RNA Thermometers for the PURExpress System

Fredrik W. Sadler, Igor Dodevski, Casim A. Sarkar

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

Cell-free synthetic biology approaches enable engineering of biomolecular systems exhibiting complex, cell-like behaviors in the absence of living entities. Often essential to these systems are user-controllable mechanisms to regulate gene expression. Here we describe synthetic RNA thermometers that enable temperature-dependent translation in the PURExpress in vitro protein synthesis system. Previously described cellular thermometers lie wholly in the 5′ untranslated region and do not retain their intended function in PURExpress. By contrast, we designed hairpins between the Shine-Dalgarno sequence and complementary sequences within the gene of interest. The resulting thermometers enable high-yield, cell-free protein expression in an inducible temperature range compatible with in vitro translation systems (30-37 °C). Moreover, expression efficiency and switching behavior are tunable via small variations to the coding sequence. Our approach and resulting thermometers provide new tools for exploiting temperature as a rapid, external trigger for in vitro gene regulation.

Original languageEnglish (US)
Pages (from-to)292-296
Number of pages5
JournalACS Synthetic Biology
Volume7
Issue number1
DOIs
StatePublished - Jan 19 2018

Bibliographical note

Funding Information:
We thank Ayako Ohoka for her advice and comments. This work was supported by the National Institutes of Health (EB022258) and the University of Minnesota.

Keywords

  • PURE system
  • RNA folding
  • RNA thermometers
  • cell-free synthetic biology
  • in vitro transcription and translation
  • temperature sensing

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