(79d) Streamlined Assessment of Membrane Permeability: A Simple and Inexpensive High-Throughput Method for Multiple Applications

Authors: 
Santoscoy, M. C., Iowa State University
Jarboe, L. R., Iowa State University
The economic viability of bio-production processes is often limited by damage to the microbial cell membrane and thus there is a demand for strategies to increase the robustness of the cell membrane. On the other hand, damage to the microbial membrane is also a common mode of action by antibiotics, therefore high-throughput and inexpensive methodologies for characterizing membrane permeability are desired for multiple applications. Membrane-impermeable DNA-binding dyes are often used to assess membrane integrity in conjunction with flow cytometry. We demonstrate that in situ assessment of the membrane permeability of E. coli to SYTOX Green is consistent with flow cytometry, with the benefit of lower experimental intensity, lower cost, and no need for a priori selection of sampling times. This method was recently reported in the Journal of Industrial Microbiology where we demonstrated its application by the characterization of four membrane engineering strategies (deletion of aas, deletion of cfa, increased expression of cfa, and deletion of bhsA) for their effect on octanoic acid tolerance, with the finding that deletion of bhsA increased tolerance and substantially decreased membrane leakage. Hence, this methodology is applicable for the membrane permeability characterization of engineered and non-engineered strains with multiple applications such as; increase cell membrane robustness, study of cell membrane permeability depending on their growth condition and growth phase, characterize entry of small molecules as antibiotics to the cell interior or leakage of valuable molecules out of the cell.