Synthetic gene networks that count

Abstract

Synthetic gene networks can be constructed to emulate digital circuits and devices, giving one the ability to program and design cells with some of the principles of modern computing, such as counting. A cellular counter would enable complex synthetic programming and a variety of biotechnology applications. Here, we report two complementary synthetic genetic counters in Escherichia coli that can count up to three induction events: the first, a riboregulated transcriptional cascade, and the second, a recombinase-based cascade of memory units. These modular devices permit counting of varied user-defined inputs over a range of frequencies and can be expanded to count higher numbers.

Fig. 1

The RTC 2-Counter and RTC 3-Counter construct designs and results. (A) The RTC 2-Counter is a transcriptional cascade with two nodes. Shown at the bottom are expected expression profiles due to 0, 1, and 2 arabinose (Ara) pulses. (B) Mean fluorescence of three replicates of RTC 2-Counter cell populations over time, measured by a flow cytometer. Shaded areas represent arabinose pulse duration. (C) The RTC 3-Counter is a transcriptional cascade with three nodes. Shown at the bottom are expected expression profiles due to 0, 1, 2, and 3 arabinose pulses. (D) Mean fluorescence of three replicates of RTC 3-Counter cell populations over time, measured by a flow cytometer. Shaded areas represent arabinose pulse duration.

Fig 2

Modeling predictions and RTC 3-Counter experimental characterization. (A) A model with fitted parameters captures the salient features of the normalized fluorescence results of the RTC 2-Counter. (B) An expanded model, again with fitted parameters, matches the normalized fluorescence results of the RTC 3-Counter. (C) Based on parameters fitted in (B), the model predicts expression output of the RTC 3-Counter across a range of pulse lengths and intervals, and these calculations were used to generate the colored contour lines. Solid circles represent experimental results, with both color and size of circles indicating the level of expression. (D) Similar to (C), except values shown are the difference in expression output after three pulses and two pulses.

Fig. 3

The single-inducer DIC 3-Counter construct design and results. (A) The single-inducer DIC 3-Counter is built by cascading SIMMs. (B) Mean fluorescence of single-inducer DIC 3-Counter cell populations over time, measured by a flow cytometer. Shaded areas represent arabinose pulse duration. (C) GFP fluorescence ratios between the single-inducer DIC 3-Counter exposed to three pulses of arabinose (N) versus two pulses of arabinose (N-1) with varying arabinose pulse lengths and intervals; experimental results are represented by black dots.

Fig. 4

The multiple-inducer DIC 3-Counter construct design and results. (A) The multiple-inducer DIC 3-Counter is similar to the single-inducer DIC 3-Counter in Fig. 3 except that each promoter is a unique inducible promoter: P_Ltet0–1, P_BAD, and P_A1lacO. These promoters respond to anhydrotetracycline (aTc), arabinose, and IPTG, respectively. (B) Mean fluorescence of multiple-inducer DIC 3-Counter cell populations over time, measured by a flow cytometer. Colored areas represent the durations of consecutive inducer pulses. (C) Flow cytometry population data showing the multiple-inducer DIC 3-Counter when exposed to the desired sequence of three inducers and to single inducers only. (D) Flow cytometry population data showing the multiple-inducer DIC 3-Counter when exposed to the desired sequence of three inducers and to all pairwise permutations of inducers.