Genetic circuit characterization and debugging using RNA-seq

Thomas Gorochowski, Amin Espah Borujeni, Yongjin Park, Alec Nielsen, Jing Zhang, Bryan Der, D Benjamin Gordon, Christopher Voigt

Research output: Contribution to journalArticle (Academic Journal)peer-review

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Abstract

Genetic circuits implement computational operations within a cell. Debugging them is difficult because their function is defined by multiple states (e.g., combinations of inputs) that vary in time. Here, we develop RNA-seq methods that enable the simultaneous measurement of: (i) the states of internal gates, (ii) part performance (promoters, insulators, terminators), and (iii) impact on host gene expression. This is applied to a three-input one-output circuit consisting of three sensors, five NOR/NOT gates, and 46 genetic parts. Transcription profiles are obtained for all eight combinations of inputs, from which biophysical models can extract part activities and the response functions of sensors and gates. Various unexpected failure modes are identified, including cryptic antisense promoters, terminator failure, and a sensor malfunction due to media-induced changes in host gene expression. This can guide the selection of new parts to fix these problems, which we demonstrate by using a bidirectional terminator to disrupt observed antisense transcription. This work introduces RNA-seq as a powerful method for circuit characterization and debugging that overcomes the limitations of fluorescent reporters and scales to large systems composed of many parts.
Original languageEnglish
Article number952
Number of pages16
JournalMolecular Systems Biology
Volume13
Issue number11
DOIs
Publication statusPublished - 9 Nov 2017

Structured keywords

  • Bristol BioDesign Institute

Keywords

  • biofab
  • combinatorial logic
  • omics
  • synthetic biology
  • systems biology

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