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Fig. 2 | Microbial Cell Factories

Fig. 2

From: A nanobody:GFP bacterial platform that enables functional enzyme display and easy quantification of display capacity

Fig. 2

Characterisation of the NB:GFP platform. a Illustration of the principal difference between displaying GFP and displaying the nanobody on the surface of the cell. GFP gives the cell a fluorescent glow whether produced intracellularly (ic) or on the surface (surf). In contrast, the only way a cell producing the nanobody can be fluorescent is if the nanobody is displayed on the surface and accessible to extracellular GFP. b Protein schemes for the OmpA and autotransporter constructs. An N-terminal signal sequence (lppss and pelBss) precedes the OmpA anchor followed by the nanobody, or the nanobody followed by the C-IgAP anchor, respectively. c Workflow of GFP assay: cells producing the nanobody are incubated with free GFP; unbound GFP is washed off and the fluorescence signal from GFP bound to nanobody on cells is assayed using SDS-PAGE, fluorescence measurements and microscopy. d In-gel fluorescence of purified GFP; purified GFP mixed with purified NB; NB; whole cells displaying OmpA-NB or NB-C-IgAP with and without GFP and with and without proteinase K treatment. The same amount of cells was loaded in each lane for whole-cell samples. e Whole-cell fluorescence measurement with and without rhamnose induction. Values are averages of three biological replicates and bars show standard error. f Bright field and fluorescence microscopy images of OmpA-NB displayed on E. coli cells, with and without rhamnose induction. g Flow cytometry profiles of pK:LppOmpA-NB and pK:NB-C-IgAP with induction (green) and without induction (red). Percentage numbers show the fraction of cells that are fluorescent. Lppss lpp signal sequence, pelBss pelB signal sequence, OmpA Outer membrane protein A, NB nanobody, C-IgAP C-terminal of IgA protease

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