We selected Lactobacillus rhamnosus (ATCC 7469) as our chassis microorganism. Upon receiving the strain, we activated it by culturing in MRS medium at 37°C and measured its growth curve. We set up three parallel experiments, manually measuring OD600 every hour for the first 8 hours after inoculation. Afterward, we transferred the original culture with an OD600 of 0.288 and continued measuring OD600 every hour for another 8 hours. The experimental data was used to fit a curve (Figure 1), demonstrating that the strain could grow normally.

Figure 1: Growth Curve of Lactobacillus rhamnosus (LGG)

Figure 2: Morphology of LGG Cells under 400× Microscope

After designing the working system, we optimized the required sequences for Lactobacillus rhamnosus and sent them to Genescript for synthesis. Once the corresponding fragments were obtained, we performed In-fusion seamless cloning based on the restriction enzyme sites reserved in the design. The ligation product was then transformed into E. coli for large-scale cultivation, and sufficient plasmids were extracted using a kit.

We used the plasmid pBS-(KS+) as the vector and inserted Glukiller and PT-acrp as the glucose operon to regulate the expression of the suicide gene MazF. The designed target fragment was linked to the vector plasmid through the Sac I restriction site at the 5' end and the Pst I restriction site at the 3' end, resulting in the construction of the suicide plasmid pBS-MazF (Figure 3), which carries an ampicillin resistance gene. After transforming Escherichia coli via heat shock, the transformants were verified by PCR (Figure 4). Colony 1 from Figure 4 was selected, and after amplifying a sufficient amount of the plasmid, it was electroporated into Lactobacillus rhamnosus, with the transformants shown in Figure5.

Figure 3 pBS-MazF plasmid

Figure 4 The PCR verification results of DH5α-pBS-MazF transformants

Figure 5 The PCR verification results of LGG+pBS-MazF transformants

Since we did not add a fluorescent protein tag, we used the most direct method, PCR. The primers used were the same as those for E. coli transformation (Primer7: TCCCATTCGCCATTCAGG; Primer8: ATCTGCTAAGGCAACACC). As shown in the figure, the electroporation efficiency was relatively low, and only transformants 6, 10, 11, 12, and 13 contained the target band. We selected transformants 6 and 13 as candidates. We conducted preliminary functional verification using transformant 13 and obtained surprising results, finding that the suicide system indeed works at relatively low glucose concentrations (Figure 6).

Figure 6 OD600 curves of engineering LGG and control group under different glucose concentrations.

We used LGG without the suicide plasmids as a control, testing 10 concentrations in each group, with three replicates per concentration. After culturing the bacteria to the logarithmic phase, 5 µL was transferred into test tubes containing 5 mL of MRS medium with different concentrations of glucose. The cultures were incubated in a constant temperature incubator at 37°C for 12 hours, and measurements were taken once for each concentration, as shown in Figure 6. Compared to the control group, the growth of LGG transformed with the glucose-sensitive suicide plasmid pBS-MazF was significantly inhibited. The lower the glucose concentration in the environment, the more pronounced the inhibitory effect, and the slower the growth and reproduction of LGG. At concentrations between 0.1 and 1.0 that we measured, the inhibition rate (ODLGG-ODLGGP/ODLGGP) was above 80%, and at lower concentrations, it was close to 100%. Based on this, we believe that in an in vitro fermentation environment where there is no glucose or the glucose concentration is very low, the glucose-sensitive death switch can function properly.

We fused the CotAGold gene with the GFP gene for co-expression and inserted them into the Sac I and Sal I sites of the plasmid vector pET-30(b+), resulting in the expression vector pCG (Figure 7), which carries a kanamycin resistance gene.

Figure 7 pCG plasmid

Figure 8. Validation of LGG transformants with pCG plasmids transformation.

Figure 9. LGG-pCG transformants

The plasmid verification method was the same as that for the suicide plasmid. After obtaining the correct plasmid, electroporation transformation of LGG was performed, and colony PCR verification was carried out (results shown in Figure 8). The bands in lanes 5, 8, 16, and 18 were slightly smaller than 1000 bp, which is close to the expected length of the correct pCG fragment (891 bp). We then selected the brightest band from lane 16 (Figure 9) for single colony preservation and used it to prepare competent cells for the subsequent transformation of the remaining 3 plasmids.

We used the paT7P-1 gene as the target gene, adding Sal I at the 5' end and Pst I at the 3' end, then ligated it into the plasmid vector pCDFDuetTM.1, resulting in the expression vector pVVD (Figure 10).

Figure 10 pVVD

Figure11 pVVD Plasmid restriction enzyme verification

From left to right, lane 1 shows pVVD, and in lane 2, the plasmid pVVD was double-digested with restriction enzymes EcoR I and Hind III, resulting in band lengths of approximately 2500 bp and 5000 bp, which are close to the theoretical fragment lengths after digestion of plasmid pVVD (2287 bp and 5027 bp). Therefore, the pVVD plasmid is correct.

Figure 12: Transformation of pVVD plasmid into E. coli for amplification.

We transformed pVVD into E. coli via heat shock. After the transformation, colony PCR verification was performed (results shown in Figure 9). All lanes showed bands of approximately 1000 bp, which is close to the expected length of the correct pVVD fragment (1041 bp). We then selected the brightest band from 10 lanes for single colony amplification and cultured it to obtain a sufficient amount of pVVD.

We linked the nanobody G8 and Gaussia luciferase using a linker and integrated them into a target gene. After adding Pst I at the 5' end and Sac I at the 3' end, the target gene was inserted into the plasmid vector pACYCDuet-1, resulting in the expression vector pGG (Figure 13), which carries chloramphenicol resistance.

Figure 13 pGG plasmid

1. The evaluation of the suicide part has been completed:

We established a glucose concentration gradient system and cultured for 12 hours, measuring the OD600 of the bacterial solution under different conditions to verify the functionality of the suicide part.

2. Next Steps:

Using the existing AFB1 detection kit, we plan to measure the AFB1 sensing sensitivity and degradation efficiency of the "Crasher" that successfully expresses the four component parts with a microplate reader and HPLC. Evaluate the safety of engineered LGG metabolites, including nutritional content and by-products of food.

We will continue to evaluate the performance of our part under different environmental conditions, aiming to improve its sensitivity, efficiency, and safety. The ultimate goal is to design a probiotic product that can be applied in industries such as grain, oil, and feed to remove AFB1.