1.Verification of the compatibility between engineered bacteria and experimental conditions
experimental conditions
The colony counting results show that there is no significant difference in the colony-forming units (CFU) per milliliter between the chassis bacteria and the engineered bacteria after 12 hours of cultivation. Additionally, we measured the bacterial growth curve (calculated by OD values), which indicates that the growth curves of the chassis bacteria and the engineered bacteria are essentially overlapping.
The results of the bacterial activity assay (CCK-8) indicate that there is no significant difference in activity between the chassis bacteria and the engineered bacteria after 12 hours of cultivation
2. Detection of target gene expression
First, we used the kanamycin resistance gene carried by the recombinant plasmid for selection to verify whether the recombinant plasmid was successfully introduced into the chassis bacteria and expressed. The results show that the chassis bacteria did not grow at kanamycin concentrations greater than 100 times the minimum inhibitory concentration, while the engineered bacteria grew normally.
We used the Western blot method to detect the expression of the recombinant protein Flab (tagged with Flag×3) and hyaluronidase (tagged with His×5). The results indicate that the engineered bacteria successfully expressed both proteins. The molecular weight of Flab is between 13-15 kDa, while the molecular weight of hyaluronidase is between 14-15 kDa, which is consistent with our expectations.
3. Hyaluronidase biological activity assay
The results of the extracellular matrix-like hydrogel degradation experiment show that incubating the hydrogel with the engineered bacteria culture supernatant for 48 hours leads to a reduction in dry weight to below 50% of the initial mass, with complete morphological collapse, indicating that the engineered bacteria culture supernatant has the ability to hydrolyze extracellular matrix. Time-course ELISA results show that the secretion of hyaluronidase by the engineered bacteria gradually increases and stabilizes within 12 hours after inoculation into the medium.
The results from the hyaluronidase activity detection kit indicate that the activity of hyaluronidase in the bacterial supernatant corresponds to a concentration of approximately 40-50 µg/ml of commercial hyaluronidase.
4. Flab immunogenicity effect test
The results show that, compared to the chassis bacteria, co-culturing the engineered bacteria with mouse macrophages significantly increased the expression of iNOS protein in the macrophages, indicating that the macrophages were polarized and transformed into the M1 anti-tumor phenotype, which aligns with our expectations.
