Calibration of fluorescence intensity as a function of GFP copy number using B. subtilis calibration strains.

A. Representative images of cells expressing known average copy numbers of sfGFP¹. B. The total intensity (sum of pixel values) per cell, plotted against copy number, after selecting cell contours using MicrobeJ software and subtracting background. Background was defined as total intensity (sum of pixel values) in wildtype cells which did not express any fluorescent protein. Data is plotted on semi-logarithmic coordinates in the inset, to show low copy number intensities. C. B. subtilis cells expressing the same average number of mEGFP (BAL038) or sfGFP (SG13) were imaged to determine the correction factor for mEGFP-FisB copy number quantification. Top: representative images using the same acquisition and display settings, bottom: quantification. On a per molecule basis, sfGFP is ~2.4±0.2-fold brighter than mEGFP. Strains were made by transforming PY79 cells with either plasmid pECE321 (encoding amyE::(Pveg_R0_sfGFP_spec)) or its variant wherein sfGFP was replaced by mEGFP (pECE321_mEGFP). On average 3.00×10⁵ GFP copies/cell are expressed¹. D. The calibration in B, rescaled with the correction factor determined in C, such that the calibration now corresponds to the copy numbers for mEGFP (the best-fit line constrained to pass through the origin has slope 22.48 (R² = 0.93). E. Distribution of background-corrected total fluorescence intensity (sum of pixel values) for cells expressing mEGFP-FisB (BAL001) that have undergone membrane fission at t = 3h. Membrane fission was assessed as in Fig 1 and S1 Fig. Background was defined as in B. N = 250 cells. F. The total intensity of ISEP and DMCs as a percentage of total cell FisB fluorescence. Using the calibration for whole-cell fluorescence shown in D, ~1300 copies of FisB per cell; ~50 FisB/ISEP and ~16 FisB/DMC are estimated. Scale bars in A, C, represent 1 μm.

(EPS)