Design of Temperature-responsive Controllers for Regulating Gene-expression
Design of Temperature-responsive Controllers for Regulating Gene-expression

Abstract

Transcription factors (TFs) are frequently used in synthetic biology for regulating gene expression. The levels of these TFs inside the cell are generally controlled using externally added small molecules. These small molecules, however, are expensive and therefore cost-prohibitive for large cultures, cannot be removed once added, and could be a potential source of contamination for cells. In this work, we propose the use of temperature-responsive elastin like polypeptides (ELPs) bound to any transcription factor (TF) as a general class of controllers to regulate the functional levels of TFs in cells. ELPs are a class of polypeptides made up of repeating units of the amino acids with sequence VPGXG, where X can be any guest amino acid. We have created a library of ELP-TF fusions that reversibly aggregate in response to a range of temperatures. These ELP-TFs allow easy, cheap, and contamination-free temperature-dependent control; fast and reversible regulation; and rapid tuning of TF levels. We demonstrate that ELP aggregation sequesters bound TFs reducing protein expression by up to 100% in E. coli. As ELPs may be engineered to control gene expression at arbitrary setpoints with any TF, these controllers are generalizable and universal for temperature-dependent gene-expression control in any organism.