Design and construction of a double inversion recombination switch for heritable sequential genetic memory

Abstract

Background: Inversion recombination elements present unique opportunities for computing and information encoding in biological systems. They provide distinct binary states that are encoded into the DNA sequence itself, allowing us to overcome limitations posed by other biological memory or logic gate systems. Further, it is in theory possible to create complex sequential logics by careful positioning of recombinase recognition sites in the sequence.

Methodology/principal findings: In this work, we describe the design and synthesis of an inversion switch using the fim and hin inversion recombination systems to create a heritable sequential memory switch. We have integrated the two inversion systems in an overlapping manner, creating a switch that can have multiple states. The switch is capable of transitioning from state to state in a manner analogous to a finite state machine, while encoding the state information into DNA. This switch does not require protein expression to maintain its state, and "remembers" its state even upon cell death. We were able to demonstrate transition into three out of the five possible states showing the feasibility of such a switch.

Conclusions/significance: We demonstrate that a heritable memory system that encodes its state into DNA is possible, and that inversion recombination system could be a starting point for more complex memory circuits. Although the circuit did not fully behave as expected, we showed that a multi-state, temporal memory is achievable.

Figure 1. How invertase site configuration defines different “machine behaviors”.

A) A single invertase can only flip one region ON or OFF. B and C) With two invertases there are two possible intercalated configurations of sites. In B, when one pair of sites is fully contained by another the resultant number of possible “states” of the system is only four, whereas in C, when the site pairs are staggered, five states are accessible and any input sequence results in a different output of the system. C also shows a configuration of promoters and terminators such that states 3 and 4 have promoters pointing outside the machine regions. This configuration is the one experimentally created. D) Possible configurations of three different pairs of recognition sites and the number of possible states. E) How both the number of such configurations (Blue curve) and the maximal number of states (Red curve) grows with number of pairs.

Figure 2. Plasmids used in the study.

A) Plasmid maps for constructs containing the invertases and the memory switch. B) Detail of structure of the double inversion switch annotated with the primers used to diagnose state as described in the text. The Supplementary Information S1 contains the complete annotated sequence of the switch.

Figure 3. Culture PCR results for the response to the different input sequences.

{FimB}, {HinB}, {FimB, HinB} and {HinB, FimB}. Each lane probes for the existence of a state or states, as shown in the legend. Because of the possible arrangements of DNA, lanes E and F could result in either short or long PCR products, indicating states {1, 3}, and {2, 4} respectively. See Figure 2 for relative location of the primers. No State 5 was observed when Hin and FimB were simultaneously expressed.