Synthetic circuits integrating logic and memory in living cells

ORIGINAL: Nature

Piro Siuti, John Yazbek & Timothy K Lu

10 February 2013

Nature Biotechnology 31, 448–452 (2013) doi:10.1038/nbt.2510 Received 25 October 2012 Accepted 17 January 2013 Published online 10 February 2013 

Logic and memory are essential functions of circuits that generate complex, state-dependent responses. Here we describe a strategy for efficiently assembling synthetic genetic circuits that use recombinases to implement Boolean logic functions with stable DNA-encoded memory of events. Application of this strategy allowed us to create all 16 two-input Boolean logic functions in living Escherichia coli cells without requiring cascades comprising multiple logic gates. We demonstrate long-term maintenance of memory for at least 90 cell generations and the ability to interrogate the states of these synthetic devices with fluorescent reporters and PCR. Using this approach we created two-bit digital-to-analog converters, which should be useful in biotechnology applications for encoding multiple stable gene expression outputs using transient inputs of inducers. We envision that this integrated logic and memory system will enable the implementation of complex cellular state machines, behaviors and pathways for therapeutic, diagnostic and basic science applications.

Figure 1: Integrated logic and memory devices. A simple rule is used to translate desired computational functions into [promoter(s)]-[terminator(s)]-[output] designs, which can be constructed with straightforward Gibson assembly. In all of the E. coli cells used in this work,

Figure 2: Recombinase-based logic gates can implement a complete set of two-input–one-output Boolean logic gates without needing to cascade multiple universal gates together. 

Figure 3: Stable memory maintenance over multiple cell generations and after cell death. 

(a) Percentage of cells maintaining GFP expression, assayed by flow cytometry after gating by forward and side scatter, in cells containing an AND gate induced to the ON state after day 0 and continuously diluted and grown without input…

Figure 4: Recombinase-based logic and memory can implement digital-to-analog converters. 

Cells were exposed to no inputs, AHL only, aTc only, or AHL and aTc simultaneously. (a–c) Various digital combinations of the input inducers result in multiple levels of analog gene expression outputs on the basis of the varying strengt…