sequences and enable precise manipulation of DNA in vitro and in vivo (8). Depending on the relative location or...

Specification and Simulation of Synthetic Multicelled Behaviors

“…How these specifications might be implemented using existing signaling molecules, enzymes, and/or transcription factors is of course a difficult and open question beyond the scope of this paper. However, we believe that many of the prerequisites have been demonstrated in the literature: noise generation, 38 concentration detection and filtering, 4,39 state control, 40,41 Boolean logic, 2 and signaling. 1 In fact, the composition of these mechanisms into more complex systems is the subject of intense research in the synthetic biology community.…”

Composability of regulatory sequences controlling transcription and translation in Escherichia coli

“…In recent years, these efforts have increased in complexity from expressing a few genes at once to optimizing multicomponent circuits and pathways (4)(5)(6)(7). To attain desired systems-level function reliably, careful and time-consuming optimization of individual components is required (8)(9)(10)(11).…”

Synthetic biology – the state of play

“…A recent example of the use of biological oscillators in another area relates to their application to liquid crystal displays [41]. Again, in the area of biologically-based digital devices, the issue of rewritable digital storage in live cells has recently been addressed [42].…”