1. Software: Image Colorimetric Detection (ICD)

In order to precisely analyze the color signals of our paper-based sensors, our modeling group developed a software called Image Colorimetric Detection (ICD) with the help of the research team from South China University of Technology.

ICD is designed to analyze the RGB values of images. To use it, the user takes a picture, and selects a region of interest of the picture. ICD gives the RBG values, including R, G, B, and IR, of the selected area (IR=blue value). For our project, after our sensor reacts with a bacteria sample, a color signal is presented and can be detected by ICD. The bacteria concentration is correlated with the color signal, and can be calculated by the formula: when a1 and b1 are predetermined in our experiment, and IR is given by ICD.
ICD can be of great value for other research teams with similar color-analysis demands in the future. ICD can be used in future researches to determine the color change of reactions with color reagents involved.

            

                          Figure 1                       Figure 2                                                Figure 3

Figure 1: App home page of Image Colorimetric Detection

Figure 2: Page for detection results of Image Colorimetric Detection

Figure 3: Blue value-concentration curve for VP sensor

2. Construction of expression vectors for RBPs and GFP-fused RBPs (GFP-RBPs)

We successfully built pET28a-TFP, pET28a-gp15, pET28a-CBD, and pET28a-TSP plasmids as expression vectors for RBPs, including TFP (tail fiber protein), gp15, CBD(C-terminal cell-binding domain), and TSP (tailspike protein). These RBPs can attach with target bacteria strains, E. coli (EC), V. parahaemolyticus (VP), S. aureus (SA), and S. enteritidis (SE), with the help of BL21(DE3) and revulsive IPTG.
Plasmid vectors of pET28a-GFP-TFP, pET28a-GFP-gp15, pET28a-GFP-CBD, and pET28a-GFP-TSP are also constructed to express GFP fusion proteins, GFP-TFP, GFP-gp15, GFP-CBD, and GFP-TSP. GFP protein is fused to RBP as a fluorescent tag to trace where and when RBP is attached when in contact with bacteria. GFP-RBPs expressed by our plasmids are great tools to investigate various characteristics and signal pathways of RBPs.

                               Figure 1                                             Figure 2                                        Figure 3

Figure 1: Plasmid vectors of pET28a-gp15 and pET28a-GFP-gp15
Figure 2: GFP-RBPs show green fluorescence under blue light (Left to right: 100% eluted gp15, 70% eluted gp15, and 10% eluted gp15)
Figure 3: The eluted proteins of CBD we collected

3. RBP-MBs for bacteria purification

We succeeded in making four RBP-nanomagnetic bead complexes (RBP-MBs) by linking nanomagnetic bead complex with the RBPs, TFP, gp15, CBD, and TSP, respectively. These RBP-MBs can be used to separate viable bacteria strains, EC, VP, SA, and SE, from other substances in solution by magnets. Bacteria purified by these complexes can be used for various research purposes.

4. Paper-based sensors for visual signals

We developed paper-based sensors that can visually indicate different concentrations of target bacteria, EC, VP, SA, and SE. The sensor is made by a fundamental paper receiver and a reaction system. The paper receiver is made of basic testing paper bought from Sangon Biotech from Shanghai, China. The reaction system is a 6.5 microliter solution containing the optimal reaction concentrations of a lysing agent and four chromogenic substrates, chlorophenol red-β-D-galactopyranoside (CPRG), X-β-glu, 4-nitrophenyl β-D-glucopyanoside (pNPG), and magenta caprylate (MC). Target bacteria strains and the bacteria concentrations can be detected by our paper-based sensors via visual signals.

                                  

         Figure 4: Detection of bacteria using RBP-MBs combined with paper based sensors (C: control)