Accidental ingestion of foodborne pathogens may result in intestinal illness or food poisoning, and in severe cases, death. Therefore, rapid real-time detection of foodborne pathogens is an important component of food safety. Colorimetric testing has attracted attention in recent years due to its low cost, intuitive detection, and high acceptability. In this study, we developed a new detection strategy for Bacillus cereus (BC) and Vibrio parahaemolyticus (VP) based on colorimetric detection of smartphone and bacterial binding protein. Therefore, we contributed the following new parts:

 

 

1. Add new experimental data to the existing part: BBa_K3521004

pET28a-backbone (BBa_K3521004) with T7 promoter (BBa_K3521000) is generally used for high bacterial (such as E. coli) expression with kanamycin resistance. This backbone is given from SubCat.

In order to obtain the protein His-tagged BC autolysin cell wall binding domain (CBD) and VP bacteriophage tail fiber protein (TFP), we chose to use pET28a (BBa_K3521004) as the vector to combine with target genes, AutolysinCBD (BBa_K4908001) and MS18 (BBa_K4908000), respectively, so as to express our expected protein through E. coli host.

To construct these plasmids, we first amplified the autolysinCBD and MS18 fragments from the synthetic plasmids and obtained the pET28a vector by enzymatic digestion reaction (Figure 1 A-B). Then, we homologous recombined the target fragment (autolysinCBD or MS18) with the corresponding pET28a vector. The constructed plasmids were transformed into E. coli and plated on LB agar containing kanamycin. The plates were incubated at 37°C overnight (Figure 1 C-D). Figure 1 showed that both pET28a-His-autolysinCBD and pET28a-His-MS18 showed the correct bands, which initially indicated that our constructs were successful.

 

Figure 1 Construction of plasmids pET28a-His-MS18 & pET28a-His-autolysinCBD.

(A) Double enzyme digestion result of pET28a.

(B) Amplification result of MS18 (495 bp) and autolysinCBD (402 bp).

(C-D) Transformation results of recombinant plasmids.

 

2. Create new parts: BBa_K4908000 and BBa_K4908002

For the detection of Vibrio parahaemolyticus (VP), we obtained the VP phage tail fibrillator protein (MS18, BBa_K4908000) and constructed the recombinant plasmid MS18-pET28a (BBa_K4908002). The constructed plasmid was transformed into E. coli and plated on LB agar containing kanamycin. The colony PCR and sequencing results confirmed the successful construction of MS18-pET28a (Figure 2).

 

 

Figure 2 Construction results of MS18-pET28a plasmid.

(A) Plasmid mapping. (B) Colony PCR results. (C) Sequencing results.

 

The plasmid was then transformed into E. coli BL21(DE3) for protein production. Cultures were grown to OD₆₀₀ 0.6-0.8 and induced with IPTG overnight. Cells were lysed by sonication and purified by Ni-affinity chromatography. SDS-PAGE analysis demonstrated successful expression and purification of MS18 at 17.84 KDa around (Figure 3).

 

 

Figure 3 Expression and purification result of MS18 (the second track).

 

To improve the detection and to obtain optimal reaction conditions, we explored the two most important experimental conditions in the MS18 detection system, i.e., the catalytic reaction time and the TMB substrate concentration. We diluted the bacterial suspension to 10⁶ CFU/mL and used different concentrations of TMB 0.01-0.05% (w/v) as the substrate. The concentration of bacteria was determined at different times (0.5-2 h). The results showed that the optimal assay conditions were at 0.04% TMB for 0.5 h for MS18 (Figure 4A).

For testing the ability of the system to VP, we diluted overnight bacterial cultures of VP to 10²-10⁷ CFU/mL using PBS, and then 1 mL of each concentration was taken for the assay. We performed curve fitting of the detection results, and the experimental results are shown in Figure 4B. For VP bacteria, the fitted curve of the detection was y = 0. 1797x - 0.1067 (R² = 0.9775), The detection values showed a favorable linear relationship with the concentration of the bacteria.

 

 

Figure 4 Testing results of MS18.

(A) Optimal reaction conditions for detection systems. (B) Testing results of different concentrations of VP.

 

3. Create new parts: BBa_K4908001 and BBa_K4908003

For the detection of Bacillus cereus (BC), we obtained the BC autolysin cell wall binding domain (AutolysinCBD, BBa_K4908001) and constructed the recombinant plasmid AutolysinCBD-pET28a (BBa_K4908003). The constructed plasmid was transformed into E. coli and plated on LB agar containing kanamycin. The colony PCR and sequencing results confirmed the successful construction of AutolysinCBD-pET28a (Figure 5).

 

 

Figure 5 Construction results of AutolysinCBD-pET28a plasmid.

(A) Plasmid mapping. (B) Colony PCR results. (C) Sequencing results.

 

The plasmid was then transformed into E. coli BL21(DE3) for protein production. Cultures were grown to OD₆₀₀ 0.6-0.8 and induced with IPTG overnight. Cells were lysed by sonication and purified by Ni-affinity chromatography. SDS-PAGE analysis demonstrated successful expression and purification of AutolysinCBD at 15.3 KDa in Figure 6.

 

 

Figure 5 Expression and purification result of AutolysinCBD (the first track on right).

 

We also explored the catalytic reaction time and the TMB substrate concentration in the AutolysinCBD detection system. We diluted the bacterial suspension to 10⁶ CFU/mL and used different concentrations of TMB 0.01-0.05% (w/v) as the colorimetric substrate. The concentration of bacteria was determined at different times (0.5-2 h). The results showed that the optimal assay conditions were at 0.01% TMB for 1 h for AutolysinCBD (Figure 7A).

To test the ability of the system to BC, we diluted overnight bacterial cultures of BC to 10²-10⁷ CFU/mL using PBS, and then 1 mL of each concentration was taken for the assay. We performed curve fitting of the detection results, and the experimental results are shown in Figure 7B. For BC bacteria, the fitted curve of the detection was y = 0.1922x - 0.1994 (R² = 0.9423), The detection values showed a favorable linear relationship with the concentration of the bacteria.

 

 

Figure 7 Testing results of AutolysinCBD.

(A) Optimal reaction conditions for detection systems. (B) Testing results of different concentrations of BC.

 

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3. Hong Bin, Li Yanmei, Wang Wenhai, Ma Yi, Wang Jufang. Separation and colorimetric detection of Escherichia coli by phage tail fiber protein combined with nano-magnetic beads[J]. Microchimica Acta,2023,190(6).