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Fig. 5 | Microbial Cell Factories

Fig. 5

From: A secretion biosensor for monitoring Sec-dependent protein export in Corynebacterium glutamicum

Fig. 5

FACS analysis of C. glutamicum K9 strains and sorting strategy for the enrichment of the best performing signal peptide. 105 cells from cultures of the respective strains were analyzed, followed by a preselection of cells to exclude cell doublets and debris by electronic gating using FSC-W against FSC-H (Table 2, gate 0). An overlay of C. glutamicum K9 carrying pEKEx2-NprE-cutinase (red) or pEKEx2-YwmC-cutinase (blue) is shown as dot plot (a) and histogram (b). In the dot plot, the fluorescence intensity (eYFP) is plotted against cell size (FSC-H), whereas the histogram shows the cell count against the fluorescence intensity (eYFP). c Cells were inoculated to an OD600 of 0.5 in CGXII medium containing 2% (w/v) glucose and cultivated at 30 °C. After 4 h of growth, IPTG (250 µM final concentration) was added to the cultures and, after 10 h of growth, cells were sampled from the respective cultures and subjected to FACS analysis. For the enrichment of the better performing signal peptide (NprE) out of a 1:1 or a 1:100 mixture with a less efficient signal peptide (YwmC), we selected a gate (gate 1) in the dot plot of the respective preselected cells such that it contains as many of the better performing cells (i.e. those containing pEKEx2-NprE-cutinase) and excludes as many of the less productive cells (i.e. those containing pEKEx2-YwmC). For the corresponding numbers of events falling in gate 1, see Table 2 and Additional file 1: Figure S4

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