Journal
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Volume -, Issue 158, Pages -Publisher
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/60829
Keywords
Bioengineering; Issue 158; Synthetic cells; cell free; emulsion; bacterial lysate; artificial cells; drug delivery; therapeutic proteins; GUVs; liposome; proteocell; protocell; proteoliposome; biologics; directed evolution; synthetic biology
Categories
Funding
- Technion Integrated Cancer Center (TICC)
- Russell Berrie Nanotechnology Institute
- Lorry I. Lokey Interdisciplinary Center for Life Sciences Engineering
- Israel Ministry of Economy [52752]
- Israel Ministry of Science Technology and Space - Office of the Chief Scientist [3-11878]
- Israel Science Foundation [1778/13, 1421/17]
- Israel Cancer Association [2015-0116]
- German-Israeli Foundation for Scientific Research and Development [I-2328-1139.10/2012]
- European Union [908049]
- Phospholipid Research Center Grant
- Rosenblatt Foundation for cancer research
- Mallat Family Foundation Grant
- Unger Family Foundation
- Alon Fellowship
- Taub Fellowship
- Sherman fellowship
- Gutwirth fellowship
- [ERC-STG-2015-680242]
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The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA-RNA-.protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.
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