Towards synthetic cells using peptide-based reaction compartments

Kilian Vogele,Tobias Pirzer,Friedrich C. Simmel,Lukas Gasser,Stephan A. Sieber,Mathias W. Hackl,Marisa A. Goetzfried,Thomas Frank

doi:10.1038/s41467-018-06379-8

Abstract

Membrane compartmentalization and growth are central aspects of living cells, and are thus encoded in every cell’s genome. For the creation of artificial cellular systems, genetic information and production of membrane building blocks will need to be coupled in a similar manner. However, natural biochemical reaction networks and membrane building blocks are notoriously difficult to implement in vitro. Here, we utilized amphiphilic elastin-like peptides (ELP) to create self-assembled vesicular structures of about 200 nm diameter. In order to genetically encode the growth of these vesicles, we encapsulate a cell-free transcription-translation system together with the DNA template inside the peptide vesicles. We show in vesiculo production of a functioning fluorescent RNA aptamer and a fluorescent protein. Furthermore, we implement in situ expression of the membrane peptide itself and finally demonstrate autonomous vesicle growth due to the incorporation of this ELP into the membrane.

Highlights

Membrane compartmentalization and growth are central aspects of living cells, and are encoded in every cell’s genome
We show that biomolecules can be enclosed in elastin-like peptides (ELP)-based vesicles, and we further demonstrate in vesiculo transcription of an RNA aptamer and translation of a fluorescent protein
Using transmission electron microscopy (TEM) we determined the peak value of the diameter distribution for 180 pM EF (Fig. 1c) to 176 nm with a dispersion of 68 nm, which is in good agreement with the dynamic light scattering (DLS) data (Supplementary Fig. 2)

Summary

Introduction

Membrane compartmentalization and growth are central aspects of living cells, and are encoded in every cell’s genome. In order to implement this in a cell-free context[10,11], transcription-translation systems based on purified components, e.g., the PURE system[7,12], or on bacterial cell extracts, e.g., the TX-TL system[13,14], can be used These systems employ the multicomponent bacterial translation machinery, to express proteins from externally added DNA in a one pot reaction. We encapsulate the TX-TL system in peptidosomes made of amphiphilic elastin-like peptides (ELP) These peptides can be expressed in cell-free systems[10,11] and they simplify the synthesis of the membrane material in comparison to lipid synthesis. We succeed in the expression of the membrane-constituting peptides inside the vesicles themselves and demonstrate their incorporation into the membrane and inherent vesicle growth

Methods

Results

Conclusion