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

ACS Synth Biol. 2021 Jun 18;10(6):1406-1416. doi: 10.1021/acssynbio.1c00024. Epub 2021 Jun 1.

Abstract

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.

Keywords: cell-free systems; coherent feed-forward loop; post-transcriptional regulation; synthetic biology; temporal decoding.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Cell-Free System / metabolism
  • Computer Simulation*
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Feedback, Physiological*
  • Gene Regulatory Networks*
  • Genes, Bacterial
  • Microfluidic Analytical Techniques / instrumentation
  • Microfluidic Analytical Techniques / methods
  • Models, Genetic
  • Protein Biosynthesis / genetics
  • Synthetic Biology / methods
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Transcription, Genetic / genetics

Substances

  • Escherichia coli Proteins
  • Transcription Factors