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Fig. 4 | Microbial Cell Factories

Fig. 4

From: Stable and selective permeable hydrogel microcapsules for high-throughput cell cultivation and enzymatic analysis

Fig. 4

SrtF transpeptidation activity can be assayed within HC-PCAMs. a Workflow of the HC-PCAMs-based method for the analysis of SrtF activity. (i) E. coli cells, harboring the plasmid encoding either the wild type Gly-SrtF (WT) or the inactive mutant Gly-SrtF-C195G (negative control), are encapsulated within HC-CAMs. (ii) Microcapsules are transferred into the growth medium to allow cells to grow to colonies that express the respective enzymes. (iii) Colonized HC-CAMs are coated with PSS and PAH to reduce the permeability and generate colonized HC-PCAMs. (iv) Upon cell lysis, the expressed N-terminally glycine-modified sortase is released from cells but retained within HC-PCAMs. (v) Microcapsules are then incubated with the fluorescent peptide. Due to the low molecular weight, the fluorescent peptide can permeate through the polyelectrolyte shell and initiate the sortase-mediated transpeptidation reaction. (vi) Upon washing, the unreacted fluorescent peptide is removed and the reaction stopped. The polyelectrolyte coating retains the fluorescent transpeptidation product within HC-PCAMs, allowing detection of sortase activity via fluorescence analysis. (vii) Microcapsules are incubated with the fluorescent DNA intercalating agent propidium iodide (PI) and analyzed for fluorescence by fluorescence microscopy and COPAS. b Bright field (upper panel) and green fluorescence (lower panel) pictures of reacted HC-PCAMs showing successful conjugation and capture of the fluorescent peptide within HC-PCAMs expressing the wild type Gly-SrtF but not within HC-CAMs expressing the inactive enzyme. c Histogram overlays of HC-PCAMs analyzed with COPAS showing green fluorescence distribution (sortase activity) of Gly-SrtF wild type (red) and Gly-SrtF-195G (cyan) populations. d Red fluorescence pictures of HC-PCAMs after the addition of propidium iodide. e Histogram overlays of COPAS analyzed HC-PCAMs showing the distribution of the two HC-PCAMs populations after normalization of the green fluorescence signal (sortase activity) for the red fluorescence signal (lysed biomass) obtained with propidium iodide

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