Optimized Internalization of Fluorescently Labeled Biomolecules into Live Bacteria

Abstract

We have recently developed a method for delivering short DNA fragments and proteins labeled with bright and stable organic fluorophores into live E. coli. Cells are electroporated with a high-voltage pulse, recovered in a rich medium and thoroughly washed to remove any non-internalized fluorescence. Internalized molecules can be tracked at the single-molecule level, and both single-cell and single-molecule FRET can be measured.We have explored the effect of electroporation voltage on the internalization efficiency of short DNA fragments and proteins, and have found a linear relationship. Hence, an appropriate voltage can be selected depending on the application of interest, such as whether single-molecule or ensemble measurements are desired. In addition, we have optimized the buffer and salt conditions for electroporation, in terms of maximizing internalization efficiency whilst preserving protein integrity.Various conditions have been tested for cell washing, including the use of salt and detergent in the washing buffers. The medium used for cell recovery after electroporation has also been noted to affect the efficiency of cell washing. A significant improvement in the removal of non-internalized fluorescence has been achieved by cell filtration. In the case of proteins prone to aggregation, cell filtration has also been found to remove any high-molecular weight species from the cell suspension.Finally, we have found free dye to be internalized at much higher efficiency than the labeled biomolecules, and hence it is important to remove any contaminating free dye from the samples used for electroporation. We have optimized the methodology for determining and minimizing the amount of free dye in our samples, and have obtained samples that show less than 1% free dye contamination, which is at the level of background autofluorescence.