Rates of Vesicle Uptake in Cells Using Vesicle Mediated Cre-Lox Transfer

Bacteria naturally produce outermembrane vesicles. These vesicles are capable of fusing other cells and in the process delivering a variety of types of biomolecular cargo, including DNA and proteins. DNA transfer via vesicle exchange is a slow process that depends on rare vesicle uptake events even within large populations of cells. Therefore, assays to measure the uptake of bacterial vesicles must be capable of capturing rare events and have a low rate of false positives.

We have developed a vesicle uptake assay that utilizes the Cre-Lox system, connecting permanent changes in a bacterial genome with the transfer of DNA inside of bacterial vesicles. The Cre protein binds to and cuts DNA that contains specific sequences known as lox sites. In our engineered strain of E. coli, Cre activity leads to the expression of GFP. The large difference in fluorescence between cells before and after Cre recombination makes this a sensitive assay with low false positives for vesicle uptake. We monitor the number of cells producing GFP in a population of cells to quantify the rate of vesicle uptake. This was used to determine how vesicle uptake is modulated by proteins expressed in the vesicle-producing and vesicle-receiving cells, as well as by the addition of membrane-binding external molecules.