Cell-Free Characterization of Coherent Feed-Forward Loop-Based Synthetic Genetic Circuits

Pascal A. Pieters, Bryan L. Nathalia, Ardjan J. van der Linden, Peng Yin, Jongmin Kim (Corresponding author), Wilhelm T.S. Huck (Corresponding author), Tom F.A. de Greef (Corresponding author)

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Regulatory pathways inside living cells employ feed-forward architectures to fulfill essential signal processing functions that aid in the interpretation of various types of inputs through noise-filtering, fold-change detection and adaptation. Although it has been demonstrated computationally that a coherent feed-forward loop (CFFL) can function as noise filter, a property essential to decoding complex temporal signals, this motif has not been extensively characterized experimentally or integrated into larger networks. Here we use post-transcriptional regulation to implement and characterize a synthetic CFFL in an Escherichia coli cell-free transcription-translation system and build larger composite feed-forward architectures. We employ microfluidic flow reactors to probe the response of the CFFL circuit using both persistent and short, noise-like inputs and analyze the influence of different circuit components on the steady-state and dynamics of the output. We demonstrate that our synthetic CFFL implementation can reliably repress background activity compared to a reference circuit, but displays low potential as a temporal filter, and validate these findings using a computational model. Our results offer practical insight into the putative noise-filtering behavior of CFFLs and show that this motif can be used to mitigate leakage and increase the fold-change of the output of synthetic genetic circuits.

Original languageEnglish
Pages (from-to)1406-1416
Number of pages11
JournalACS Synthetic Biology
Issue number6
Publication statusPublished - 1 Jun 2021

Bibliographical note

Funding Information:
We thank Emilien Dubuc, Maaruthy Yelleswarapu, and Roel Maas for helpful discussions. W.T.S.H. was supported by a TOPPUNT grant from The Netherlands Organization for Scientific Research (NWO). T.F.A.d.G. was supported by the NWO-VIDI grant from The Netherlands Organization for Scientific Research (NWO, 723.016.003). P.A.P., A.J.v.d.L., and T.F.A.d.G. were supported by an ERC starting grant by the European Research Council (project No. 677313 BioCircuit) and funding from the Ministry of Education, Culture and Science (Gravity programs, 024.001.035 and 024.003.013). J.K. was supported by the National Research Foundation of Korea (NRF-2019R1A2C1086830) grant funded by the Korean government (MSIT). P.Y. was supported by NSF CBET-1729397.


  • cell-free systems
  • coherent feed-forward loop
  • post-transcriptional regulation
  • synthetic biology
  • temporal decoding


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