Targeting module

1. Amplify the target gene fragment of commercially synthesized Lpp-OmpA-scFv by PCR. Identify PCR products by agarose gel electrophoresis. Purify PCR products.
2. Obtain the linearized vector by double digestion of vector pFPV25.1-GFP with endonucleases XbaI and MfeI.

shRNA silencing module

1. Prepare competent cells of S. typhimurium VNP20009.
2. Transfer the synthetic plasmids pSilence-SLC7A11 and VB230-shRNA-GFP into VNP20009 by electroporation.

GOx killing module

1. Amplify the target gene fragment of commercially synthesized SopE-FLAG-GOx by PCR. Identify PCR products by agarose gel electrophoresis. Purify PCR products.
2. Ligate the above PCR products with linearized vector pFPV25.1 by homologous recombination to obtain the vector pFPV25.1-SopE-FLAG-GOx.

Anaerobic expression module

1. Amplify the target fragment of nirB promoter (pnirB) from the genome of E. coli K12. Identify PCR products by agarose gel electrophoresis. Purify PCR products.
2. Ligate the above PCR product with linearized vector pFPV25.1 by homologous recombination to obtain the vector pFPV25.1-pnirB::HlyA-6×His.

Targeting module

1. Ligate PCR product Lpp-OmpA-scFv with linearized vector pFPV25.1-GFP by homologous recombination.
2. Transfer the vector pFPV25.1-[Lpp-OmpA-scFv]-GFP into E. coli by the chemical transformation method. Conduct colony PCR and Sanger sequencing.
3. Incubate the pFPV25.1-[Lpp-OmpA-scFv]-GFP plasmids with correct sequence in E. coli. Extract plasmids by alkaline lysis. Transfer the plasmids into VNP20009 by electroporation.

shRNA silencing module

Analyze the expression of gene HlyA in VNP20009 by conducting prokaryotic Western blot.

GOx killing module

1. Transfer the vector pFPV25.1-[SopE-FLAG-Gox] into E. coli by the chemical transformation method. Conduct colony PCR and Sanger sequencing.
2. Incubate the pFPV25.1-[SopE-FLAG-Gox] plasmids with correct sequence in E. coli. Extract plasmids by alkaline lysis. Transfer the plasmids into VNP20009 by electroporation.

Anaerobic expression module

1. Transfer the vector pFPV25.1-pnirB::HlyA-6×His into E. coli by the chemical transformation. Conduct colony PCR and Sanger sequencing.
2. Incubate the pFPV25.1-pnirB::HlyA-6×His plasmids with correct sequence in E. coli . Extract plasmids by alkaline lysis. Transfer the plasmids into VNP20009 by electroporation.

Targeting module

Analyze the expression of fusion protein Lpp-OmpA-scFv-GFP in VNP20009 by prokaryotic Western blot.

shRNA silencing module

1. Resuscitate and culture BGC-823 cells.
2. Transiently transfect eukaryotic expression vector of GFP into BGC-823.
3. Infect BGC-823 transfected with GFP by the VB230-shRNA-GFP engineered bacteria.

GOx killing module

1. Analyze the expression of fusion protein SopE-FLAG-GOx in VNP20009 by conducting prokaryotic Western blot.
2. Infect BGC-823 by the SopE-FLAG-GOx engineered bacteria (VNP-SopE-GOx).
3. Collect the cells after bacterial infection. Verify the engineered bacteria are able to inject FLAG-GOx into tumor cells via type III secretion system by conducting eukaryotic Western blot analysis.

Anaerobic expression module

1. Induce the expression of gene HlyA driven by the pnirB in the engineered bacteria.
2. Analyze the effect of expression by prokaryotic Western blot.

Targeting module

1. Resuscitate and culture NUGC-3 and BGC-823 cells.
2. Infect BGC-823 by the pFPV25.1-[Lpp-OmpA-scFv]-GFP engineered bacteria.
3. Infect NUGC-3 by the pFPV25.1-[Lpp-OmpA-scFv]-GFP engineered bacteria.

shRNA silencing module

1. Infect BGC-823 by the pSilence-SLC7A11 engineered bacteria (VNP-SLC7A11-shRNA).
2. Collect cells after bacterial infection. Extract total RNA from the cells. Synthesize cDNA using total RNA as templates.
3. Analyze the expression of gene SLC7A11.

GOx killing module

1. Analyze the expression of fusion protein SopE-FLAG-GOx in VNP20009 by conducting prokaryotic Western blot.
2. Infect BGC-823 by the SopE-FLAG-GOx engineered bacteria (VNP-SopE-GOx).
3. Collect the cells after bacterial infection. Verify the engineered bacteria are able to inject FLAG-GOx into tumor cells via type III secretion system by conducting eukaryotic Western Blot analysis.

Anaerobic expression module

1. Induce the expression of gene HlyA driven by the pnirB in the engineered bacteria.
2. Analyze the effect of expression by prokaryotic Western blot.

Safety module

1. Obtain the linearized vector by double-digestion of vector pGEN with endonucleases SalI and BamHI.
2. Amplify the target fragment prpsM::tetR from the commercially synthesized pFPV25.1-tetR by PCR. Identify PCR products by agarose gel electrophoresis. Purify PCR products.
3. Ligate the target fragment prpsM::tetR with the linearized pGEN vector by homologous recombination to obtain the vector pGEN-prpsM::tetR.
4. Amplify the target fragment prpsM::RFP from the commercially synthesized pFPV25.1-RFP by PCR. Identify PCR products by agarose gel electrophoresis. Purify PCR products.
5. Obtain the linearized vector by double-digestion of vector pET-T7::Hok-Sok-LacI with endonucleases SphI and BamHI.
6. Ligate PCR product prpsM::RFP with the linearized vector pET-T7::Hok-Sok-LacI by homologous recombination to obtain the vector pET-RFP-LacI.

Targeting module

1. Transfer the plasmids pFPV25.1-RFP into VNP20009 by electroporation.
2. Stably transfect the plasmid carrying GFP into VNP20009 .
3. Transfer the plasmids pFPV25.1-[Lpp-OmpA-scFv]-GFP into VNP20009 transfected with GFP by the electric shock method.

shRNA silencing module

1. Infect BGC-823 by the pSilence-SLC7A11 engineered bacteria (VNP-SLC7A11-shRNA).
2. Collect cells after bacterial infection. Extract total RNA from the cells. Synthesize cDNA using total RNA as templates.
3. Analyze the expression of gene SLC7A11.

GOx killing module

1. Infect BGC-823 by VNP-SopE-GOx.
2. Detect cell viability after bacterial infection by using the CCK-8 reagent.

Safety module

1. Detect the plasmid loss rate without selection pressure by culturing the bacteria with pGEN and pET-RFP-LacI.
2. Verify the effect of the toxin Hok using the bacteria with pET-T7::Hok-Sok-LacI.
3. Amplify the target gene GFP from pFPV25.1-GFP by PCR. Identify PCR products by agarose gel electrophoresis. Purify PCR products.
4. Obtain the linearized vector of vector pET-T7::Hok-Sok-LacI by reverse PCR.
5. Connect PCR product GFP with linearized vector pET-T7::Hok-Sok-LacI by homologous recombination to obtain the vector pET-GFP-LacI.
6. Transfer the vector pET-GFP-LacI into E. coli by chemical transformation. Conduct colony PCR and Sanger sequencing.
7. Transfer the vector pJKR-L-tetR commercially synthesized into E. coli by the chemical transformation method. Conduct colony PCR and Sanger sequencing.

Targeting module

1. Infect BGC-823 by the pFPV25.1-Lpp-OmpA-scFv-GFP engineered bacteria transfected with GFP (VNP-Lpp-OmpA-scFv-GFP).
2. Infect NUGC-3 by VNP-Lpp-OmpA-scFv-GFP.
3. Infect BGC-823 by pFPV25.1-RFP bacteria (VNP-RFP).
4. Infect NUGC-3 by VNP-RFP.
5. Infect BGC-823 by VNP-Lpp-OmpA-scFv-GFP and VNP-RFP.
6. Infect NUGC-3 by VNP-Lpp-OmpA-scFv-GFP and VNP-RFP.

shRNA silencing module

1. Infect BGC-823 by VNP-SLC7A11-shRNA.
2. Detect cell viability after bacterial infection using the CCK-8 reagent.

GOx killing module

1. Add purified GOx to the BGC-823 cells.
2. Collect cells. Detect lipid peroxide level in tumor cells.

Safety module

1. Verify the working effect of the lac operator by adding IPTG when culture the bacteria with pET-GFP-LacI.
2. Verify the working effect of the pLtetO by adding Dox when culture the bacteria with pJKR-L-tetR.

shRNA silencing module

1. Infect BGC-823 by VNP-SLC7A11-shRNA.
2. Detect glutathione level in tumor cells after bacterial infection.

Safety module

1. Amplify the target fragment trp promoter by PCR. Identify PCR products by agarose gel electrophoresis. Purify PCR products.
2. Obtain the linearized vector pET-T7::Hok-Sok-LacI by removing the T7 promoter by reverse PCR.
3. Ligate the PCR product trp promoter with linearized vector pET-T7::Hok-Sok-LacI by homologous recombination to obtain the vector pET-trp::Hok-Sok-LacI.
4. Transfer the vector pET-trp::Hok-Sok-LacI into E. coli by the chemical transformation. Conduct colony PCR and Sanger sequencing.
5. Verify the effect of the toxin Hok using the bacteria with pET-trp::Hok-Sok-LacI.