# Genetic circuit characterization and debugging using RNA-seq

Article · Gorochowski TE, Espah Borujeni A, Park Y, Nielsen AA, Zhang J, Der BS, Gordon DB, Voigt CA

- Archive id: doi:10.15252/msb.20167461
- Published: 2017-11-09
- Venue: Molecular systems biology
- DOI: https://doi.org/10.15252/msb.20167461
- PMID: 29122925
- PMCID: PMC5731345
- Citations: 73
- Source URL: https://doi.org/10.15252/msb.20167461
- PDF: https://europepmc.org/articles/PMC5731345/pdf
- Provenance: europepmc

## 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.

_Abstract reproduced under CC-BY (article license as recorded by Europe PMC; abstract reproduced unmodified apart from markup removal)._

Topics: [Synthetic Biology & Genome Engineering](https://space-kitty.com/topics/synthetic-biology)

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