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Notebook
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Our project explores the groundbreaking potential of genetically engineered Salmonella in cancer therapy. By selectively targeting tumor cells and inducing apoptosis, we aim to create a more effective and less invasive treatment option.
Engineered Salmonella Beat Cancer
Experiment Record
24.04.27
Reagent Preparation
  1. Prepare 1 L SOC medium, among which is 5×100 mL SOC medium.
  2. Prepare 200 mL 10 mM HEPES (0.47 g HEPES with 1 L ddH2O), neutralized by 10 mM KOH, to pH 7.0.
  3. Prepare 50 mL 10% glycerol.
  4. Sterilize at 121℃ for 20 minutes.
  5. Prepare plates (SOC medium + Ampicillin (100 μg/mL) + arabinose): 0.05% arabinose ×10 plates, 0.2% arabinose ×10 plates.
  6. Culture 2 tubes of E.coli and 1 tube of negative control.
5.30
pGLO Plasmid Transformation
Protocol: Beyotime DH5α Super Competent Cells
Modification:
  1. At step 2, 0.5 μL pGLO was added into 100 μL DH5α and 100 μL E.coli Nissle. Incubation: 2 LB1917.
  2. At step 3, the time for 42℃ heating is 90 seconds.
  3. At step 4, 500 μL LB was added and was recovered i7,29.
  4. At step 5, 300 μL culture was plated.
7.16
pTD103 (luxI-sfGFP) Amplification
Protocol: Offered by Beyotime DH5α Super Competent Cells
Modification:
  1. At step 2, 2 μL plasmid was mixed with 100 μL competent cells and ice bathed for 20 minutes.
  2. At step 3, the mixture was heated at 42℃ for 90 seconds.
  3. At step 4, 500 μL antibiotic-free LB medium was added and then recovered in a 220 rpm shaker for 30 minutes.
  4. At step 5, centrifuge at 4000 rpm for 1 minute.
Plate: Kan+ LB
7.17
Single Colony Pick
Pick single colony from the pTD103 transformed competent DH5α bacteria colony and incubate.
7.18
pTD103 (luxI-sfGFP) Plasmid Extraction
Protocol: Beyotime Plasmid Mini Preparation Kit
7.22
pYX00004 (pBAD202-mCherry-2aa) Plasmid Extraction
Protocol: Offered in Beyotime Plasmid Mini Preparation Kit
Modification: In step 9, solution Ⅴ was replaced with ddH2O.
7.23
lux System and lyseE PCR
7.24
Purification of lyseE and GFP Genes
lyseE gene
Protocol: Protocol A offered in Thermo Scientific GeneJET PCR Purification Kit
Modification: 50 μL elution buffer was replaced with 20 μL ddH2O.
GFP gene
Protocol: Protocol A offered in Thermo Scientific GeneJET PCR Purification Kit (Group 6)
Protocol offered in TIANGEN TIANgel Mini Purification Kit (Group 7~10)
Modification: EB buffer was replaced with 30 μL 65℃ pre-warmed ddH2O.
7.25
Bax Gene PCR
CD513B-NLS-mCherry digestion: 37℃, 1 h
1% agarose gel electrophoresis:
7.26
Purification and Digestion of BAX Gene and CD513B-NLS-mCherry
Protocol: Offered in TIANGEN TIANgel Midi Purification Kit
Modification: At step 8, 40 μL EB buffer was added into each column
BAX Gene Fragment Digestion: 37℃, 1.5 h
CD513B-NLS-mCherry Digestion: 37℃, 1.5 h
1% agarose gel electrophoresis
Purification of BAX Gene Fragment and Digested CD513B-NLS-mCherry
Protocol: Offered in TIANGEN TIANgel Midi Purification Kit
Modification: At step 8, 40 μL EB buffer was added into each column
BAX Gene Fragment and Digested CD513B-NLS-mCherry Ligation
Ligation reaction: Room temperature, 2 h
Competent DH5α Cells Preparation
Protocol: Offered in Beyotime DH5α Super Competent Cells
Plate all culture media onto LB medium and cultivate overnight at 37℃
7.29
DH5α-CD513B-Bax Competent Cells Preparation
  1. Prepare 50 mL culture medium of LB + Ampicillin in a ratio of 1000:1.
  2. Transfer 500 μL culture medium into 8 centrifuge tubes.
  3. Pick the colony into 8 tubes.
  4. Transfer the tubes into shaker (37℃, 220 rpm).
DH5α-CD513B-Bax PCR
mCherry Digestion
37℃, 1 h
7.30
Purification of Lpp-OmpA Plasmid
B16-F10 Cell Thawing
  1. Gentle agitation in 37℃ water bath for 2 minutes to thaw.
  2. Remove the cryopreservation tubes from the water bath as soon as thawed.
  3. Decontaminate with 75% ethanol.
  4. Centrifuge with 1 mL medium, 500 g, 5 minutes.
  5. Discard the supernatant.
  6. Resuspend in 10% FBS DMEM and dispense into 10 cm² dish.
  7. Incubation at 37℃, 5% CO₂.
7.31
DH5α-CD513B-Bax Competent Cells Storage
80% sterilized glycerol: bacteria culture = 1:1. Store at -80℃.
DH5α-CD513B-Bax Plasmid Amplification
10 μL bacteria culture + 5 mL LB + Ampicillin medium. 37℃ overnight.
Purification of Lpp-OmpA
Protocol: Offered in TIANGEN TIANgel Midi Purification Kit
Modification:
  1. At step 7, the column was put in the fume hood to allow ethanol to evaporate completely.
  2. At step 8, EB buffer was replaced with 30 μL ddH2O.
8.1
CD513B-Bax Plasmid Extraction
Protocol: offered in TIANGEN Endo Free Mini Plasmid KitⅡ
Modification:
  1. At step 2, we centrifuged 10 mL culture at 4000 rpm for 6 min.
  2. At step 5, the solutions were centrifuged twice.
  3. At step 13, the solutions were centrifuged for 2 min.
Concentration: tube 1: 87.900 ng/μL; tube 2: 112.80 ng/μL (The concentrations were too low for following experiments).
DH5α-CD513B-Bax Plasmid Amplification
10 μL bacteria culture + 5 mL LB + Amp medium
Incubate at 37℃ overnight.
PCR Amplification of Non-OmpA, Lpp-2-OmpA, and BPK764 Lpp-1-OmpA Fragments
  1. Centrifuge chemically synthesized Non-OmpA, Lpp-2-OmpA, BPK764 Lpp-1-OmpA-scFv at 5,000 ×g.
  2. Add 20 μL ddH2O to chemically synthesized Non-OmpA (500 ng), Lpp-2-OmpA (500 ng), BPK764_Lpp-1-OmpA-scFv (2 μg).
  3. PCR Non-OmpA with NcoI-forward-2 and EcoRI-backward-2 (50 ng template), Lpp-2-OmpA with NcoI-forward-3 and EcoRI-backward-3 (50 ng template).
8.2
100 bp Lpp-OmpA Purification
Protocol: Protocol A of Thermo Scientific GeneJET PCR Purification Kit (250 preps)
Modifications:
  1. At step 1, the volume of each component: 50 μL PCR mixture + 50 μL Binding Buffer + 50 μL isopropanol.
  2. At step 6, the empty column was placed in a fume hood to allow ethanol to evaporate completely.
  3. At step 6, 50 μL Elution Buffer was replaced with 20 μL ddH2O.
mCherry Plasmid Extraction
Protocol: Offered in Beyotime Plasmid Mini Preparation Kit
Modification: At step 9, Solution V was replaced with 50 μL ddH2O.
Concentration was lost (unfortunately).
PsseJ Promoter PCR
PCR and Electrophoresis of BPK764-Vector and Lpp-OmpA Construction:
  1. PCR BPK764-vector with BPK764-1 and BPK764-2 (200 ng template).
  2. Perform electrophoresis of BPK764-vector, Lpp-OmpA-2, Non-OmpA.
CD513B-Bax Plasmid Extraction
Protocol: Offered in TIANGEN Endo Free Mini Plasmid KitⅡ
Modification: At step 2, we centrifuged 5 mL culture at 4000 rpm for 6 min.
8.3
Inoculation of DH5α
  1. Inoculate DH5α (BPK764_Lpp-1-OmpA-HER2_scFv) into 5 mL LB (Chl+).
  2. DH5α-CD513B-Bax plasmid amplification: 10 μL bacteria culture + 5 mL LB + Amp medium.
  3. Incubate at 37℃ overnight.
8.4
BPK764_Lpp-1-OmpA-HER2_scFv Extraction
Extract plasmids from DH5α (BPK764_Lpp-1-OmpA-HER2_scFv) using the Plasmid Miniprep Kit (Beyotime).
CD513B-Bax Plasmid Extraction
Protocol: Offered in TIANGEN Endo Free Mini Plasmid KitⅡ
Modification: At step 2, we centrifuged 5 mL culture at 4000 rpm for 6 min.
Performed 1.5% agarose gel electrophoresis.
8.5
Lpp-OmpA-HER2 Plasmid Extraction
Bacteria OD: late 2.870; early 3.435
Protocol: Offered in Beyotime Plasmid Mini Preparation Kit
Modification: At step 1, we transferred a total of 4.5 mL culture.
mCherry Digestion
Incubate at 37℃ for 1 h.
1% Agarose Gel Electrophoresis
PCR and Gel Electrophoresis of BPK764-Vector Amplification and DNA Extraction
  1. PCR BPK764-vector with BPK764-1 and BPK764-2 (200 ng template).
  2. Gel electrophoresis and extraction with DNA Gel Extraction kit (Beyotime).
  3. 1.5% agarose gel electrophoresis.
Lpp-OmpA-HER2 Plasmid Extraction
Bacteria OD: late 2.870; early 3.435
Protocol: Offered in Beyotime Plasmid Mini Preparation Kit
Modification: At step 1, we transferred a total of 4.5 mL culture.
B16-F10 Cell Subculture (P2)
  1. Remove the medium. Rinse the cell layer with 1.0 mL PBS.
  2. Add 1.0 mL 0.25% Trypsin-0.53 mM EDTA. Wait until dispersed.
  3. Add 1.0 mL medium to stop digestion.
  4. Centrifuge at 500 g for 5 minutes and discard the supernatant, then resuspend in 1 mL medium.
  5. Add 0.25 mL cell suspension into each 10 cm² dish, then add 10.0 mL medium to each dish.
  6. Incubate the 4 dishes.
8.6
PCR Amplification and Cloning of BPK764, Non-OmpA, and PsseJ Genes in DH5α Cells
  1. PCR BPK764-vector with BPK764-1 and BPK764-2 (200 ng template).
  2. PCR Non-OmpA with NcoI-forward-2 and EcoRI-backward-2 (50 ng template), Lpp-2-OmpA with NcoI-forward-3 and EcoRI-backward-3 (50 ng template).
  3. PCR PsseJ with PsseJ-1 and PsseJ-2 (50 ng template).
  4. Inoculate DH5α (BPK764_Lpp-1-OmpA-HER2_scFv).
  5. 1.5% agarose gel electrophoresis.
8.7
BPK764_Lpp-1-OmpA-HER2_scFv Extraction
  1. Extract plasmids from DH5α (BPK764_Lpp-1-OmpA-HER2_scFv) with Plasmid Miniprep Kit (Beyotime).
  2. 1.5% agarose gel electrophoresis.
pYX00004 Plasmid Extraction and Restriction Enzyme Cutting Site Test
1.5% agarose gel electrophoresis.
CD513B-SV40 Plasmid Transformation
Protocol: offered in Beyotime DH5α Super Competent Cells
Modification:
  1. At step 4, the cell was recovered for 30min.
  2. At step 5, 300μl culture was taken for plating and another 200μl was mixed with 5ml LB+Amp medium and put in a 37℃ shaker.
CD513B-SV40 Plasmid Extraction
Protocol: offered in TIANGEN Endo Free Mini Plasmid KitⅡ
Modification:
  1. At step 2, centrifuge 5ml culture at 4000rpm for 6min.
8.8
Verification of BPK764-Vector and PsseJ
  1. Gel electrophoresis and extraction of BPK764-vector, PsseJ with DNA Gel Extraction kit (Beyotime).
  2. Inoculate DH5α (BPK764_Lpp-1-OmpA-HER2_scFv).
  3. PCR BPK764-vector with BPK764-1 and BPK764-2 (200 ng template).
  4. 1.5% agarose gel electrophoresis.
DH5α-CD513B-Bax Plasmid Amplification
10μl bacteria culture + 5ml LB+Amp medium
37℃ overnight
CD513B-SV40 Plasmid Extraction
Protocol: offered in TIANGEN Endo Free Mini Plasmid KitⅡ
Modification:
  1. At step 2, centrifuge 5ml culture in 4000rpm for 6min.
8.9
pYX00004 Plasmid PCR
1.5% agarose gel electrophoresis.
Plasmid Extraction, Gel Electrophoresis, and Restriction Digestion of scFv Part
  1. Extract plasmids from DH5α (BPK764_Lpp-1-OmpA-HER2_scFv) with TIANprep Mini Plasmid Kit (TIANGEN).
  2. Gel electrophoresis and extraction of Non-OmpA, Lpp-2-OmpA, and BPK764-vector.
  3. Digest BPK764, Lpp-2-OmpA, Non-OmpA with EcoRI-HF, NcoI-HF.
B16-F10 Cell Substrating (P3) and Cryopreservation
  1. Refer to the substrating to 8.5 for future use.
  2. Prepare the cryoprotectant as 60% DMEM + 30% FBS + 10% DMSO.
  3. Remove the medium and rinse the cell layer with 1mL PBS to wash off the trypsin inhibitor.
  4. Add 1.0mL 0.25% Trypsin-0.53mM EDTA to digest off the cell. Then add 1.0mL medium to stop digesting.
  5. Centrifuge at 500g for 5 minutes and discard the supernatant, then resuspend in 2mL cryoprotectant.
  6. Put the cryopreservation tubes in a cell-freezing container, then put the container at -80°C.
8.10
BPK764, Lpp-2-OmpA, Non-OmpA Gel Electrophoresis and Extraction
Gel electrophoresis of digestion products (BPK764, Lpp-2-OmpA, Non-OmpA) and extract them with TIANgel Midi Purification Kit (TIANGEN).
8.11
pYX00004 Plasmid Purification
Protocol: TIANGEN TIANgel Mini Purification Kit
Modification: Do not wait for 2min at step 5.
Lpp-OmpA-HER2_scfv PCR
Purification of scFv Plasmid:
Purify digestion products BPK764, Lpp-2-OmpA, Non-OmpA with GeneJET PCR Purification Kit (Thermo Scientific)
pYX00004 Digestion
37℃, 2h
Preliminary Experiment of CD513B-BAX Cell Transfection:
  1. Seed the B16-F10 cells in a 12-well plate one day before.
  2. Transfection Reagent Preparation Protocol: YEASEN Hieff Trams Liposomal Transfection Reagent (diluted by DMEM or 1640).
  3. Incubation: 37℃, 5% CO₂.
  4. Observe fluorescence signal and cell death condition every 6h/12h.
8.12
Construction of HER2_scFv
  1. PCR HER2_scFv with HER2_scFv-EcoRI and HER2_scFv-XhoI (100 ng template).
  2. Digest HER2_scFv (1.6 μg).
B16-F10 Culture Renew
Refer to the experiment on 8.2.
8.13
Ligation and Transformation of scFv Plasmids
  1. Ligate: BPK764 + Lpp-2-OmpA, BPK764 + Non-OmpA.
  2. Transform the ligation products to Top10 super-competent cells (Beyotime).
  3. Plate all competent cells to LB(Chl+) plates, and incubate at 37℃ overnight.
pYX00004 Plasmid PCR: 1% agarose gel electrophoresis.
1% agarose gel electrophoresis
LuxR-LuxI-lyseE digestion.
8.14
PCR and Purification of Lpp-2-OmpA and Non-OmpA Fragments for BPK764 Vector Construction
  1. PCR Lpp-2-OmpA with XXX-OmpA-1 and Lpp-OmpA(BBa_K103006)-2, and PCR Non-OmpA-2 with XXX-OmpA-1 and Non-OmpA-2.
  2. Purify PCR products Lpp-2-OmpA-T and Non-OmpA-T with GeneJET PCR Purification Kit (Thermo Scientific).
  3. PCR BPK764-vector with Lpp-2-OmpA-T, and PCR BPK764-vector with Non-OmpA-T.
pYX00004 Plasmid Purification: The same as 8.11.
Mcherry Digestion: 37℃, 3h.
1% agarose gel electrophoresis 40min.
DH5α-CD513B-Bax Plasmid Amplification: 10μl bacteria culture + 5ml LB+Amp medium, 37℃ overnight.
8.15
Gel Electrophoresis and Digestion of scFv Plasmids
  1. 1% agarose gel electrophoresis of HER2_scFv, BPK764-Non-OmpA, and BPK764-Lpp-2-OmpA.
  2. Digest BPK764-vector with NcoI-HF and EcoRI-HF, and digest HER2_scFv with XhoI and NdeI.
Mcherry and PsseJ Digestion: 1% agarose gel electrophoresis.
DNA extraction: Protocol: offered in TINGEN TIANgel Midi Purification Kit.
CD513B-SV40 and CD513B-Bax Plasmid Extraction
Protocol: offered in TIANGEN EndoFree Mini Plasmid KitⅡ.
Modification: At step 2, centrifuge 5ml culture at 4000rpm for 6min.
8.16
Ligation and Transformation of scFv Plasmids into Competent Cells
  1. Ligation: HER2_scFv + pET28(a), Non-OmpA + BPK764-vector, Lpp-2-OmpA + BPK764-vector.
  2. Transform pET28(a)-HER2_scFv, BPK764-Non-OmpA, BPK764-Lpp-2-OmpA to Top10 super-competent cells (Beyotime) and DH5α competent cells (CWBIO).
  3. Plate all competent cells to LB (Kana+ for pET28(a) and Chl+ for BPK764) plates, and incubate at 37℃ overnight.
pYX00004 and PsseJ promoter Ligation
16℃
8.18
Co-transformation and Incubation of BPK764 and pET28(a) Plasmids in BL21 Cells
  1. Inoculate cells with BPK764-Non-OmpA (Homologous recombination), BPK764-Non-OmpA (Ligation), BPK764-Lpp-2-OmpA (Homologous recombination), BPK764-Lpp-2-OmpA (Ligation) to LB (Chl+) liquid medium, and inoculate cells with pET28(a)-HER2_scFv to LB (Kana+) liquid medium, and incubate at 37℃, 220 rpm overnight.
  2. Co-transform pLH001 and BPK764-Lpp-1-OmpA to BL21 competent cells (Vanzyme) and transform BPK764-Lpp-1-OmpA to BL21 competent cells (Vanzyme).
  3. Plate BL21 with pLH001 and BPK764-Lpp-1-OmpA to LB(Kana+, Chl+) plate, plate BL21 with BPK764-Lpp-1-OmpA to LB(Chl+) plate and incubate at 37℃ overnight.
8.19
Sequencing and Transformation of BPK764 and pET28(a) Plasmids in BL21 Cells
  1. Inoculate BL21 cell with pLH001 and BPK764-Lpp-1-OmpA to LB (Kana+, Chl+) liquid medium, and incubate at 37℃, 220 rpm overnight.
  2. Sequence BPK764-Non-OmpA (Homologous recombination), BPK764-Non-OmpA (Ligation), BPK764-Lpp-2-OmpA (Homologous recombination), BPK764-Lpp-2-OmpA (Ligation), pET28(a)-HER2_scFv (Done by Tsingke).
  3. Extract BPK764-Lpp-2-OmpA (with successful and correct result of sequencing) from Top10 super-competent cells with TIANprep Mini Plasmid Kit (TIANGEN).
  4. Transform pET28(a)-HER2_scFv to BL21 competent cells (Vanzyme).
  5. Plate BL21 with pET28(a)-HER2_scFv to LB(Kana+) plate, and incubate at 37℃ overnight.
Preliminary Experiment of CD513B-SV40 and CD513B-BAX Cell Transfection:
  1. Seed the B16-F10 cells one day before.
  2. Transfection Reagent Preparation Protocol: YEASEN Hieff Trams Liposomal Transfection Reagent (use DMEM or 1640 for dilution).
  3. Incubation: 37℃, 5% CO₂.
  4. Observe fluorescence signal after 24h.
8.20
Induction and Expression of pET28(a) in BL21 Cells
  1. Inoculate BL21 with pET28(a) to 600 mL LB(Kana+) liquid medium, and incubate at 37℃, 220 rpm.
  2. Add IPTG (0.1 mM as final concentration) to the culture after 3.5 hours, and incubate at 16℃, 220 rpm overnight.
Non-OmpA-HER2_scfv, lyseE and GFP Gene Fragments PCR and Purification
PCR product purification:
Protocol: GeneJET PCR Purification Kit
Modification: 50 μl elution buffer was replaced with 20μl ddH2O.
8.21
Non-OmpA-HER2_scfv, lyseE and GFP Gene Fragments Extraction
  1. 1.5% agarose gel electrophoresis of the DNA fragments from 8.20.
  2. Extract DNA from the gel.
Protocol: TINGEN TIANgel Midi Purification Kit
Plasmid Extraction from BL21 Cells: Extract plasmid from the BL21 cells (LuxI, sfGFP, pET-HER2, scFv).
Extraction and Expression of scFv Protein & Digestion of Non-OmpA and pYX00004
  1. Do protein purification of HER2_scFv-6xHis.
  2. Digest Non-OmpA with EcoRI-HF and NcoI-HF overnight at 37℃, and digest pYX00004 with NsiI and amHI overnight at 37℃.
  3. Inoculate BL21 with BPK764-Lpp-1-OmpA to 600 mL LB(Chl+) liquid medium, and incubate at 37℃, 220 rpm.
  4. Add IPTG (0.1 mM as final concentration) to the culture after 3.5 hours, and incubate at 16℃, 220 rpm overnight.
8.22
PCR Purification, Ligation, Homogenous Recombination of Non-OmpA, PsseJ-mCherry, and pTD103-lyseE and Protein Purification of Lpp-1-0mpA-HER2_SCFV.6xHis
  1. Purify PCR products of pTD103-vector Non-OmpA-T, lyseE-T, and digestion product of Non-OmpA with GeneJET PCR Purification Kit (Thermo Scientific).
  2. Ligate Non-OmpA+BPK764.
  3. PCR Non-OmpA-T with XXX-OmpA-1 and Non-OmpA-2.
  4. Purify PCR products of Non-OmpA-T with GeneJET PCR Purification Kit (Thermo Scientific).
  5. PCR Non-OmpA-T with BPK764-vector, PsseJ with pYX00004-vector, BPK764-vector and pTD103-T with pTD103-Forward and pTD103-Reverse.
  6. Do protein purification of Lpp-1-OmpA-HER2_scFv-6xHis.
8.23
Transformation of Recombinant Construction
  1. Transform Homogeneous Recombination product Non-OmpA-T in BPK764, PsseJ-mCherry in pYX00004, and lyseE in pTD103-Forward to DH5α competent cell (Vanzyme).
  2. Transform Ligation product Non-OmpA in BPK764 to DH5α competent cell (Vanzyme).
  3. Plate transformed DH5α to LB (Chl+ or Kana+) plate, and incubate at 37℃ overnight.
8.24
Inoculation of DH5α Competent Cells
Inoculate DH5α with BPK764 (Non-OmpA-T), DH5α with pYX00004 (PsseJ-mCherry), and DH5α with pTD103-luxI (lyseE) to 2ml LB (Chl+ or Kana+) culture medium, and incubate at 37℃, 220 rpm overnight.
8.25
Sequencing
Sequence BPK764 (Non-OmpA-T), pYX00004 (PsseJ-mCherry), pTD103-luxI (lyseE). (Done by Tsingke)
8.30
Amplification, Purification, Digestion and Ligation of lyseE and pYX00004 and Transformation
  1. Using protocol from TIANGEN, plasmid pYX00004 (PsseJ-mCherry) was extracted from bacteria.
  2. Inverse PCR of pYX00004 plasmid and PCR of lyseE.
  3. 1% agarose gel electrophoresis of amplified pYX00004.
  4. Using protocol from TIANgel Midi Purification Kit (Spin Column), extract plasmids from gel.
  5. Digestion of lyseE.
  6. Using protocol A from Thermo Scientific GeneJET PCR Purification Kit.
  7. Ligation at 37℃ for 2h.
  8. Transform pYX00004-lyseE into DH5α competent cell.
Procedure:
  1. Place the competent cells on ice.
  2. Add 10μl ligation product into 100μl competent DH5α cell, mix them by gently tapping the tube wall. Let it stand on ice for 30 min.
  3. Heat shock in a 42℃ water bath for 90s. Then quickly place it on ice and let it stand for 2 min.
  4. Add 900μl SOC into each tube. Place in a shaker at 37℃ and 220 rpm for 1h for recovery.
  5. Centrifuge at 5000rpm for 3min. Discard 900 μl of the supernatant and resuspend the cell with the remaining culture. Coat the cell on LB-Kana+ solid culture medium plates.
  6. Place the flat plates in a 37℃ incubator for 10 min. Then invert the plates and incubate them.
8.31
lyseE PCR
pTD103 PCR
lyseE purification
Protocol: Thermo Scientific GeneJET PCR Purification Kit.
pTD103 plasmid digestion
overnight 37℃.
9.1
pTD103 Purification
Protocol: protocol A in GeneJET PCR purification kit.
Modification: 50ml Elution Buffer was replaced by 20μl ddH2O.
Homogeneous Recombination for pTD103-lyseE.
PCR procedure.
pTD-lyseE Ligation
Put the ligation system at room temperature for 2h.
Transformation of pTD103-lyseE into DH5α Competent Cells
Follow the Vezyme protocol of chemical competent cells.
CD513B-SV40 and CD513B-Bax Extraction and Cell Transfection
  1. Inoculate the DH5α cells overnight.
  2. Protocol: TIANGEN Endofree Mini Plasmid Kit. In step 2: centrifuge 4000rpm, 6min.
  3. Measure the concentration.
  4. Cell Transfection: Pre-seed cells in 12-well plate a day ahead. Protocol: YEASEN Hieff Trans Liposomal Transfection Reagent.
  5. Incubate in 37℃, 5% CO2, fluorescence confocal after 24h.
9.2
Transformation of pSL582, pSL816, and pCMV-T7-SB100 Plasmids into DH5α Competent Cells
  1. Transform pSL582 (Amp+), pSL816 (Amp+), and pCMV-T7-SB100 (Chl+) to DH5α competent cells respectively.
  2. Plate transformed DH5α to LB (Chl+ or Amp+) plate, and incubate at 37℃ overnight.
9.3
Inoculation of DH5α Competent Cells
Inoculate DH5α with pSL582 (Amp+), pSL816 (Amp+), and pCMV-T7-SB100 (Chl+) to 2ml LB (Chl+ or Amp+) culture medium, and incubate at 37℃, 220 rpm overnight.
9.4
Sequencing
Sequence pSL582 (Amp+), pSL816 (Amp+), and pCMV-T7-SB100 (Chl+). (Done by Sangon Biotech)
pTD103 plasmid purification and digestion. Protocol: Thermo Scientific GeneJET PCR Purification Kit.
9.5
Extraction of pSL816, pSL586, and SB100 Plasmid from DH5α
Protocol: EndoFree Mini Plasmid Kit II.
PCR of lyseE and Digestion of pYX00004:
  1. PCR lyseE with PsseJ-lyseE-F and PsseJ-lyseE-R.
  2. Digest pYX00004 (PsseJ-mCherry) with XhoI and HindIII.
Construction and Transformation of pTD103-lyseE Plasmid:
  1. Purify pTD103 digestion product. Protocol: GeneJET PCR Purification Kit.
  2. PCR with purified pTD103 and purified lyseE.
  3. Ligation at room temperature for 1h.
  4. DMT enzyme digestion 37℃ for 1h.
  5. Transform pTD103-lyseE into DH5α competent cells.
Subculturing of B16-F10 Cells
  1. Discard the old culture medium.
  2. Add PBS to wash and then add 0.25% trypsin to digest the cells.
  3. Add DMEM (10%FBS, 1%p/s) and triturate with pipette.
  4. Centrifuge at 800rpm, 5mins.
  5. Discard the supernatant and resuspend with pure DMEM.
  6. Seed the suspension into pure DMEM.
  7. 37℃, 5%CO2 incubation.
9.6
Construction and Transformation of pYX00004 Plasmid
  1. PCR lyseE product with digestion product of pYX00004.
  2. Transform PCR product to DH5α competent cell (Vanzyme).
  3. Plate transformed DH5α to LB(Kana+) plate, and incubate at 37℃ overnight.
Verification of BAX function: B16-F10 Cell Death Condition Detection by Flow Cytometry:
  1. Set up four groups: cell (untreated cells), lipo (cells added with transfection reagent), CD513B (cells transfected with CD513B-SV40), and Bax (cells transfected with CD513B-Bax).
  2. Transfected the CD513B-SV40 and CD513B-Bax into cells. Protocol: YEASEN Hieff Trams Liposomal Transfection Reagent (use DMEM for dilution).
  3. 37℃, 5%CO2 incubation.
  4. After 12h and 24h, use Annexin-FITC and PI double stain to conduct flow cytometry, choosing the PerCP and FITC channel. Protocol: Annexin V-FITC Apoptosis Detection Kit, NovoCyte Quick Operation Guide.
9.7
Extraction of PYX00004-PsseJ and LuxI-GFP
Protocol: TIANGEN Mini Plasmid Kit.
Concentration: LuxI-GFP: 143.95ng/μl, PYX00004-PsseJ: 87.6ng/μl.
Inoculation of DH5α:
Inoculate DH5α with pYX00004 (PsseJ-lyseE) to 2ml LB (Kana+) culture medium, and incubate at 37℃, 220 rpm overnight.
Co-transformation of pLH001 and BPK764
  1. Co-transform pLH001 (pBAD-sfGFP) and BPK764 (Lpp-2-OmpA) or BPK764 (Non-OmpA) to E.coli BL21 and SHuffle.
  2. Plate transformed BL21 and SHuffle to LB(Chl+ and Kana+) plate, and incubate at 37℃ overnight.
Extraction of pSL826, SB100, pSL582 Plasmids
Protocol: TIANGEN Endofree Mini Plasmid Kit II.
Plasmid Extraction of pGLO-scFv SHuffle
Protocol: TIANGEN Endofree Mini Plasmid Kit II.
MCF10A and SK-BR-3 Cell Thawing
  1. Gentle agitation in 37°C water bath for 2min to thaw.
  2. Remove the cryopreservation tubes from the water bath as soon as thawed.
  3. Decontaminate with 75% ethanol.
  4. Centrifuge with 1mL medium, 300g, 12min (Due to the specificity of the cells).
  5. Discard the supernatant.
  6. Resuspend in corresponding medium and dispense into T25 bottles.
  7. Incubation at 37℃, 5%CO2.
9.8
Inoculation of BL21 and SHuffle
Inoculate BL21 and SHuffle with pLH001 (pBAD-sfGFP) and BPK764 (Lpp-2-OmpA) or BPK764 (Non-OmpA) to 2ml LB (Kana+) culture medium, and incubate at 37℃, 220 rpm overnight.
B16-F10 cell recovery (P2) (done by Dr. Jia YOU, our biology lab teacher)
  1. Transfer the cell from -80℃ to 37℃ water bath.
  2. Add DMEM (10%FBS, 1%p/s) to it and triturate with pipette.
  3. Centrifuge at 800rpm, 5mins, and discard the supernatant.
  4. Add DMEM (10%FBS, 1%p/s) and mix thoroughly.
  5. 37℃, 5%CO2 incubation.
9.9
Sequencing
Sequence BL21 and SHuffle with pLH001 (pBAD-sfGFP) and BPK764 (Lpp-2-OmpA) or BPK764 (Non-OmpA). (Done by Tsingke)
9.10
Transformation of pSL776, pSL886 and pCMV-T7-SB100
  1. Inoculate pSL582 (Amp+), pSL816 (Amp+) and pCMV-T7-SB100 (Chl+) to 50 ml LB culture medium (Amp+ or Chl+).
  2. Transform pSL776 (Amp+) and pSL886 (Amp+) to DH5α, respectively.
  3. Plate transformed DH5α to LB(Amp+) plate, and incubate at 37℃ overnight.
B16-F10 Cell Passage (P3)
  1. Discard the old medium and then add PBS.
  2. Discard PBS and then add 0.25% trypsin.
  3. Add DMEM (10%FBS, 1%p/s) and triturate with pipette.
  4. Collect all the liquid mixture into a tube, and then centrifuge at 800rpm, 5mins.
  5. Discard the supernatant, then add DMEM (10%FBS, 1%p/s) to resuspend.
  6. Take part of the suspension to seed into new DMEM (10%FBS, 1%p/s).
  7. 37℃, 5%CO2 incubation.
MCF10A and SKBR3 Solution culture Renew
  1. For MCF10A, remove the supernatant gently, then add 5mL new medium.
  2. For SK-BR-3, collect all the supernatant, centrifuge at 300g for 12 minutes. Resuspend in 5mL new medium. Add the medium back to the original T25 bottle.
9.11
Extraction of pSL776, pSL886 and pCMV-T7-SB100
  1. Extract pSL582, pSL816 and pCMV-T7-SB100 endo-free plasmids from overnight culture with TIANGEN Endo Free Mini Plasmid KitⅡ.
  2. Inoculate DH5α with pSL776 (Amp+) or pSL886 (Amp+) to 2ml LB (Amp+) culture medium, and incubate at 37℃, 220rpm overnight.
Subculturing of B16-F10 Cells
  1. Discard the old culture medium.
  2. Add PBS to wash and then add 0.25% trypsin to digest the cells.
  3. Add DMEM (10%FBS, 1%p/s) and triturate with pipette.
  4. Centrifuge at 800rpm, 5mins.
  5. Discard the supernatant and resuspend with pure DMEM.
  6. Seed the suspension into pure DMEM.
  7. 37℃, 5%CO2 incubation.
9.12
Sequencing
Sequence pSL776 (Amp+) and pSL886 (Amp+). (Done by Sangon Biotech)
Preliminary Experiment of CD513B-mCherry, pYFP-N1 Transfection, and pSL816 and SB100 Cell Co-transfection
  1. Protocol: YEASEN Hieff Trams Liposomal Transfection Reagent (use DMEM for dilution).
  2. After 24h, observe the fluorescence signal under inverted microscope.
9.13
B16-F10 Cell Passage (P4)
Same as 9.10.
B16-F10 Cell Cryopreservation (P3)
  1. Discard the old medium and then add PBS.
  2. Discard PBS and then add 0.25% trypsin.
  3. Add DMEM (10%FBS, 1%p/s) and triturate with pipette.
  4. Collect all the liquid mixture into a tube, and then centrifuge at 800rpm, 5mins.
  5. Discard the supernatant, then add 2mL cell cryopreservation solution (10%DMSO, 30%FBS, 60%DMEM) to resuspend.
  6. Transfer the suspension into cryogenic vials then put into cell cryopreservation box with enough isopropanol in it.
  7. Put the cell cryopreservation box into -80℃.
  8. After >4h, let the cryogenic vials remain at -80℃ and take out the cell cryopreservation box to store under room temperature.
MCF10A and SKBR3 Solution culture Renew
Refer to the experiment at 9.10.
9.15
Inoculation of DH5α with pSL776 or pSL886 Plasmid
Inoculate DH5α with pSL776 (Amp+) or pSL886 (Amp+) to 50ml LB (Amp+) culture medium, and incubate at 37℃, 220 rpm overnight.
9.16
Extraction of DH5α with pSL776 or pSL886 Plasmid
Extract pSL776 (Amp+) and pSL886 (Amp+) from overnight culture with TIANGEN EndoFree Mini Plasmid KitⅡ.
MCF10A and SKBR3 Subculturing (P2)
  1. Remove the medium.
  2. Rinse the cell layer with 1.0mL PBS.
  3. Add 1.0mL 0.25% Trypsin-0.53mM EDTA. Wait until dispersed. 2 minutes at room temperature for SK-BR-3 and 10 minutes at 37℃ for MCF10A.
  4. Add 1.0mL medium to stop digesting.
  5. Centrifuge at 300g for 12 minutes and discard the supernatant, then resuspend in 1mL medium.
  6. Add 0.5mL cell suspension in each T25 bottle, then add 5.0mL medium to each bottle.
  7. Incubation.
9.18
B16-F10 cell recovery (P3)
  1. Transfer the cell from -80℃ to 37℃ water bath.
  2. Add DMEM (10%FBS, 1%p/s) to it and triturate with pipette.
  3. Centrifuge at 800rpm, 5mins, and discard the supernatant.
  4. Add DMEM (10%FBS, 1%p/s) and mix thoroughly.
  5. 37℃, 5%CO2 incubation.
9.20
B16-F10 cell passage (P4)
Same as 9.10.
MCF10A and SKBR3 Subculturing (P3)
Refer to the experiment at 9.16. Now we have 4 T25 bottles for each cell line.
9.21
Verification of BAX function: B16-F10 Cell Death Condition Detection by Flow Cytometry
  1. Set up four groups: cell (untreated cells), lipo (cells added with transfection reagent), CD513B (cells transfected with CD513B-SV40), and Bax (cells transfected with CD513B-Bax).
  2. Transfect the CD513B-SV40 and CD513B-Bax into cells using YEASEN Hieff Trams Liposomal Transfection Reagent (use DMEM for dilution).
  3. 37℃, 5%CO2 incubation.
  4. After 12h and 24h, use Annexin-FITC and PI double stain to conduct flow cytometry, choosing the PerCP and FITC channel using the Annexin V-FITC Apoptosis Detection Kit, NovoCyte Quick Operation Guide.
9.22
B16-F10 cell passage (P5)
Same as 9.10.
Co-transfection of pSL816 and SB100 Plasmids
  1. Transfect plasmids with a mass ratio of pSL816:SB100 = 100:1 using YEASEN Hieff Trams Liposomal Transfection Reagent (use DMEM for dilution).
  2. 37℃, 5%CO2 incubation.
9.24
Verification of BAX and Bio-switch function: B16-F10 Cell Western Blot Material Preparation
For BAX function verification:
  1. Set up two groups: cell (cells not transfected with CD513B-Bax) and Bax (cells transfected with CD513B-Bax).
  2. Transfect CD513B-Bax into cells using YEASEN Hieff Trams Liposomal Transfection Reagent (use DMEM for dilution).
  3. 37℃, 5%CO2 incubation. After 24h, digest cells and collect cell samples for Western blot.
For bio-switch function verification:
  1. Set up two groups: OFF (bio-switch + cells without NS3a) and ON (bio-switch + cells with NS3a).
  2. Transfect plasmids with a mass ratio of pSL886:pSL776:pSL582 = 1:1:1 using ThermoFisher Lipofectamine™ 3000 Reagent Protocol (use DMEM for dilution).
  3. 37℃, 5%CO2 incubation. After 24h, digest cells and collect cell samples for Western blot.
MCF10A and SKBR3 Subculturing (P4) and Cryopreservation
2 T25 bottles were subcultured. This part refers to the experiment at 9.16.
  1. 2 T25 bottles were cryopreserved:
  2. Prepare the cryoprotectant as 60% Complete growth medium + 30% FBS + 10% DMSO.
  3. Remove the medium and rinse the cell layer with 1mL PBS to wash off the trypsin inhibitor.
  4. Add 1.0mL 0.25% Trypsin-0.53mM EDTA to digest the cells. Then add 1.0mL medium to stop digestion.
  5. Centrifuge at 300g for 12 minutes and discard the supernatant, then resuspend in 2mL cryoprotectant.
  6. Put the cryopreservation tubes (2 tubes of MCF10A and 2 tubes of SK-BR-3) in a cell-freezing container, then put the container at -80°C.
9.25
ELISA on HER2_scFv purified
  1. Double dilute the purified anti-HER2 scFv into 100ng/mL, 50ng/mL, 25ng/mL, and 12.5ng/mL.
  2. Add 100 μL HER2 (2ng/ml) standard to each well.
  3. Incubate for 120 min, RT, 160 rpm.
  4. Add 100 μL of purified anti-HER2 scFv per well.
  5. 37 °C, 60 min incubation.
  6. Wash 5 times with 100 μL/well wash buffer.
  7. Add His-tag-HRP (MC10) Mouse Monoclonal Antibody (LABLEAD H1104).
  8. Incubate for 120 min, RT, 160 rpm.
  9. Wash 5 times with 100 μL/well wash buffer.
  10. Add 100 μL/well TMB substrate, then RT in the dark for 20 mins.
  11. Add 100 μL/well stop solution.
  12. Within 30 minutes, perform a dual-wavelength measurement using a microplate reader to determine the OD values at the maximum absorption wavelength of 450 nm and 570 nm or 630 nm.
Verification of BAX and Bio-switch function: Western Blot Analysis
  1. Wash the sample with PBS 2-3 times.
  2. Resuspend by Ripa (1mM PMSF), 1h on ice.
  3. Add loading dye (dilute from 4× to 1×), 98°C, 15 mins.
  4. Centrifuge at 800rpm, 5 mins, and collect the supernatant.
  5. Load 20μL/well supernatant and 5μL/well protein ladder #26619 into a 4-20% SDS-PAGE gel.
  6. Run at 140V for 60 mins.
  7. Transfer to a 0.22μm PVDF membrane by semi-dry method.
  8. Incubate the membrane with blocking buffer (5% BSA, 1×TBST) at RT for 1h.
  9. Incubate the membrane with anti-β-ACTIN (BioLegend 643802), 1:500 diluted in 5% BSA, 1×TBST.
  10. Wash the membrane three times with 1×TBST, 10 mins each.
  11. Incubate the membrane with HRP-conjugated Goat Anti-Mouse IgG (H+L) (Proteintech SA00001-1), 1:2000 diluted in 5% BSA, 1×TBST.
  12. Wash the membrane three times with 1×TBST, 10 mins each.
  13. Discard the 1×TBST and preserve it at -20°C.
B16-F10 cell passage (P6)
Same as 9.10
B16-F10 Cell Cryopreservation (P6)
  1. Discard the old medium and then add PBS.
  2. Discard PBS and then add 0.25% trypsin.
  3. Add DMEM (10% FBS, 1% p/s) and triturate with pipette.
  4. Collect all the liquid mixture into a tube, and then centrifuge at 800rpm, 5 mins.
  5. Discard the supernant, then add 2mL cell cryopreservation solution (10%DMSO, 30%FBS, 60%DMEM) to resuspend.
  6. Transfer the suspension into cryogenic vials than put into cell cryopreservation box with enough isopropanal in it.
  7. Put the cell cryopreservation box into -80℃.
  8. After >4h, let the cryogenic vials remain at -80℃ and take out the cell cryopreservation box to store under room temperature.
9.26
Reagent Preparation
  1. Prepare 1L SOC medium, among which is 5*100 mL SOC medium.
  2. Prepare 200mL 10mM HEPES (0.47g HEPES with 1L ddH2O), neutralized by 10mM KOH, to pH 7.0.
  3. Prepare 50 mL 10% glycerol.
  4. 121℃ 20min sterilize.
  5. Prepare plates (SOC medium + Ampicillin (100 μg/mL) + arabinose)
    1. 0.05% arabinose *10 plates,
    2. 0.2% arabinose *10 plates.
  6. Culture 2 tubes of Salmonella and 1 tube of negative control.
16s Sequencing and Gene Deletion Sequencing for VNP20009
Primers:
Procedure:
  1. 16s PCR and gene PCR for VNP20009.
  2. PCR details (Es Taq, 2kb/min) for 16s PCR
    1. purL gene: 3888 bp.
    2. msb gene: 982 bp.
  3. 1% agarose gel electrophoresis.
Chemiluminescence of Western Blot results
  1. Wash the PVDF membrane from 9.25 with 1×TBST.
  2. Cover the membrane with superpico ECL (1:1).
  3. Scan and merge the figure under chemiluminescent/colormetric mode.
Growth Curve Measurement of VNP20009 and E.coli
  1. Refer to the protocol "Bacteria Growth Curve Measurement Protocol".
9.27
pGLO transformation to Salmonella
  1. (14:10) 1:100 dilute the overnight Salmonella culture with SOC medium. (1 mL to 100 mL)
    1. 18:15 – OD600 1.5
  2. (18:26) 1:100 dilute the overnight Salmonella culture with SOC medium. (1 mL to 100 mL)
    1. 19:46 – OD600 0.13
    2. 20:28 - OD600 0.26
    3. 21:11 - OD600 0.634
  3. Do electroporation and heat shock transformation on Salmonella. (See protocol).
pTD103-luxI and pYX00004 Transformation
  1. Prepare Salmonella VNP20009 competent cells using Supercompetent Cell Preparation Kit (Beyotime).
  2. Transform 100 ng pTD103-luxI and pYX00004 (PsseJ-mCherry) to VNP20009 competent cells.
  3. Incubate VNP20009 competent cells at 37℃, 220 rpm for 1 hour.
  4. Plate VNP20009 with pTD103-luxI or pYX00004 (PsseJ-mCherry) to LB (Kana+) plates, and incubate at 37℃ overnight.
Co-transformation to Salmonella
  1. Prepare Salmonella VNP20009 competent cells for electroporation. (See Salmonella Electroporation Protocol).
  2. Add 100 ng pTD103-luxI and 100ng BPK764-Lpp-1-OmpA, BPK764-Lpp-2-OmpA, or BPK764-Non-OmpA to 50 μL VNP20009 competent cells, respectively.
  3. Electroporate VNP20009. (See Salmonella Electroporation Protocol).
  4. Incubate VNP20009 with pTD103-luxI and BPK764-Lpp-1-OmpA, BPK764-Lpp-2-OmpA, or BPK764-Non-OmpA at 37℃, 220 rpm for 1 hour.
  5. Plate VNP20009 to LB (Kana+ and Chl+) plates, and incubate at 37℃ overnight.
B16-F10 cell passage (P7)
  1. Same as 9.10.
Growth Curve Measurement of VNP20009 and E.coli
  1. Count the colonies' number and get the growth curve.
  2. Decide the harvest point of VNP20009 and E.coli
    • VNP20009: 6h OD600: 0.63-0.69, CFUs: 50M CFUs/mL.
    • E.coli: 6h OD600: 0.62-0.65, CFUs: 15M CFUs/mL.
9.28
Salmonella Inoculating
  1. Inoculate VNP20009 with pTD103-luxI and BPK764-Lpp-1-OmpA, BPK764-Lpp-2-OmpA, or BPK764-Non-OmpA to LB (Kana+ and Chl+) liquid culture medium.
VNP20009 Sequencing
  1. Sequence VNP20009 to verify plasmids pTD103-luxI, BPK764-Lpp-1-OmpA, BPK764-Lpp-2-OmpA, BPK764-Non-OmpA, or pYX00004 (PsseJ-mCherry). (Done by Tsingke).
  2. Do 16s assay on the VNP20009 transformed with these plasmids.
Co-transfection of pSL816 and SB100 Plasmids
  1. Transfect plasmids with a mass ratio of pSL816:SB100 = 100:1
    Protocol: YEASEN Hieff Trams Liposomal Transfection Reagent (use DMEM for dilution)
  2. 37℃, 5% CO₂ incubation.
9.29
Verification of BAX and Bio-switch function: B16-F10 Cell Western Blot Material Preparation
For BAX function verification:
  1. Set up two groups: cell (cells don't transfected with CD513B-Bax) and bax (cells transfected with CD513B-Bax).
    Transfect the CD513B-Bax into cells.
    Protocol: YEASEN Hieff Trams Liposomal Transfection Reagent (use DMEM for dilution)
  2. 37℃, 5% CO₂ incubation. After 24h, digest cells and collect cell samples for Western blot.
For bio-switch function verification:
  1. Set up two groups: OFF (bio-switch+cells without NS3a) and ON (bio-switch+cells with NS3a).
    Transfect plasmids with a mass ratio of pSL886:pSL776:pSL582 = 1:1:1.
    Protocol: ThermoFisher Lipofectamine™ 3000 Reagent Protocol (use DMEM for dilution)
  2. 37℃, 5% CO₂ incubation. After 24h, digest cells and collect cell samples for Western blot.
MCF10A and SK-BR-3 Plate Seeding
  1. Remove the medium and rinse the cell layer with 1 mL PBS to wash off the trypsin inhibitor.
  2. Add 1.0 mL 0.25% Trypsin-0.53 mM EDTA to digest off the cell. Then add 1.0 mL medium to stop digesting.
  3. Incorporate all cell suspensions, and centrifuge at 300g for 12 minutes and discard the supernatant, then resuspend in 1 mL medium.
  4. Add 10 μL cell suspensions to blood cell counting plate and count the cell number under the microscope, get the concentration of cell suspension: MCF10A: 60M/mL, SK-BR-3: 15M/mL.
  5. Dilute the cell suspension to 5M/mL. Add 100 μL cell suspension and 900 μL medium to each well in 12 well plates.
9.30
Verification of BAX and Bio-switch function: Western Blot Analysis
  1. Wash the sample with PBS 2~3 times.
  2. Resuspend by Ripa (1mM PMSF), 1h on ice.
  3. Add loading dye (dilute from 4×~1×), 98°C, 15mins.
  4. Centrifuge at 800rpm, 5mins, collect the supernant.
  5. Load 20μL/well supernant and 5μL/well protein ladder #26619 into 4~20% SDS-Page gel.
  6. 140V, 60mins.
  7. Transfer to 0.22μm PVDF membrane by semi-dry method.
  8. Incubate the membrane with blocking buffer (5% BSA, 1×TBST) at RT, 1h.
  9. Incubate the membrane with Rb anti-BAX (ab32053), 1:1000 diluted in 5% BSA, 1×TBST.
  10. Wash the membrane three times with 1×TBST, 10mins each.
  11. Incubate the membrane with Goat Anti-Rabbit IgG H&L (HRP) (ab205718), 1:2000 diluted in 5% BSA, 1×TBST.
  12. Wash the membrane three times with 1×TBST, 10mins each.
  13. Discard the 1×TBST.
  14. Cover the membrane with superpico ECL (1:1).
  15. Scan and merge the figure under chemiluminescent/colormetric mode.
10.1
Co-culturing of bacteria with cells and FACS analysis
  1. Inoculate the overnight bacteria solution 100 μL into 10 mL medium, static in 37°C for 3h.
  2. Add 20 μL 1000x IPTG and 100 μL 20% w/v L-Arabinose to each tube, static for another 3h.
  3. Harvest Bacteria to the needed concentration:
    1. Centrifuge at 4000g for 10 minutes and resuspend in 200 μL PBS.
    2. Prepare Far Red dye by adding 1 μL 100x CellTrace Far Red to 20 μL DMSO.
    3. Add 1 μL Far Red DMSO to each tube. Incubate at 37°C for staining.
    4. Centrifuge at 4000g for 10 minutes and resuspend in 200 μL 10% FBS containing PBS. Repeat the step for 3 times to wash off the excess dye.
  4. Inoculate 10 μL bacteria in each well. Incubate at 37°C, 5% CO₂ for 3h.
  5. Collect the supernatant, and replace it with new 1 mL medium. Observe under fluorescence microscope.
  6. Digest off the cell and combine all the cell suspension.
  7. Centrifuge the cell suspension at 300g for 12 minutes and resuspend in 500 μL PBS.
  8. Add 5 μL 100x DAPI to each tube. Analyzed by FACS.