Abstract
A longstanding goal of synthetic biology has been the programmable control of cellular functions. Central to this is the creation of versatile regulatory toolsets that allow for programmable control of gene expression. Of the many regulatory molecules available, RNA regulators offer the intriguing possibility of de novo design - allowing for the bottom-up molecular-level design of genetic control systems. Here we present a computational design approach for the creation of a bacterial regulator called Small Transcription Activating RNAs (STARs) and create a library of high-performing and orthogonal STARs that achieve up to ∼ 9000-fold gene activation. We demonstrate the versatility of these STARs - from acting synergistically with existing constitutive and inducible regulators, to reprogramming cellular phenotypes and controlling multigene metabolic pathway expression. Finally, we combine these new STARs with themselves and CRISPRi transcriptional repressors to deliver new types of RNA-based genetic circuitry that allow for sophisticated and temporal control of gene expression.
| Original language | English (US) |
|---|---|
| Article number | 1051 |
| Journal | Nature communications |
| Volume | 8 |
| Issue number | 1 |
| DOIs | |
| State | Published - Dec 1 2017 |
Funding
The authors gratefully acknowledge the gift of TX-TL extract and buffers used in this work from Vincent Noireaux’s laboratory (University of Minnesota). The authors gratefully acknowledge the gift of dCas9 expression plasmids used in this work from Stanley Qi’s laboratory (University of Stanford). The authors gratefully acknowledge the gift of a deoxyviolacein expression plasmid from Robert Egbert of Adam Arkin’s laboratory (University of California, Berkley). The authors also thank John Dueber (University of California, Berkley) for helpful conversations about the deoxyviolacein pathway. The authors also thank Niles Pierce’s laboratory (California Institute of Technology) for helpful conversations about NUPACK and RNA design. This work was supported by an NSF CAREER Award (1452441 to J.B.L.), the Defense Advanced Research Projects Agency (contract HR0011-16-C-0134 to J.B.L.), and Searle Funds at The Chicago Community Trust (to J.B.L.).
ASJC Scopus subject areas
- General Chemistry
- General Biochemistry, Genetics and Molecular Biology
- General Physics and Astronomy