(582cf) A Flow-Cytometry Method for Optimizing Transformation Conditions in Bacteria

The application of metabolic engineering to non-traditional host organisms is often limited by the lack of transformation protocols for these organisms. Developing such protocols requires optimization of the method used for DNA delivery. However, there are few well-developed assays specifically for analyzing the efficiency of DNA uptake, rather than the transformation process as a whole. The objective of this study was to develop an assay to track and optimize the delivery of plasmid DNA to bacteria cells. Plasmid DNA covalently labeled with the Cy5 fluorophore was used for electroporation and sonoporation of competent cells, and the proportion of cells that had taken up plasmid determined by flow cytometry (FC). To address the issue of cell viability following transformation, cells were treated with propidium iodide (PI) just prior to FC. Fluorescence microscopy was used to confirm the presence of labeled plasmid within the cytoplasm of the cells. The assay was first validated in E. coli, then used to establish optimal transformation conditions for the gram-positive anaerobe Moorella thermoacetica. The optimized protocol allowed DNA delivery to up to 3% of the cells viable after electroporation. The assay is applicable to a variety of organisms, and will thus be useful in expanding the range of hosts amenable to metabolic engineering.