Fig. 3
A microscopy-based method to measure MHETase cell surface display efficiency. A Outline of the microscopy-based method to quantify GFP signal on the cell surface (see text for details). B Mean fluorescence intensities at each pixel coordinate for the indicated strains. Bars indicate standard deviation. Analysis was performed on at least 40 cells in each replicate. conA-A594: n = 98, Mrh1-GFP and intra-cellular MHETase (intra-M): n = 4, Tif2-GFP and Rrp1A-GFP: n = 2. C Comparison of mean fluorescence intensities for the − 5, − 4, and + 1 pixel coordinates for the indicated strains (grey shading in B). Bars indicate standard deviation. D, E Representative fluorescence micrographs for the strains in B and C and for the MHETase surface display chimeras. Scale bar: 5 μm. F Fraction of MHETase chimeras displayed at the cell surface. Cells were induced for 4 h, labelled with conA-A594 and imaged. The fraction of displayed chimera is plotted. Horizontal bars indicate the means of the replicates (n = 6). Each replicate included at least 200 cells. G Abundance of the MHETase chimeras at the cell surface. The fraction of chimera displayed from panel F was used to calculate the cell surface abundance in molecules per cell. Theoretical construct molarity is indicated for a cell density of 108 cells/mL. Horizontal bars indicate the means of the replicates (n = 6)