Our experiments aimed to construct attenuated Salmonella and recombinant plasmids to precisely target tumor tissues and exert anti-tumor effects.

In addition, we completed a reference manual on microbial-mediated tumor therapy in synthetic biology with BNUZH-China, Guangxi-U-China, PekingHSC, Peking, and Tsinghua. Our goal is to provide practical and meaningful solutions to common challenges faced by iGEM teams working on future projects for microbiome-mediated therapeutics in tumors, including safety, ethics, and engineering design. Our work is expected to provide a new understanding of bacterial treatment of tumors and propose a new strategy for co-silencing in iGEM.

Cell line culture is the foundation. We performed a series of experiments such as CT26 cell resuscitation, cell exchange, cell passage, cell cryopreservation, and so on. After that, the recombinant plasmid was transfected into murine colon cancer CT26 cell line to verify the recombinant plasmid in vitro. The recombinant plasmids added were shControl plasmid, shSTAT3 plasmid, shPD-L1 plasmid, and shSTAT3/shPD-L1 recombinant plasmid.

Then the mRNA expression was detected by qPCR, the concentration of RNA and cDNA was detected, the cell protein was extracted and collected, and the protein concentration was determined. The protein concentration standard curve was drawn according to the protein standard value, and the concentration of the sample to be tested was calculated. The expression of the target protein was detected by Western blotting. We used CCK-8 to detect cell proliferation and soft agar colony formation assay to detect cell proliferation ability.

We then constructed recombinant suicide plasmids, including asnB-pRE112 and PtrcflaB-asnB-pRE112, and selected them by PCR with single exchange between the suicide plasmid homology arm and the genomic target gene or double exchange of the suicide plasmid homology arm. The recombinant plasmid was identified, including bacterial activation, Escherichia coli χ7213 competent cell preparation, plasmid extraction, restriction enzyme recovery, ligation, transformation, enzyme digestion and PCR identification.

After plasmid construction, we constructed and identified the ΔasnBχ11802 attenuated Salmonella engineering strain. The χ7213 E. coli (asnB-pRE112) was used as the donor strain and Salmonella χ11802 as the recipient strain for plasmid transfer. The ΔasnB χ11802 attenuated Salmonella strain was screened and identified as Salmonella when agglutination occurred. Then, the lines were respectively delimited on LB plate (containing arabinose and DAP) and chloramphenicol plate (containing arabinose and DAP). PCR performed identification of recombinant strains.

Then χ7213 E. coli (PtrcflaB-asnB-pRE112) was used as the donor strain and Salmonella ΔasnB χ11802 strain as the recipient strain to construct the ΔasnB:: PtrcflaB χ11802 attenuated Salmonella engineering strain. The method was the same as above. The selected strains were identified by PCR with primers asnB-F and asnB-R to determine whether the amplified fragments increased in size, and then specific PCR was performed with primers flaB-F and flaB-R. Thus, the flaB gene was introduced into the strain to enhance the invasion of tumor cells and elicit immunocompetent responses.

The cell structure was observed under the transmission electron microscope, the growth curve of the attenuated Salmonella mutant strain was determined, the migration ability of the attenuated Salmonella mutant strain was determined by observing the diameter of the colony, the biofilm formation ability of the attenuated Salmonella mutant strain was selected, and the sensitivity of the attenuated Salmonella mutant strain to polymyxin B was determined. The adhesion and invasion of mutant attenuated Salmonella to epithelial cells was then determined.

Preparation of electroconversion receptive cells for electroconversion and seed preservation and electroconversion of attenuated Salmonella χ11802 mutant strains. Then the attenuated Salmonella solution after electroporation was sent to Sangon Biotechnology Company for sequencing to detect the presence of the target gene fragment.

Finally, the biological function of Salmonella containing the recombinant plasmid was tested, including flow cytometry to detect cell apoptosis: Annexin V-FITC/PI apoptosis detection kit was used to detect cell apoptosis. Transwell assay was used to detect cell migration ability: OD value (570 nm) was measured on a microplate reader to reflect the difference in the number of migrating cells between groups. Cell scratch assay was used to detect cell migration ability: 24 hours after transfection, scratch healing was observed. Flow cytometry was used to detect cell cycle changes, and finally statistical analysis was performed.

1.Experimental cell lines

The mouse colon cancer CT26 cell line and human intestinal epithelial cell INT407 were both preserved in our laboratory.

2.Plasmids and strains

The shControl plasmid, shSTAT3 plasmid, shPD-L1 plasmid, and shSTAT3/shPD-L1 recombinant plasmid containing PLKO skeleton were purchased from Saixin Biological.

χ7213 Escherichia coli, χSalmonella11802, pRE112 suicide plasmid (CmR), and asd-STAT3-shRNA plasmids were all preserved in our laboratory. According to the published sequence of Salmonella asnB gene (ID: 1252200) and Vibrio vulnificus flagellar protein flaB (ID: WP_017790082.1) on GenBank. Primers asnB-F/R and flaB-F/R were used to amplify homologous arm fragments and flaB sequence fragments of the asnB sequence, synthesized by Bioengineering (Shanghai) Co., Ltd. The plasmids asnB-arms-pUC57 (AmpR) containing homologous arm fragments of the asnB gene and PtrcflaB-pUC57 (AmpR) containing flaB gene fragments were synthesized by Nanjing Jinsrui Company.

The shRNA sequence information is as follows:

shSTAT3 (5'-GCAGCAGCTGAACAACATG-3')

shSTAT3 (shPD-L1 (5'-CCGAAATGATACACAATTCGA-3'))