A Fast Assay of Bacteria Cell Permeability for Genetic Transformation

Abstract

Bacterial cell genetic engineering is fundamental for research aiming to learn more about bacterial species for a broad range of applications. One method of intracellular delivery of foreign DNA during the genetic engineering process is the use of electroporation to create pores along the bacterial cell membrane. Current methods for assessing pore formation do not directly measure cell permeabilization or enable same-day assessment. In this thesis, a novel fast-screening protocol combining SYTOX green, microfluidics, and fluorescence imaging is evaluated for its capability to assess multiple conditions for cell permeabilization within a single day. By imaging bulk suspensions of post-electroporated cells stained with intracellularly delivered SYTOX, multiple electroporation conditions can be rapidly screened for cell permeabilization. This fast-screening protocol utilizes standard microbiology equipment and low-cost microfluidic imaging chambers, lowering the barrier to adoption and significantly reducing experimental time compared to conventional protocols involving foreign DNA delivery. Importantly, by decoupling permeabilization assessment from foreign DNA uptake, this method isolates the effect of membrane permeabilization from confounding factors such as restriction-modification systems. As a result, it provides a more accurate qualitative and quantitative assessment of bacterial membrane disruption. This approach enables same-day evaluation of electroporation conditions regardless of bacterial growth rate, potentially accelerating the optimization process for intracellular delivery in gene editing applications.