DNA-mediated fusion of spheroplasts with synthetic liposomes

This project aims to engineer controlled adhesion and fusion of artificial liposomes to spheroplast cells.

The Idea

We have very good control over phospholipid liposome (vesicle) formation, transport, and fusion; we also know how to lyse the external cell membrane of gram negative bacteria, yeast and single cell algae, all of which then form a ‘spheroplast’ state, from which the whole cell can be recovered under appropriate culture. Removing the external cell membrane/wall is indeed a standard step in various protocols for uptake of material into the cells. The key idea of our proposal is to demonstrate a ‘hybrid’ system, engineering controlled adhesion and fusion of artificial liposomes to spheroplast cells. This could represent a new high throughput and selective tool for delivering cargo into cells, not limited to genetic material and very flexible in terms of size and chemical nature of the cargo (Jones, Seeman & Mirkin, 2015 in Science 347).

 

The Team

Dr Lorenzo di Michele,
Research Fellow, The Cavendish Laboratory, Department of Physics, University of Cambridge

Prof Pietro Cicuta,
Head of Biological and Soft Systems, The Cavendish Laboratory, Department of Physics, University of Cambridge

Prof Martin Howard,
Research Group Leader, Computational and Systems Biology, John Innes Centre, Norwich

Dr Emma Talbot,
Research Fellow, Department of Plant Sciences, University of Cambridge

Mr Ryan Brady,
Graduate researcher, NanoCDT, University of Cambridge

Mr Sho Takamori,
Graduate researcher, The Cavendish Laboratory, Department of Physics, University of Cambridge


Project Outputs

Project Report

Summary of the project's achievements and future plans

Project Proposal

Original proposal and application

software.png

Project Resources


DNA-mediated fusion of spheroplasts with synthetic liposomes

Summary

The project aims at controlling the fusion of spheroplasts or mammalian cells with artificial liposomes using amphiphilic DNA nanostructures. These complexes mimic the function of SNARE proteins (soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptors), inducing membrane fusion by bridging across two lipid bilayers and driving them to close proximity. Liposomes can be used to deliver efficiently large amounts of cargoes into the live cells, needed for instance for genetic editing or otherwise interfering with the cell’s metabolism, mechanical properties etc. Various candidate DNA nanostructures were designed, assembled and characterised. Then their ability of inducing fusion of liposomes of different size with other liposomes was successfully demonstrated. In parallel, a protocol for the preparation of giant spheroplasts from filamentous E. coli was implemented and the possibility of functionalising the spheroplasts with the DNA nanostructures demonstrated. We are currently attempting the fusion of spheroplasts with liposomes, but preliminary results are inconclusive. In parallel, we started exploring the possibility of functionalising mouse stem cells with the same DNA complexes, with the objective of delivering CRISPR machinery for genetic editing. This part of the project is done in collaboration with Dr A. Russel (CRUK).