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The outer membrane of Gram-negative bacteria acts as an additional diffusion barrier for solutes and nutrients. It is perforated by outer membrane proteins (OMPs) that function most often as diffusion pores, but sometimes also as parts of larger cellular transport complexes, structural components of the cell wall, or even as enzymes. These OMPs often have large loops that protrude into the extracellular environment, which have promise for biotechnological applications and as therapeutic targets. Thus, understanding how modifications to these loops affect OMP stability and folding is critical for their efficient application. In this work, the small outer membrane protein OmpX was used as a model system to quantify the effects of loop insertions on OMP folding and stability. The insertions were varied according to both hydrophobicity and size, and their effects were determined by assaying folding into detergent micelles in vitro by SDS-PAGE and in vivo by isolating the outer membrane of cells expressing the constructs. The different insertions were also examined in molecular dynamics simulations to resolve how they affect OmpX dynamics in its native outer membrane. The results indicate that folding of OMPs is affected by both the insert length and by its hydrophobic character. Small insertions sometimes even improved the folding efficiency of OmpX, while large hydrophilic inserts reduced it. All the constructs that were found to fold in vitro could also do so in their native environment. One construct that could not fold in vitro was transported to the OM in vivo , but remained unfolded. Our results will help to improve the design and efficiency of recombinant OMPs used for surface display. 

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressingE. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals  <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.