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. 2020 Aug 27;19:170. doi: 10.1186/s12934-020-01427-9

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© The Author(s) 2020

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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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