Confocal microscopy images of <i>E</i>. <i>coli</i> (A–G, a–g), <i>Salmonella enterica</i> (H, h) and <i>Enterobacter cloacae</i> (I, i) fluorescent labeling tests.

<p>Each group of 4 images correspond to a different chimeric biosensor fluorescent protein. Images from “A” to “I” correspond to confocal microscopy, showing the fluorescent labelled cells, and images from “a” to “i” are the same confocal images but fused with an optical image generating a single photo (merged snapshot for testing that fluorescent spots actually correspond to labelled cells, and not to background fluorescence). The symbol “✽” means that the sample has been filtered in order to get rid of unbound chimera protein. A-B, images of <i>E</i>. <i>coli</i> labelled with GFP-hadrurin fluorescent chimera protein, where labeling is lost after a filtering step. C-D, images of <i>E</i>. <i>coli</i> labelled with GFP-pb5 fluorescent chimera protein, where labeling is also lost after a filtering step. Only the fluorescent biosensor chimera protein GFP-colS4 (E,F) is able to maintain its strong binding to the <i>E</i>. <i>coli</i> surface after the filtration step. Also, the other two bacterial species (negative controls) are not labelled at all (H, I). Images G and g are negative controls for <i>E</i>. <i>coli</i>, where no fluorescent biosensor chimera protein has been added, as a method to test that this bacterial cells do not show autofluorescence under these conditions.</p

Functional blocks of the measurement system at the portable <i>E</i>. <i>coli</i> Analyzer device.

<p>Functional blocks of the measurement system at the portable <i>E</i>. <i>coli</i> Analyzer device.</p

MUSCLE multiple sequence alignment of the OmpW proteins from <i>E</i>. <i>coli</i>, <i>S</i>. <i>enterica</i> var. <i>arizonae</i> and <i>E</i>. <i>cloacae</i>, highlighting in yellow the region encompasing the the Asp¹¹⁶, His¹¹⁷ and Glu¹²⁰ amino acids of OmpW (in blue letters: D, H and E), which are freely exposed to the extracellular space and act as binding motifs for colicin S4 bacteriocin (as well as for GFP-colS4 chimera protein in this biosensor).

<p>As it is shown, the other two enterobacteria species do not have the conserved motif DH—E as in <i>E</i>. <i>coli</i>. Accesion numbers: SAI89619 (<i>E</i>. <i>cloacae</i>), BAA14788 (<i>E</i>. <i>coli</i>), OSE54138 (<i>S</i>. <i>enterica</i>).</p

Development of a biosensor protein bullet as a fluorescent method for fast detection of <i>Escherichia coli</i> in drinking water - Fig 2

<p>A: Hypothetical spatial structure of the fluorescent biosensor chimera protein GFP-hadrurin. B: Diagram showing the attachement process of the fluorescent biosensor chimera protein to the membrane of <i>E</i>. <i>coli</i>. PMT: photomultiplier (part of the <i>E</i>. <i>coli</i> Analyzer device in charge of detecting and amplifying the fluorescence signal produced by the GFP domain of each biosensor chimera protein, after receiving the 395 nm excitation light from the LED source).</p