Genetic circuit characterization and debugging using RNA‐seq

• Main Authors: Gorochowski, Thomas Edward (Contributor), Espah Borujeni, Amin (Contributor), Park, YongJin (Contributor), Nielsen, Alec Andrew (Contributor), Der, Bryan S. (Contributor), Gordon, David B (Contributor), Voigt, Christopher A. (Contributor)
• Other Authors: Massachusetts Institute of Technology. Department of Biological Engineering (Contributor)
• Format: Article
• Language: English
• Published: Nature Publishing Group, 2018-03-30T18:02:40Z.
• Subjects: Article
• Online Access: Get fulltext

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.
United States. Defense Advanced Research Projects Agency. Living Foundries Program (award HR0011-13-1-0001)
United States. Defense Advanced Research Projects Agency. Living Foundries Program (award HR0011-12-C-0067)
United States. Defense Advanced Research Projects Agency. Living Foundries Program (award HR0011-15-C-0084)
National Institute of Standards and Technology (U.S.) (award 70NANB16H164 )
United States. Office of Naval Research. Multidisciplinary University Research Initiative (grant N00014-13-1-00740
Samsung Scholarship Foundation