Engineering Escherichia coli Membrane Vesicles to Transfect Mammalian Cells in Vitro

Abstract

Membrane vesicles (MVs) are the nano-sized structures membrane lipid formed from budding off of the outermost membrane of a cell. MVs are known to package a variety of cargo, including small molecules, peptides, proteins and genetic material. Based on their assorted cargo, MVs are known to transport DNA, RNA, and Proteins across the extracellular space. The unique characteristics could be utilized for therapeutic applications. In this study, Escherichia coli (E. coli) BW25113 strain will be engineered to mass produce DNA containing MVs by knocking out the NlpI gene and transforming with Red Fluorescent Protein(mCherry). Also, INV (gene for invasion)/LLO (gene synthesizing listeriolysin O) plasmid will be transformed into the E. coli strains for active transport of the MVs into the target mammalian cell. The characteristic of these vesicles will be analyzed using Qubit 3 fluorometer system to measure the concentration of the MVs produced and DNAs in the vesicle. We will also analyze the transfect ability of the MVs produced to the mammalian cell lines such as Hela, the human cervical cancer cell, and HEK293, the human embryonic kidney cell, which are good models for transfection. The toxicity and the immunogenicity of invasive vesicles will be characterized using MTT assay and Enzyme-Linked Immunosorbent Assay(ELISA). Throughout the study, we will demonstrate the MV as a transfecting agent.