One promise of synthetic biology is the creation of genetic circuitry that enables the execution of logical programming in living cells. Such ‘wet programming’ is positioned to transform a wide and diverse swathe of biotechnology ranging from therapeutics and diagnostics to water treatment strategies. Although progress in the development of a library of genetic modules continues apace1,2,3,4, a major challenge for their integration into larger circuits is the generation of sufficiently fast and precise communication between modules5,6. An attractive approach is to integrate engineered circuits with host processes that facilitate robust cellular signalling7. In this context, recent studies have demonstrated that bacterial protein degradation can trigger a precise response to stress by overloading a limited supply of intracellular proteases8,9,10. Here we use protease competition to engineer rapid and tunable coupling of genetic circuits across multiple spatial and temporal scales. We characterize coupling delay times that are more than an order of magnitude faster than standard transcription-factor-based coupling methods (less than 1 min compared with ∼20–40 min) and demonstrate tunability through manipulation of the linker between the protein and its degradation tag. We use this mechanism as a platform to couple genetic clocks at the intracellular and colony level, then synchronize the multi-colony dynamics to reduce variability in both clocks. We show how the coupled clock network can be used to encode independent environmental inputs into a single time series output, thus enabling frequency multiplexing (information transmitted on a common channel by distinct frequencies) in a genetic circuit context. Our results establish a general framework for the rapid and tunable coupling of genetic circuits through the use of native ‘queueing’ processes such as competitive protein degradation.
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Gardner, T. S., Cantor, C. R. & Collins, J. J. Construction of a genetic toggle switch from Escherichia coli. Nature 403, 339–342
Siuti, P., Yazbek, J. & Lu, T. K. Synthetic circuits integrating logic and memory in living cells. Nature Biotechnol. 31, 448–452 (2013)
Tigges, M., Marquez-Lago, T., Stelling, J. & Fussenegger, M. A tunable synthetic mammalian oscillator. Nature 457, 309–312 (2009)
Xie, Z., Wroblewska, L., Prochazka, L., Weiss, R. & Benenson, Y. Multi-input RNAi-based logic circuit for identification of specific cancer cells. Science 333, 1307–1311 (2011)
Moon, T. S., Tamsir, A., Stanton, B. C. & Voigt, C. A. Genetic programs constructed from layered logic gates in single cells. Nature 491, 249–253 (2012)
Del Vecchio, D., Ninfa, A. J. & Sontag, E. D. Modular cell biology: retroactivity and insulation. Mol. Syst. Biol. 4, 161 (2008)
Nandagopal, N. & Elowitz, M. B. Synthetic biology: integrated gene circuits. Science 333, 1244–1248 (2011)
Fredriksson, Å. et al. Decline in ribosomal fidelity contributes to the accumulation and stabilization of the master stress response regulator σs upon carbon starvation. Genes Dev. 21, 862–874 (2007)
Merrikh, H., Ferrazzoli, A. E., Bougdour, A., Olivier-Mason, A. & Lovett, S. T. A DNA damage response in Escherichia coli involving the alternative sigma factor, RpoS. Proc. Natl Acad. Sci. USA 106, 611–616 (2009)
Cookson, N. A. et al. Queueing up for enzymatic processing: correlated signaling through coupled degradation. Mol. Syst. Biol. 7, 561 (2011)
Keiler, K. C., Waller, P. & Sauer, R. Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA. Science 271, 990–993 (1996)
Goldbeter, A. & Koshland, D. E. An amplified sensitivity arising from covalent modification in biological systems. Proc. Natl Acad. Sci. USA 78, 6840–6844 (1981)
Rosenfeld, N. & Alon, U. Response delays and the structure of transcription networks. J. Mol. Biol. 329, 645–654 (2003)
Hooshangi, S., Thiberge, S. & Weiss, R. Ultrasensitivity and noise propagation in a synthetic transcriptional cascade. Proc. Natl Acad. Sci. USA 102, 3581–3586 (2005)
Mika, F. & Hengge, R. A two-component phosphotransfer network involving ArcB, ArcA, and RssB coordinates synthesis and proteolysis of σs (RpoS) in E. coli. Genes Dev. 19, 2770–2781 (2005)
Pruteanu, M. & Hengge-Aronis, R. The cellular level of the recognition factor rssb is rate-limiting for σs proteolysis: implications for rssb regulation and signal transduction in σs turnover in Escherichia coli. Mol. Microbiol. 45, 1701–1713 (2002)
Danino, T., Mondragón-Palomino, O., Tsimring, L. & Hasty, J. A synchronized quorum of genetic clocks. Nature 463, 326–330 (2010)
Unger, M. A., Chou, H.-P., Thorsen, T., Scherer, A. & Quake, S. R. Monolithic micorfabricated valves and pumps by multilayer soft lithography. Science, 288, 113–116 (2000)
Stricker, J. et al. A fast, robust and tunable synthetic gene oscillator. Nature 456, 516–519 (2008)
Press, W. H. in Numerical Recipes: The Art of Scientific Computing 3rd edn (Cambridge Univ. Press, 2007)
Prindle, A. et al. A sensing array of radically coupled genetic ‘biopixels’. Nature 481, 39–44 (2012)
Grünberg, R. & Serrano, L. Strategies for protein synthetic biology. Nucleic Acids Res. 38, 2663–2675 (2010)
McGinness, K. E., Baker, T. A. & Sauer, R. T. Engineering controllable protein degradation. Mol. Cell 22, 701–707 (2006)
Griffith, K. L. & Grossman, A. D. Inducible protein degradation in Bacillus subtilis using heterologous peptide tags and adaptor proteins to target substrates to the protease ClpXP. Mol. Microbiol. 70, 1012–1025 (2008)
Burger, A., Walczak, A. M. & Wolynes, P. G. Abduction and asylum in the lives of transcription factors. Proc. Natl Acad. Sci. USA 107, 4016–4021 (2010)
Mukherji, S. et al. MicroRNAs can generate thresholds in target gene expression. Nature Genet. 43, 854–859 (2011)
Buchler, N. E. & Louis, M. Molecular titration and ultrasensitivity in regulatory networks. J. Mol. Biol. 384, 1106–1119 (2008)
Strogatz, S. Nonlinear Dynamics and Chaos: with Applications to Physics, Biology, Chemistry and Engineering (Perseus Books, 2001)
Ferry, M. S., Razinkov, I. & Hasty, J. Microfluidics for synthetic biology from design to execution. Methods Enzymol. 497, 295–372
Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240–2246 (1998)
This work was supported by the National Science Foundation (MCB-1121748) and by the San Diego Center for Systems Biology (NIH Grant P50 GM085764) and the US Department of Defense National Defense Science and Engineering Graduate Fellowship (A.P.). We would like to thank T. Danino, M. Jin, C. Rivera, O. Din and J. De Friel for critical reading of the manuscript.
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Increasing the length of the TS linker sequence results in increasing downstream module degradation delay.
a, Detailed breakdown of single fluorescent trajectory analysis. Peaks are identified in red, troughs in green, upslope 10% points in purple and downslope 10% points in dark grey. The two period measurements are peak to peak and the time between two successive 10% upslope points. b, Top, sfGFP does not show bleed-over into the CFP fluorescence channel. Induction of sfGFP with 10 nM acyl-homoserine lactone (AHL, dashed line) showed increase in fluorescence of sfGFP, which was not detected in the CFP channel. Bottom, the use of the published AAV degradation tag30 shows delay in the downstream module degradation of 15 min. c, Without the TS linker sequence, there is very little delay in downstream module degradation. d, Single TS linker sequence results in 10 min delay. e, Double TS linker sequence results in 16 min delay, similar to that of AAV degradation sequence. f, 5-TS linker sequence results in 25 min delay (data shown in c–f were used to generate Fig. 1c).
a, Individual ‘leader’ cells show early activation of quorum clock proteins relative to the mean population response. b, In a two-cell simulation, cells 1 and 2 start out unlinked with slightly different constitutive production of AiiA and LuxI. At t = 100 min the two cells are linked through external AHL in the media, showing the cell with slower dynamics (cell 2) linking up to cell 1 with shorter periods. c, Cells 1 and 2 start out unlinked with cell 1 including intracellular clock dynamics (green) that result in higher frequency oscillations in cell 1. When the cells are linked (t = 100), the slower cell 2, without the intracellular clock, links on to the faster cell through external AHL communication between the cells. d, Trajectories of 20 cells (different colour traces) with noisy constitutive production at lux promoter synchronize when their external AHL pool is mixed at t = 400 min. Mean trajectory is shown in black. e, Period variability after cell synching (red) is lower than in individual cells (blue). QS, quorum-sensing oscillator.
Extended Data Figure 3 The intracellular clock increases robustness in the coupled oscillator system by reducing the period of the quorum clock.
a, Removal of IPTG, which increases intracellular clock strength, leads to more regular oscillations (experimental). b, The decrease in variability of the inter-pulse time of the coupled oscillator without IPTG suggests that the intracellular clock plays an important role in the inter-pulse dynamics (experimental). c, At very high flow rates, the quorum clock oscillates irregularly. Tuning up the intracellular clock reduces the quorum clock period, restoring regular oscillations and allowing for global level synchronization between colonies due to H2O2 biopixel coupling. Genetic addition of the intracellular clock (0.1 mM IPTG) helps synchronize the quorum clock at high flows (430 µm s−1). Increasing the strength of the intracellular clock with removal of IPTG further enhances H2O2 inter-colony synchronization (experimental, black lines indicate the mean of experimental races).
Extended Data Figure 4 H2O2 increases the degradation rate by ClpXP, and this in combination with transcriptional increase at the lux promoter decreases variability in the oscillator period.
a, There is a significant decrease in the degradation time due to H2O2 (experimental). b, Decrease in the degradation time due to H2O2 is due to effective increase in ClpXP degradation rate (experimental). c, H2O2 activation of lux promoter alone would only increase the amplitude of quorum clock oscillations. Similarly, H2O2-dependent increase in ClpXP activity results only in steeper degradation and longer inter-pulse duration. Combination of the two effects leads to increase in amplitude and decrease in inter-pulse duration, which matches experiments (model). d, Individually, the two H2O2 effects do little to lower the quorum clock period CV, which is reduced when both are present (model).
This file contains Supplementary Text and Supplementary References. (PDF 124 kb)
In small devices (100 cells), we observed fast and asynchronous intracellular clock oscillations without quorum clock contribution, since the quorum clock requires a critical cell density to function. (MP4 10314 kb)
In larger devices (5,000 cells), we followed individual colonies from single cells to full density, observing a marked transition from asynchronous intracellular clock oscillations to synchronized intracellular/quorum clock oscillations at the point of ClpXP saturation. (MP4 5222 kb)
Frequency contributions from both clocks (left: intracellular clock, right: quorum clock) can be found in the intracellular clock time series. (MP4 5104 kb)
In devices housing many identical colonies (500 biopixels of 5,000 cells), we observed global synchronization between colonies via H2O2. This video illustrates the process of analyzing trajectories taken in the presence (right, red) and absence (left, blue) of H2O2 to discern subtle host regulation in response to H2O2. (MP4 7858 kb)
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Prindle, A., Selimkhanov, J., Li, H. et al. Rapid and tunable post-translational coupling of genetic circuits. Nature 508, 387–391 (2014). https://doi.org/10.1038/nature13238
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