Expanding Chemical Interface of Artificial Cells By Synthetic Riboswitch

Lipid vesicles encapsulating cell-free transcription/translation system are studied as artificial cell models that can be genetically programmed by DNA. Like natural cells, it is crucial for such artificial cells to be able to regulate gene expression in response to chemcial signals to adapt to dynamic environment. Researchers have transplanted natural regulatory elements based on transcription factors (TFs) to artificial cells to interface them with the chemical environment. For example, the quorum sensing systems based on acyl homoserine lactone (AHL) signals and their associated TFs, as well as other bacterial TFs and their small molecule inducers such as IPTG, tetracycline, and arabinose have been adapted to artificial cells. However, TF-based gene switches require multiple parameters (amounts of TF and DNA, promoter sequence, etc.) to be optimized, and it is not trivial to reengineer TFs to respond to new chemical inputs. Riboswitches that regulate gene expression via direct interaction between the 5' untranslated region (UTR) of the mRNA and the targeted molecule could significantly expand the chemical interface of artificial cells because RNA aptamers that recongize various molecules can be discovered by in vitro selection. We demonstrated the process of building artificial cells that genetically respond to a new chemical signal through: 1) selecting a novel RNA aptamer that recognizes histamine, a biogenic amine; 2) engineering cell-free riboswitches through semi-rational design/test cycles; 3) constructing artificial cells that respond to histamine by expressing three distinct proteins that confer various phenotypes (e.g. release of small molecule payload, self-destruction) to the artificial cells. Additionally, the riboswitches we have engineered exhibit high ON/OFF ratios (up to ~30), therefore, could be used as generally applicable gene switches for artificial cells and cell-free systems.