Engineering cell free protein synthesis and synthetic cells

Thesis

Mimicking the structure and function of natural cells, synthetic cells hold significant potential in various applications. These synthetic cells, which come in numerous forms, are developed from different materials, including lipids and proteins, as well as synthetic materials such as polymers. The method of producing synthetic cells depends largely on their intended use and the materials used in their formation. Expression of genes within a synthetic cell can allow its usage in many fields to either study minimal cellular behaviour and function or use them as cargo holding vehicles for controlled delivery to target sites.

This thesis focuses on the production of lipid-based synthetic cells using a cell-free protein expression system and a DNA template. The primary objective of this study was to generate synthetic cells capable of producing a target protein, which can be released in a controlled manner upon the application of heat. Heat is a promising stimulus for future applications in SCs due to its thermal control to allow precise manipulation, biocompatibility, non-invasiveness, versatility, scalability, and integration with existing technologies.

To produce the SCs, we initially optimized the inverted-emulsion method to create giant unilamellar vesicles. These vesicles successfully encapsulated the cell-free protein expression system, enabling the production of a fluorescent protein or a reporter enzyme. However, incorporating heat-sensitive lipids into the synthetic cells using this approach proved unsuccessful. Consequently, we pursued an alternative strategy to produce SCs known as the freeze-dried empty liposomes (FDEL) method. Through optimization, this method enabled us to incorporate thermosensitive materials such as DPPC, lysolipids, and PEG into the SCs at specific compositions, facilitating controlled release upon heating.

Furthermore, we investigate a genetic engineering method to enhance the protein yield in the E. coli cell-free protein expression system, PURExpress, by introducing a short sequence referred to as a "booster." We identify specific cell penetrating peptide nucleotide sequences and their derivatives which allow higher expression of the fluorescent protein, mVenus.

Authors: Chowdhry, R

• DOI: 10.5287/ora-obanmewqq
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Increasing protein yield in IVTT; Thermoresponsive vesicles; IVTT; Thermosensitive vesicles; Synthetic cells; Liposome; Lipid vesicles
• Subjects: Synthetic biology; Chemical biology