Synthetic cells, expressing proteins using cell-free transcription-translation (TXTL), is a technology utilized for a variety of applications, such as investigating natural gene pathways, metabolic engineering, drug development or bioinformatics. For all these purposes, the ability to precisely control gene expression is essential. Various strategies to control gene expression in TXTL have been developed; however, further advancements on gene-specific and straightforward regulation methods are still needed. Here, we present a method of control of gene expression in TXTL using a “silencing oligo”: a short oligonucleotide, designed with a particular secondary structure, that binds to the target messenger RNA. We demonstrated that silencing oligo inhibits protein expression in TXTL in a sequence-dependent manner. We showed that silencing oligo activity is associated with RNase H activity in bacterial TXTL. To complete the gene expression control toolbox for synthetic cells, we also engineered a first transfection system. We demonstrated the transfection of various payloads, enabling the introduction of RNA and DNA of different lengths to synthetic cell liposomes. Finally, we combined the silencing oligo and the transfection technologies, demonstrating control of gene expression by transfecting silencing oligo into synthetic minimal cells.
Bibliographical noteFunding Information:
This work was supported by: NASA award 80NSSC18K1139 Center for the Origin of Life—Translation, Evolution And Mutualism, John Templeton Foundation award 61184 Exploring the Informational Transitions Bridging Inorganic Chemistry and Minimal Life, NSF award 1844313 RoL: RAISE: DESYN‐C3: Engineering multicompartmentalised synthetic minimal cells, NSF award 2123465 Synthetic P‐bodies: Coupling gene expression and ribonucleoprotein granules in synthetic cell vesicles for sensing and response, and Hackett Royalty Fund award. Wakana Sato was supported by the Funai Overseas Scholarship of The Funai Foundation for Information Technology.
© 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.
- gene regulation
- synthetic biology
- synthetic cells