Synthetic analog and digital circuits for cellular computation and memory

Abstract

Biological computation is a major area of focus in synthetic biology because it has the potential to enable a wide range of applications. Synthetic biologists have applied engineering concepts to biological systems in order to construct progressively more complex gene circuits capable of processing information in living cells. Here, we review the current state of computational genetic circuits and describe artificial gene circuits that perform digital and analog computation. We then discuss recent progress in designing gene networks that exhibit memory, and how memory and computation have been integrated to yield more complex systems that can both process and record information. Finally, we suggest new directions for engineering biological circuits capable of computation.

Figure 1

A. A 4-input genetic AND gate formed by coupling the output of two 2-input genetic AND gates into the input of a third 2-input genetic AND gate. Inducers 1, 2, 3, and 4 induce expression from promoters p1, p2, p3, and p4 respectively. Each AND gate comprises a transcription factor (e.g. TF1) and a corresponding chaperone (i.e. Ch1). Both are required together to activate transcription from the target promoter. The output of each of the left pair of genetic AND gates is used to induce expression from promoters in the right genetic AND gate. The output is only ‘1’ when the inducers are all present. B. A genetic AND gate formed from 3 different E. coli colonies connected via cell-cell communication modules. The cells of colony 1 all possess an identical genetic NOR gate. Colony 2 contains a genetic NOR gate that is different from the gate in colony 1. Colony 3 contains a genetic NOT gate. Inducers 1 and 2 induce expression from promoters p1 and p3, respectively. Promoters p2 and p4 are not induced in the operation of the AND gate. The output from colonies 1 and 2 is a diffusible quorum-sensing molecule that serves as the input to the promoter of the NOT gate. Thus, the output is only ‘1’ when inducers 1 and 2 are present. Figure 1A is adapted from Moon et al.

Figure 2

A. Digital versus analog computation. Digital logic uses the extremes of an input-output transfer curve to represent ‘0’ and ‘1’ signals, while the analog paradigm uses the entire range in between the extremes for computation based on mathematical laws which determine how the output value(s) depends on the input value(s). B. Circuit design of the positive-feedback-and-shunt circuit, which implements a scaled positive-logarithmic transformation of the input inducer concentration to the output reporter (RP) protein concentration. The transcription factor (‘TF’) induces its own expression via a positive- feedback loop located on a low-copy plasmid and that of a reporter gene (‘RP’) from a high-copy plasmid. C. Two orthogonal versions of the circuit in B are used to make a genetic adder circuit. Each circuit uses a different inducible TF (TF1 and TF2) and inducer (Inducer 1 and Inducer 2). The output is the same for both circuits, and thus the net output is the sum of the outputs from both positive-logarithm circuits. The graph is an approximation of the result from Daniel et al, which shows the additive nature of the analog circuit (x and y axes are in log scale, whereas the z axis is in linear scale).

Figure 3

Strategies for building memory, counters, and integrated logic and memory in living cells. A. Design of a positive-feedback loop circuit where a transcription factor activates its own transcription. To control switching, the promoter is also controlled by an external activator or repressor input. B. In an example of recombinase-mediated memory, the Bxb1 recombinase recognizes the att sites denoted as triangles and inverts the intervening DNA, forming two new att sites which can no longer be recognized by Bxb1 for further inversion. C. Multiple-inducer 3-counter built by cascading recombinase-invertible memory units. Addition of Inducer 1 induces expression from promoter p1, producing recombinase R1 which flips DNA located between the att1 sites (triangles). This inverts promoter p2 so it is now capable of expressing recombinase R2. Subsequent addition of Inducer 2 induces expression from promoter p2, producing recombinase R2, which flips DNA between the att2 sites (square brackets). This inverts promoter p3 so it is now capable of expressing GFP upon addition of Inducer 3. D. Genetic OR gate based on recombinase logic. Triangles denote one pair of recombinase-recognition sites recognized by Bxb1 recombinase while square brackets denote another pair of recombinase-recognition sites recognized by phiC31 recombinase. E. Digital-to-analog converters can be implemented using multiple recombinase-invertible units within a single cell, each of which activates expression of a common output gene with a different expression level when flipped by their respective inputs. Thus, digital combinations of inputs lead to programmable levels of analog gene expression inside of living cells.