iGEM 2024 — Team westlake

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Notebook

“

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.

#### Westlake IGEM 2024

The Westlake iGEM team is a dedicated group of undergraduate researchers focused on advancing synthetic biology. Our project employs genetically engineered Salmonella for targeted cancer therapies. Committed to innovation and collaboration, we strive to make a meaningful impact in the fight against cancer while inspiring our community through research and education.

#### Links

* [Description](./description)
* [Model](./model)
* [Engineering](./engineering)
* [Education](./education)
* [integrated Human Practice](./human-practices)

#### Contact

No. 600 Dunyu Road,
XihuDistrict, Hangzhou,
Zhejiang, 310030, P.R. China

(+86)15058007607

gudingqi@westlake.edu.cn

---

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![Salmonella](https://static.igem.wiki/teams/5363/images/killer-salmonella.png)

Engineering

“

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.

Boost thoughts and action

Overview

* Original innovation is important across all sciences but is especially important in synthetic biology. Novel designs and projects require a comprehensive understanding of underlying principles to weave together ideas and bring a project to life. Whether considering the physiological ramifications of a signaling cascade or transforming bacteria, synthetic biology covers a wide breadth of expertise, and each aspect is important to consider when constructing new systems.
* Similar to how established engineering disciplines such as aerospace engineering have evolved, we too are advancing towards greater predictability and control in biology. As participants in iGEM, we are thrilled to be at the forefront of this endeavor, contributing to the evolution of tools and methodologies in biological engineering. With determination and commitment, we strive to exemplify engineering brilliance and further extend the limits of synthetic biology's potential.
* The T-SAT project aims to develop a novel therapeutic strategy utilizing engineered *Salmonella* strains to target and induce apoptosis in cancer cells. Critical components of this system include the recognition system, the delivery system, and the killing system coupled with a bio-switch. The recognition system contains a Human Epidermal growth factor Receptor 2 (HER2) single-chain variable fragment (scFv) （BBa\_K5363017) expressed by a *Salmonella* surface display system. The delivery system, which can induce *Salmonella's* self-lysis after *Salmonella* invades human cells, uses a *PsseJ-lyseE* gene circuit. Lastly, the killing system consists of an effector gene to induce cancer cell apoptosis and a synthetic translation initiation factor (STIF)-based bio-switch that ensures the expression of effector genes is confined exclusively to cancer cells. This section details the engineering efforts undertaken to verify the functionality and specificity of our project.

Project Design

Cycle 1

Design

* We set out to create a new therapeutic for treating solid tumors. Solid tumors are notoriously hard to treat owing to several properties. They are typically immunosuppressive, necrotic, nonvascularized, and hypoxic. All of these features block traditional treatments. However, the necrotic, hypoxic, and immunologically under-surveilled core is a perfect home for many bacteria. Taking advantage of this, we decided to use Salmonella typhimurium strain *VNP20009*. This strain was modified not to produce LPS or purines, making it much less immunogenic and pathogenic. (Clairmont et al., 2000)
* In our first iteration, we aimed to add a single-chain variable fragment (scFv) to a *Salmonella* expression system to recognize tumor-specific antigens. We found a fusion protein of a human epidermal growth factor receptor 2 (HER2) scFv(BBa\_K5363017) with the *Salmonella* Lpp-OmpA expression system. We also sought to add a fluorescence marker to visualize the bacterial colonization of tumors. To supplement bacterial colonization, we also looked for a signal transduction system with the scFv, much like the CAR-T system, to add a customizable response. (Francisco et al., 1992; Jeiranikhameneh et al., 2017)

Build

* We started by designing the recognition system in *E.coli*. We use the Lpp-OmpA surface display system to express the HER2-scFv(BBa\_K5363017) on the surface of *E.coli*.
![Recognition System](https://static.igem.wiki/teams/5363/engineering/1.png)

Lpp-OmpA-HER2\_scFv Plasmid Construction

* To better demonstrate the population distribution of *Salmonella*, we utilize the *LuxI-LuxR*(BBa\_K5363021) circuit to express the superfolder green fluorescent protein (*sfGFP*)(BBa\_K5363023). We aim to visualize *Salmonella* by introducing a plasmid named pTD103(BBa\_K5363031). The *GFP* gene within the plasmid enables the modified bacteria to emit green fluorescence. The plasmid also contains a two-component system: *LuxI-LuxR*(BBa\_K5363021), which is controlled by the promoter *pLuxI*(BBa\_K5363018). This promoter induces the expression of an autoinducer, AHL. AHL binds to *LuxR*(BBa\_K5363021) and transcriptionally activates the promoter, thereby inducing the expression of GFP. AHL can diffuse to neighboring cells, providing a mechanism for intercellular synchronization. Consequently, the concentration of AHL reflects the size of the bacterial population, and the intensity of green fluorescence can, to some extent, indicate the bacterial population density.
![Population Distribution of Salmonella](https://static.igem.wiki/teams/5363/engineering/2.png)

LuxI-LuxR-GFP Circuit Construction

Test

* We first tested the recognition and reporter system in *E. coli*. We expressed HER2 scFv(BBa\_K5363017) separately without a surface display system to get its recognition efficacy to HER2 antigen on human cells. Then we confirmed the fluorescence of the *luxI-sfGFP*(BBa\_K5363031) transformed *E. coli* compared to that of *E. coli* that was transformed with *sfGFP*(BBa\_K5363023) expressed under *pBAD* promoter without arabinose.

Learn

* With confirmation that the Lpp-OmpA-HER2\_scFv expression system was successful, we decided to push forward the *Salmonella* design. Improving on this initial design, we looked for a method to kill cancer cells upon invasion by bacteria. We also reviewed the literature for bacterial signal transduction systems that might streamline the recognition of cancer cells; however, no clear options were available. Given our current system has increased recognition of cancer cells, we also read literature for ways to improve bacterial invasion into cancer cells.

Cycle 2

Design

* We were already aware of *Salmonella's* strong invasive action toward epithelial cells. Our next step was to determine how to utilize *Salmonella* to introduce plasmids into cancer cells and identify the specific plasmids for introduction. After discussions with Dr. Yanbo MAO, we devised a creative method involving two types of *Salmonella*: Helper Salmonella (HS) and Killer Salmonella (KS).
* Killer Salmonella is engineered with an scFv recognition system and a killing system plasmid to target and eliminate cancer cells. However, it lacks the ability to invade cells due to a deletion of the flagellum gene. In contrast, Helper Salmonella retains its invasive capability.
* During the treatment process, Killer Salmonella is first injected into the tumor, where it binds to the surface of target cancer cells. Subsequently, Helper Salmonella is injected and invades cells randomly. Killer Salmonella then follows Helper Salmonella into the target cancer cells, disrupting the endosomal compartment and directly releasing the plasmid containing the killer gene.
* Regarding the killing system, our objective was to induce programmed cell death, thereby reducing the risk of cytokine storms and other toxicities associated with current cancer treatments. Extensive research has demonstrated that the *BAX* gene (Bcl-2 Associated X-protein)(BBa\_K5363000), a pro-apoptotic member of the Bcl-2 gene family, can effectively induce apoptosis in various types of cancer (Liu et al., 2016). Therefore, we constructed a plasmid containing the *BAX* gene(BBa\_K5363000) under the control of a constitutive *CMV* promoter.

Build

* Firstly, to delete the flagellum gene in *Salmonella enterica serovar Typhimurium* strain *VNP20009* of KS, we employed the CRISPR-Cas9 method to specifically delete the *flhD* and *fliE* genes. We inserted gRNA sequence into vector plasmid that containing *Cas9* gene to construct pU6-Cas9 plasmid. This plasmid was then transfected into *VNP20009* for selection and subsequent sequencing verification to confirm the deletion. (Chen et al., 2021)
![Flagellum Gene Deletion](https://static.igem.wiki/teams/5363/engineering/3.png)

*flhD* and *fliE* Gene Deletion Plasmid Construction

![gDNA Sequence](https://static.igem.wiki/teams/5363/engineering/4.png)

gDNA Sequence

* To verify the functionality of the *BAX* gene(BBa\_K5363000), we amplified the *BAX* gene(BBa\_K5363000) from the plasmid pSL886 PMusAFP-MusBax-(MS2)24-HHR-pA (Shao et al., 2024). Then, to mimic the plasmid-delivery function of killer Salmonella, we inserted the amplified gene into CD513B-SV40 to construct CD513B-Bax. The plasmid contains a *CMV* promoter that constitutively expresses genes in mammalian cells.
![BAX Gene Amplification](https://static.igem.wiki/teams/5363/engineering/5.png)

CD513B-Bax Plasmid Construction

Test

* After constructing the CD513B-Bax plasmids, we transfected it into the B16-F10 cell line. Subsequently, we observed that the cells transfected with the CD513B-Bax plasmids exhibited a lack of continuous growth, and also a higher rate of cell death compared to the control groups, as determined by flow cytometer analysis. Additionally, Western blot analysis confirmed elevated *BAX* gene(BBa\_K5363000) overexpression in the transfected cells, indicating that the overexpression of *BAX* protein led to the death of B16-F10 cells.

Learn

* We had uncertainties regarding the effectiveness of this method and were attentive to the specificity of the two strains of *Salmonella*. Therefore, there is a need to enhance the efficiency and safety of these two types of *Salmonella* to ensure improved functionality.

Cycle 3

Design

* Extensive literature reviews revealed the absence of an appropriate system for signal transduction in Gram-negative bacteria. Consequently, we required a promoter capable of facilitating bacterial expression upon invasion into cells. Researchers at the University of Massachusetts have utilized the *PsseJ* promoter(BBa\_K5363013), which is activated intracellularly following the invasion of cancer cells by *Salmonella*, to deliver drugs to host cells (cancer cells) through intracellular self-lysis. (Raman et al., 2021)
* Moreover, to enhance the safety level, we added Synchronized lysis circuit (SLC) circuit to *Salmonella* *VNP20009* to control its population under a safe level.

Build

* To induce autonomous self-lysis of cancer cells following invasion by *Salmonella*, we constructed the plasmid pYX00004, which contains the *PsseJ* promoter and the *φ*X174E (also called *lyseE*) gene(BBa\_K5363015). *φ*X174E is a gene that codes for the E protein of the Phix174 microvirus (Sinsheimervirus), which induces host cell lysis. Initially, the *PsseJ* promoter(BBa\_K5363013) was synthesized based on its known nucleotide sequence. The *lyseE*(BBa\_K5363015) gene was then amplified via polymerase chain reaction (PCR) from the phiX174 plasmid. Both the synthesized *PsseJ* promoter(BBa\_K5363013) and the amplified *lyseE* gene(BBa\_K5363015) were inserted into the pYX00004 plasmid vector. Consequently, the self-lysis plasmid pYX00004 was successfully constructed.
![Self-Lysis Plasmid](https://static.igem.wiki/teams/5363/engineering/6.png)

*PsseJ*-*lyseE* Plasmid (pYX00004) Construction

* To ensure biosafety, it is necessary to control the population density of both extracellular and intracellular helper Salmonella. Therefore, we constructed a genetic circuit to insert specific genes into the system, thereby achieving regulation of *Salmonella* population density.
![Population Density Control](https://static.igem.wiki/teams/5363/engineering/7.png)

*LuxI*-*LuxR*-*lyseE* Circuit Construction

Test

* After constructing pYX00004 (*PsseJ-mCherry*)(BBa\_K5363026) and pYX00004 (*PsseJ-lyseE*)(BBa\_K5363027), we transform them and the original plasmid pYX00004 (*pBAD-mCherry*) into *Salmonella*, respectively. We verified that *PsseJ* promoter(BBa\_K5363013) would not be activated when *Salmonella* is out of cells, and mCherry(BBa\_K5363020) would be expressed when *Salmonella* is in human cells.

Learn

* In the third cycle, the successful construction of the self-lysis plasmid pYX00004 represented a significant advancement toward autonomous Salmonella-mediated killing of cancer cells. This cycle emphasized the necessity for a more sophisticated and tightly regulated gene expression system to attain efficient and specific targeting of cancer cells. The challenges encountered with the *PsseJ-lyseE* system(BBa\_K5363027) offered valuable insights that steered the development of the Bio-switch approach in the fourth cycle.

Cycle 4

Design

* In eukaryotic cells, protein translation is initiated when a preinitiation complex, consisting of a 40S ribosome and initiation factors (eIFs), is recruited to the untranslated region (UTR) at the guanine-rich 5′ cap of mature mRNA molecules that have been exported to the cytoplasm. Cooperative activity by cap-binding protein eIF4E, RNA helicase eIF4A, central scaffolding protein eIF4G, and helicase enhancers eIF4B and eIF4H subsequently triggers RNA unwinding, ribosome attachment, and codon scanning.
* It is well known that the 3′ poly-adenine (poly(A)) tail enhances mRNA stability in living cells, but the underlying mechanism is still a subject of debate. Nevertheless, numerous studies support a "closed-loop" model in which poly(A)-binding protein (PABP) functions as another canonical eIF capable of simultaneously binding both poly(A) and eIF4G to induce a circularized mRNA configuration that is favorable for mRNA scanning, ribosome recycling, and protein translation.
* To engineer trigger-inducible translational devices, the incorporation of RNA-binding protein (RBP)-specific aptamers into the UTRs of target gene mRNA is a popular starting point, as it enables site-specific recruitment of RBP-containing regulatory proteins designed to control mRNA stability or eIF4E recruitment. With all these ideas, we constructed an intracellular signal-activated killing system, derived from Shao et al., 2024.

![Killing System Overview](https://static.igem.wiki/teams/5363/engineering/13.png)

Bio-switch Mechanism

Build

* To achieve efficient translational control over the expression of the effector gene (*BAX*)(BBa\_K5363000) in human cells, we utilize synthetic translational initiation factors (STIFs) developed by Prof. Xie Mingqi’s team. (Shao et al., 2024) Following the "closed-loop" model of translation, it is believed that manipulating the mRNA circularization process can regulate translation.
* Firstly, an expression vector is required for a synthetic mRNA transcript containing a control region with RBP-specific aptamers in the 3'-UTR, with MS2-box motifs selected for this purpose. To reduce background activity from *BAX*(BBa\_K5363000) mRNA translation independent of STIFs, trans-acting shRNA-binding sites are replaced with cis-acting hammerhead ribozyme (HHR)(BBa\_K5363002) motifs. These HHR motifs should induce spontaneous self-excision of the natural poly(A) signal either before or immediately after nuclear mRNA export.
* Secondly, an antibody linked to rotaviral non-structural protein 3 (NSP3)(BBa\_K5363007), which is an eIF4F-binding protein (eIFBP), is employed to recognize the intracellular signal. Thirdly, another antibody recognizing the intracellular signal is linked to bacteriophage-derived MS2 coat protein (MCP)(BBa\_K5363003), which can bind to tandem repeats of cognate MS2-box motifs(BBa\_K5363001). These three plasmids combine to form a killing system activated by STIF. The intracellular signal plays a crucial role in this system. If the two antibodies recognize the specific signal, forming a loop with *BAX* mRNA connected to NSP3-antibody-signal-antibody-MCP, translation of BAX protein will occur.
* Regarding the specific intracellular signal, EGFP(BBa\_K5363011) is fused to NS3a(H1)(BBa\_K5363005) for system verification. NS3a(H1):ANR represents a constitutive protein association pair, while LaG16(BBa\_K5363004) is a high-affinity GFP nanobody. Consequently, ANR repeats ((ANR)8)(BBa\_K53363006) are linked to NSP3(BBa\_K5363007), LaG16(BBa\_K5363004) is linked to MCP(BBa\_K5363003), and the killing system coupled with STIF is activated.

Plasmid Construction

* In the vector, NS3a(H1)(BBa\_K5363005) was linked to EGFP(BBa\_K5363011) to mimic the intracellular signal for validating our killing system. To optimize the efficiency of this process, it was crucial to initially establish a cellular environment conducive to strong NS3a(H1) expression.
![Intracellular Signal Plasmid](https://static.igem.wiki/teams/5363/engineering/8.png)

Intracellular Signal Plasmid (pSL816 EGFP-NS3a(H1))(BBa\_K5363010)

* A plasmid containing the *BAX* gene(BBa\_K5363000) under the control of the Bio-switch was constructed. The *BAX* gene(BBa\_K5363000) is positioned after the synthetic control region containing the RBP-specific aptamers.
![Bax Plasmid](https://static.igem.wiki/teams/5363/engineering/9.png)

Bax Plasmid (pSL886 PMusAFP-MusBax-(MS2)24-HHR-pA)

![ANR-NSP3 Plasmid](https://static.igem.wiki/teams/5363/engineering/11.jpg)

pSL582 PhCMV-(ANR)8-NSP3(B)-pA)

![MCP-LaG16 Plasmid](https://static.igem.wiki/teams/5363/engineering/12.jpg)

pSL776 PhCMV-MCP-LaG16-pA

Test

* Before introducing the three bio-switch plasmids (pSL886, pSL582, pSL776) into B16-F10 cells, we first expressed the target protein (NS3a protein) in this cell line by transfecting pSL816 along with the expression-enhancing vector SB100. After 24 hours, we observed the fluorescence signal of the cells. With a transfection efficiency exceeding 70%, we then co-transfected the pSL886, pSL582 and pSL776 plasmids.
* Subsequently, we observed a significant difference in cell growth conditions between experimental groups. Specifically, cells expressing both NS3a protein and bio-switch showed a much higher death rate compared to control groups, which included cells without NS3a and those without three bio-switch plasmids. Additionally, Western blot method was employed to verify the expression levels of *BAX* gene(BBa\_K5363000). We confirmed that the *BAX* gene(BBa\_K5363000) is overexpressed in the presence of NS3a protein but has very low expression levels in its absence.

Learn

* The Bio-switch demonstrated high specificity and efficiency in controlling the expression of therapeutic genes. In vitro assays confirmed that *BAX*(BBa\_K5363000) translation was strictly dependent on the presence of STIFs. (See more details in Results) Further analysis and experimentation are needed to gain a deeper understanding of the killing system and its application in cancer therapy. This study provides a foundation for future research in this area, and continued investigation may lead to valuable insights into the potential applications of the systems.

References

1. Clairmont, C., Lee, K. C., Pike, J., Ittensohn, M., Low, K. B., Pawelek, J., Bermudes, D., Brecher, S. M., Margitich, D., Turnier, J., Li, Z., Luo, X., King, I., & Zheng, L. M. (2000). Biodistribution and Genetic Stability of the Novel Antitumor Agent VNP20009, a Genetically Modified Strain of *Salmonella typhimuvium*. *The Journal of Infectious Diseases*, 181(6), 1996-2002. <https://doi.org/10.1086/315497>
2. Francisco, J. A., Earhart, C. F., & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of *Escherichia coli*. *Proceedings of the National Academy of Sciences*, 89(7), 2713-2717. <https://doi.org/10.1073/pnas.89.7.2713>
3. Jeiranikhameneh, M., Razavi, M. R., Irani, S., Siadat, S. D., & Oloomi, M. (2017). Designing novel construction for cell surface display of protein E on *Escherichia coli* using non-classical pathway based on Lpp-OmpA. *AMB Express*, 7(1), 53. <https://doi.org/10.1186/s13568-017-0350-0>
4. Liu, Z., Ding, Y., Ye, N., Wild, C., Chen, H., & Zhou, J. (2016). Direct Activation of Bax Protein for Cancer Therapy. *Med Res Rev*, 36(2), 313-341. <https://doi.org/10.1002/med.21379>
5. Chen, J., Qiao, Y., Chen, G., Chang, C., Dong, H., Tang, B., Cheng, X., Liu, X., & Hua, Z. (2021). Salmonella flagella confer anti-tumor immunological effect via activating Flagellin/TLR5 signalling within tumor microenvironment. *Acta Pharmaceutica Sinica B*, 11(10), 3165-3177. <https://doi.org/10.1016/j.apsb.2021.04.019>
6. Raman, V., Van Dessel, N., Hall, C. L., Wetherby, V. E., Whitney, S. A., Kolewe, E. L., Bloom, S. M. K., Sharma, A., Hardy, J. A., Bollen, M., Van Eynde, A., & Forbes, N. S. (2021). Intracellular delivery of protein drugs with an autonomously lysing bacterial system reduces tumor growth and metastases. *Nature Communications*, 12(1). <https://doi.org/10.1038/s41467-021-26367-9>
7. Shao, J., Li, S., Qiu, X., Jiang, J., Zhang, L., Wang, P., Si, Y., Wu, Y., He, M., Xiong, Q., Zhao, L., Li, Y., Fan, Y., Viviani, M., Fu, Y., Wu, C., Gao, T., Zhu, L., Fussenegger, M., . . . Xie, M. (2024). Engineered poly(A)-surrogates for translational regulation and therapeutic biocomputation in mammalian cells. *Cell Research*, 34(1), 31-46. <https://doi.org/10.1038/s41422-023-00896-y>
8. Shi, L., Yu, B., Cai, C.-H., Huang, W., Zheng, B.-J., Smith, D. K., & Huang, J.-D. (2016). Combined prokaryotic–eukaryotic delivery and expression of therapeutic factors through a primed autocatalytic positive-feedback loop. *Journal of Controlled Release*, 222, 130-140. <https://doi.org/10.1016/j.jconrel.2015.12.005>

#### Westlake IGEM 2024

The Westlake iGEM team is a dedicated group of undergraduate researchers focused on advancing synthetic biology. Our project employs genetically engineered Salmonella for targeted cancer therapies. Committed to innovation and collaboration, we strive to make a meaningful impact in the fight against cancer while inspiring our community through research and education.

#### Links

* [Description](./description)
* [Model](./model)
* [Engineering](./engineering)
* [Education](./education)
* [integrated Human Practice](./human-practices)

#### Contact

No. 600 Dunyu Road,
XihuDistrict, Hangzhou,
Zhejiang, 310030, P.R. China

(+86)15058007607

gudingqi@westlake.edu.cn

---

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![Salmonella](https://static.igem.wiki/teams/5363/images/killer-salmonella.png)

Experiments

“

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

Primers

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Protocol

Molecule Experiment

Salmonella Electroporation Protocol

1. Pick one clone from LB plate stored at 4 ℃.
2. Inoculate the clone in 2 mL LB broth, 37℃, 220 rpm, overnight.
3. Inoculate the overnight culture at 1:100 ratio to 100 mL LB.
4. Incubate at 37℃, 220 rpm, until OD(600)=0.6.
5. Chill the tubes on ice-water bath for 15 min.
6. (Pre-cool the centrifuge) Centrifuge at 4℃, 4000 xg, centrifuge for 15 min to collect the bacteria, and discard the supernatant.
7. Gently resuspend pellets in 100 mL pre-cold Double-Distilled Water. Stay on ice for 5 min.
8. Centrifuge at 4℃, 4000 xg, centrifuge for 10 min to collect the bacteria, and discard the supernatant.
9. Gently resuspend pellets in 50 mL pre-cold Double-Distilled Water. Stay on ice for 5 min.
10. Centrifuge at 4℃, 4000 xg, centrifuge for 10 min to collect the bacteria, and discard the supernatant.
11. Gently resuspend pellets in 2 mL pre-cold 10% glycerol. Stay on ice for 5 min
12. Centrifuge at 4℃, 4000 xg, centrifuge for 10 min to collect the bacteria, and discard the supernatant.
13. Gently resuspend pellets in 50 μL pre-cold 10% glycerol.
14. Add 100 ng plasmid DNA. Stay on ice for 30 min.
15. Transfer the cells into the pre-cold cuvette.
16. Wipe moisture from the cuvette and insert the cuvette into the device, with the parameters below:
![Electroporation parameters for the Multiporator/Eppendorf Eporator device](https://static.igem.wiki/teams/5363/protocol/salmonella-electroporation.png)17. Immediately add 1 mL pre-warmed SOC medium.
18. Transfer the cells to 1.5 mL EP tube. Incubate at 37℃, 220 rpm, 1h.
19. Centrifuge at 5,000 rpm, 3 min. Resuspend the pellets in 100 μL LB.
20. Spread the cells on pre-warmed LB+Amp plates. Incubate at 37℃, overnight

Bacteria Growth Curve Measurement Protocol

1. Inoculate the bacteria in 1.5ml EP tubes and incubate for more than 12 hours to make sure that the concentration of bacteria tends to be stable
2. Prepare multiple (the number is decided by how many time points you want) 10ml tubes and liquid medium, each tube is inoculated with 100uL bacterial solution from Step 1, and incubation.
3. Put the tube on the ice once it reaches the set time.
4. Measure its OD600 with spectrophotometer
5. Measure its CFUs:
   1. Dilute the solution with different magnifications
   2. Choose 3 appropriation dilution multiples and plate 100ul bacteria solution onto solid culture plates (“appropriate” means each plate has 30-300 colonies) Incubated them at 37°C
   3. Count the colony's number and determine the original CFUs
6. Repeat Step 4-5 for all the tubes.

Beyotime Plasmid Mini Preparation Kit

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TIANGEN EndoFree Mini Plasmid Kit II

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TIANprep Mini Plasmid Kit

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2×Es Taq MasterMix (Dye)

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2×Phanta Flash MasterMix

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2×Phanta Max MasterMix

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Agarose gel electrophoresis

1. Add an appropriate amount of agarose and TAE to the beaker. The ratio of agarose mass to TAE volume is shown in the following figure.
![Ratio of Agarose and TAE](https://static.igem.wiki/teams/5363/protocol/ratio-of-agarose-and-tae.png)2. Bring slurry to a boil in a microwave (heat about 2 minutes on high power).
3. After the agarose solution is slowly cooled (but still liquid), add GELRED at a ratio of 1:20000.
4. Set up the gel tray in the gel box (in the casting orientation, which is perpendicular to the long axis of the gel box) and insert a comb. Pour molten agarose into the gel box. The agar is completely set when it becomes opaque (< 20 min).
5. Add the loading buffer/dye to the DNA samples to a final concentration of 1×.
6. Load samples and marker on the gel.
7. Run the gel at constant voltage of 120~150V. When the gel has run far enough (the bromophenol blue should still be in the gel), view the gel using the gel documentary.

DNA Marker

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GeneJET PCR Purification Kit

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TIANgel Midi Purification Kit (Spin Column)

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In-Fusion Snap Assembly User Manual

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pEASY®-Basic Seamless Cloning and Assembly Kit

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Mut Express II Fast Mutagenesis Kit V2

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DMT Enzyme

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T4 DNA Ligase (Takara)

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E.coli Competent Cell Protocol

1. **Plate Coating**
   To achieve optimal sensory efficiency, glycerol bacteria or other preserved bacterial strains must be coated on LB plates and cultured overnight.
2. **Vaccination**
   Place freshly cultured bacterial strains on a weighing plate, and perform subsequent operations in a clean bench. Dip the tip of the tweezers in 70% alcohol and slightly burn it on an alcohol lamp to sterilize the tip. Use the tweezers to pick up a sterile plastic tip or toothpick, select a monoclonal colony from the plate, and then place the plastic tip or toothpick dipped in the bacterial strain into a bacterial culture tube containing 3 mL of LB broth. This operation can also be performed using an inoculation loop.
3. **Cultivation**
   Cultivate the bacteria overnight at 37℃ and 200 rpm, controlling the cultivation time to approximately 16 hours, not exceeding 18 hours.
4. **Re-inoculation Culture**
   Inoculate fresh overnight bacteria into a fresh culture in a 1:500 ratio based on the amount of supercompetent bacteria required. For instance, add 100 μL of fresh overnight bacteria to 50 mL of the supercompetent bacterial culture medium provided in the kit. Cultivate at 37°C and 200 rpm for approximately 2.5-3.5 hours until the OD600 reaches 0.6-0.75.
5. **Preparation of Competent Bacteria**
   1. When the OD600 of the cultured bacteria reaches 0.6-0.75, place the bacterial culture in an ice bath and cool for 15 minutes. Note: All subsequent operations must be performed at 4°C or in an ice bath.
   2. Centrifuge at 4000 g at 4°C for 5 minutes to collect bacteria, and discard the supernatant. Note: The centrifuge must be pre-cooled.
   3. If the initial bacterial culture volume is 50 mL, follow the subsequent procedures. Adjust volumes proportionally for other volumes.
   4. Resuspend the bacterial pellet gently using 20 mL of pre-cooled supercompetent preparation reagent. Suspend very lightly and slowly to avoid disrupting the bacteria.
   5. Ice bath for 15 minutes.
   6. Centrifuge at 4000 g at 4°C for 5 minutes to collect bacteria, and discard the supernatant.
   7. Gently resuspend the bacterial pellet using 2 mL of pre-cooled supercompetent preparation reagent B. Suspend very lightly and slowly to avoid disrupting the bacteria.
   8. Ice bath for 15 minutes.
   9. On an ice bath, divide the suspension into 50-200 μL aliquots as needed.
   10. Use immediately or freeze in liquid nitrogen or an ethanol dry ice bath and store at -70°C. Efficiency typically remains stable for up to 6 months.
6. **Use of Competent Bacteria**
   1. For Determining Competent Bacteria Efficiency or Converting Ligation Products:
      1. Slowly melt the competent bacteria on ice (freshly prepared competent bacteria can be used directly). To test the efficiency of competent bacteria, add 10 pg-1 ng plasmid, but the volume of plasmid should not exceed 10% of the volume of competent bacteria. For converting ligation products, add 2-10 μL of ligation product for every 50 μL of competent bacteria.
      2. Gently flick the centrifuge tube with your fingers to mix the bacteria and plasmids or ligation products. Incubate in an ice bath or ice water bath for 30 minutes.
      3. Heat shock in a 42°C water bath for 2 minutes.
      4. Immediately after heat shock, place in an ice water bath for 2 minutes.
      5. Add 900 μL of LB broth and incubate at 37°C and 200 rpm for 1 hour.
      6. For efficiency testing, plate 100 μL onto LB plates containing the appropriate antibiotics. For ligation products, centrifuge at 2000-3000 g for 1 minute or longer to allow the bacteria to fully precipitate. Remove approximately 900-950 μL of LB culture medium. Resuspend the bacterial pellet in the remaining LB culture medium and plate onto LB plates containing the appropriate antibiotics. Incubate overnight at 37°C.
   2. For Plasmid Transformation:
      1. Dissolve the competent bacteria and add at least 50 ng of plasmid to 30-50 μL of competent bacteria. The amount of plasmid can be 1 μg or more, but the volume should not exceed 10% of the volume of competent bacteria.
      2. Gently flick the centrifuge tube with your fingers to mix the bacteria and plasmid.
      3. Incubate in an ice bath or ice water bath for 10 minutes.
      4. Plate all of the bacterial suspension directly onto LB plates containing the appropriate antibiotics and incubate overnight at 37°C.

Multiporator/Eppendorf Eporator Salmonella typhimurium

Salmonella Electroporation Protocol

1. Pick one clone from LB plate stored at 4 ℃.
2. Inoculate the clone in 2 mL LB broth, 37℃, 220 rpm, overnight.
3. Inoculate the overnight culture at 1:100 ratio to 100 mL LB.
4. Incubate at 37℃, 220 rpm, until OD(600)=0.6.
5. Chill the tubes on ice-water bath for 15 min.
6. (Pre-cool the centrifuge) Centrifuge at 4℃, 4000 xg, centrifuge for 15 min to collect the bacteria, and discard the supernatant.
7. Gently resuspend pellets in 100 mL pre-cold Double-Distilled Water. Stay on ice for 5 min.
8. Centrifuge at 4℃, 4000 xg, centrifuge for 10 min to collect the bacteria, and discard the supernatant.
9. Gently resuspend pellets in 50 mL pre-cold Double-Distilled Water. Stay on ice for 5 min.
10. Centrifuge at 4℃, 4000 xg, centrifuge for 10 min to collect the bacteria, and discard the supernatant.
11. Gently resuspend pellets in 2 mL pre-cold 10% glycerol. Stay on ice for 5 min
12. Centrifuge at 4℃, 4000 xg, centrifuge for 10 min to collect the bacteria, and discard the supernatant.
13. Gently resuspend pellets in 50 μL pre-cold 10% glycerol.
14. Add 100 ng plasmid DNA. Stay on ice for 30 min.
15. Transfer the cells into the pre-cold cuvette.
16. Wipe moisture from the cuvette and insert the cuvette into the device, with the parameters below:
![Electroporation parameters for the Multiporator/Eppendorf Eporator device](https://static.igem.wiki/teams/5363/protocol/salmonella-electroporation.png)17. Immediately add 1 mL pre-warmed SOC medium.
18. Transfer the cells to 1.5 mL EP tube. Incubate at 37℃, 220 rpm, 1h.
19. Centrifuge at 5,000 rpm, 3 min. Resuspend the pellets in 100 μL LB.
20. Spread the cells on pre-warmed LB+Amp plates. Incubate at 37℃, overnight

Super Competent Cell Preparation Kit by Beyotime

Ice box needed

1. Dilute the overnight culture with medium provided by the kit with ratio of 1:500, and cultivate at 37℃, 200 rpm. After 2.5-3.5 hours, OD600 can reach 0.6-0.7.
2. Put the bacterial fluid on ice and cool it for 15 min. (The following steps should all be on ice)
3. (Pre-cool the centrifuge) 4℃, 4000 xg, centrifuge for 5 min, discard the supernatant.
4. Resuspend the bacteria pellet with 20 mL Reagent A softly and slowly.
5. Ice bath for 15 min.
6. (Pre-cool the centrifuge) 4℃, 4000 xg, centrifuge for 5 min to collect the bacteria, and discard the supernatant.
7. Use 2 mL pre-cooled Reagent B to resuspend bacteria pellet softly and slowly.
8. Ice bath for 15 min.
9. Package on ice, 50-200 μL per tube according to the need.
10. (For storage) Freeze with liquid nitrogen or ethanol in a dry ice bath and keep at -80℃.

Beyotime DH5α Super Competent Cells

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Trans1-T1 Phage Resistant Chemically Competent Cell

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Protocol for Maintaining Salmonella Culture

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WB protocol

Before we begin:

1. Confirm the objective.
2. Verify the dilution buffer and blocking buffer.
3. Calculate dilution and set up your layout according to the protein extracted and your objective.

1. Protein preparation :

1. Digest and collect cells and use PBS to wash 2~3 times
2. Add Ripa into cell pellet and resuspend, put it on ice for 1 h
3. Add loading dye (dilute from 4× to 1×)
4. 95℃ 5~10min

2. Loading and running the gel

1. Select an appropriate SDS-PAGE gel. (15% or 12%)
2. Loading equal mass of protein in a inverted order (loading the ladder lastly to make up with wrong loading)
3. Run the gel (Use lower voltage (80mV) at first to make a line when running gel. Then increase the voltage to higher, like 120mV. After finishing running and before getting out the gel, set a low voltage(30mV) in case of diffusion.)
   Prepare cooling water when running gel!(for semi-dry method)

3. Transferring from the gel to the membrane (semi-dry method)

1. Soak the membrane in methanol, if using a PVDF membrane. Then in a balance buffer (semi-dry method needs to use balanced buffer. Methanol is necessary)
2. Assemble the SDS-PAGE gel and the membrane in the transfer cassette. Heavy band on the bottom of the transfer cassette (higher temperature and ion concentration)
3. Run the transfer
4. Cooling it in water

4. Blocking and antibody incubation

1. Prepare blocking buffer and antibody dilution buffer: dissolve 5% BSA into 1×TBST
2. Place the membrane in a container and cover with blocking buffer. For chemiluminescent western blot, incubate the membrane with gentle rocking overnight at 4 °C, or for 1 h at room temperature
3. Cover the membrane with diluted primary antibody (Incubate with gentle rocking overnight at 4 °C, or 1~2h at room temperature.)
4. Wash the membrane three times with 1×TBST, 10mins each
5. Cover the membrane with second antibody (1h at RT)
6. Wash the membrane three times with 1×TBST, 10mins each

5. Detection

1. Clean the imaging scanning bed (clean it every time between different membranes to detect!)
2. Cover the membrane with ECL.
3. Adjust the mode (chemiluminescent/ colormetric) and exposure time and number of photos
4. After scanning, merge and rename photos. Export photos.

6. Preservation

Wash down the ECL with TBST and put in -20℃

Attention:

1. PVDF is very easy to dry
2. Add secondary antibody next to the fridge.

ScFv Purification Protocol

Expression

1. Culture at least 10mL LB broth with Carbenicillin(50 μ g/mL)from glycerol bacteria, 37℃ 220 rpm, for no more than 12h.
2. Add 10ml cultured bacteria into 1L LB broth with Carbenicillin(50 μ g/mL), then grow in the shaker under 37℃220 rpm, meansure the OD600 by the Ultraviolet spectrophotometer, until the OD600 reaches 0.6-0.8.
3. Add 10ml ultrapure water dissolved-20% Arabinose solution (mass fraction), then incubate at 30℃ in the shaker with 220 rpm.

Collection and Sonic

1. After 20h, collect the bacteria by centrifuging at 6,000g, 15min, 4℃.
2. Discard the supernatant and resuspend the pellet by 80ml pre-cooled TBS buffer in total. Add PMSF solution, which is dissolved in 100% ethanol, to final concentration at 0.8mM.
3. Sonic the TBS-bacteria solution under 600W, work 5s following stop 3s, 30min total.
4. Centrifuge the soniced solution uner 14,000g for 45min, 4℃. Collect the supernatant.

Purification

1. Add 2mL Ni-charged resin into the supernatant. Incubate in 4℃ on Rotating mixing instrument, overnight.
2. Use gravity column to flow through, and wash by TBS with 40mM immidazole for 20mL. Elute by TBS with 300mM immidazole for 12mL.
3. Use a 10kDa concentration tube to concentrate 5mL of it to 500μ L.
4. Load the concentrated solution to chromatographic protein purifier.

SDS-PAGE

1. Mix the samples and 4×NuPAGE loading buffer together, respectively.
2. Boil the mixture on heating block at 98℃ for at least 15min. Then put into a pre-cooled centrigue, 6,000g, 5min at 4℃.
3. Load the samples into pre-cast 4-20% gels, run in MOPS buffer, constant voltage at 125V, for 60min.
4. Take the gel out and stain with Comassie blue.

Western-Blot

1. Run the SDS-PAGE gel as discribed previously.
2. Transfrom the brand to 0.45μm PVDF membrane by the Genescript®eblot L1.
3. Block the membrane in 5% TBST-dissolve BSA, for at least 1h under room temperature.
4. Transform the membrane to the HRP-linked His tag antibody solution, which is 1:2000 diluted in 1×TBST. Incubate overnight at 4℃ on 40 rpm horizontal shaker.
5. Wash the membrane with TBST for 3 times, 15min each.
6. Use enhanced chemiluminescence to detect the brands.

ELISA (MULTI SCIENCES EK1182) for scFv

1. 100 μL 2ng/ml of HER2 standard is added to each well.
2. Incubate 120 min at RT on shaker, 160 rpm.
3. 100 μL of purified anti-HER2 scFv per each well (10 and 100 ng/μL) was bound to the HER2 antigen.
4. Incubating at 37 °C for 60 min.
5. Wash 5 times with 100 μL/well wash buffer.
6. Add His-tag-HRP（MC10）Mouse Monoclonal Antibody (LABLEAD H2104).
7. Incubate 120 min at RT on shaker, 160 rpm.
8. Wash 5 times with 100 μL/well wash buffer.
9. Add 100 μL/well TMB substrate, and incubate at room temperature in the dark for 20 minutes.
10. Add 100 μL/well stop solution.
11. 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 the reference wavelength of 570 nm or 630 nm. The calibrated OD value is the measurement at 450 nm minus the measurement at 570 nm or 630 nm.

Human ErbB2/HER2/CD340 ELISA Kit

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Guidelines for Selection of Flow Fluorescent Dyes and Cell Function Testing Reagents

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Annexin V-FITC Apoptosis Detection Kit

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Media Recipes For MCF10A Cells: <https://brugge.hms.harvard.edu/protocols>

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CellTrace™ Cell Proliferation Kits

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Cell Experiment

Coculture Protocol:

Preparation:

The Growth Curve of bacteria

Passage the cell into needed plates

1. Inoculate the overnight bacteria solution 100ul into 10mL medium, static in 37°C for needed culturing time (usually 5-6h)
2. Harvest Bacteria to the needed concentration: the volume should be less than bacteria medium 1:cell medium 100, and the number should be cell number : bacteria CFUs at ratio 1:25 to 1:50
   1. Centrifuge at 4000g for 10 minutes, or 12000rpm for 2 minutes for 1.5mL EP tube
   2. Resuspend in appropriate volume of PBS
   3. (optional) Dye the bacteria and wash off the excess dye
3. Inoculate bacteria in the cell. Incubate at 37°C, 5%CO2 for an appropriate time
4. Observe under microscope, do FACS or other experiment

Cancer Cell Protocol

Basic rules:

1. Label on all the tubes and dishes: date + kind + passage number + other required things.
2. Different flasks/culture dishes/wells have different capacities of culture medium.
3. Always add things including cells against the wall of the container (unless you need to mix in culture dish).
4. Always count cells and observe cells to determine its confluence and vitality before adding mixture.
5. Always add culture medium and rinse before adding cells.
6. The condition of cell incubation and passage can be found on ATCC.
7. Be gentle when opening and closing the incubation box to prevent cell uneven growth. Don’t put your dish on the edge of the box to prevent it from dropping when you reach the dishes deep inside.
8. Discard pipette under right hand instead of above the dish.
9. Put your dish cover far away from operating area. Don’t operate above dish and dish cover. The best method is to open a crack with left hand.
10. When triturating, don’t let all liquid go out of pipette to prevent bubbles.

B16-F10:

* Medium: 90% DMEM +10% FBS +1% Antibiotics
* Subcultivation Ratio: 1:5 to 1:10
* Medium Renewal: Depend on the daily observation.

SK-BR-3:

* Medium: 90% McCoy's 5A +10% FBS +1% Antibiotics
* Subcultivation Ratio: 1:2 to 1:4
* Medium Renewal: 3-4 Days
* Notice:
  + The cell's adhesion ability is quite weak. Few floating cells were widely observed even after weeks of culturing, especially after thawing or subculturing.
  + The culturing in the first week needs to be very careful. Do not disturb the cells too often. To avoid cell loss, all the medium should not be discarded before centrifuge in the first week.

MCF10A:

* Medium: See the protocol "Media Recipes For MCF10A Cells"
* Subcultivation Ratio: 1:3, no more than 1:4
* Medium Renewal: 3-6 Days
* Notice:
  + The adhesion process of the cell is quite slow, do not disturb it for at least 3 hours after subculturing.
  + The adhesion force of the cell is quite strong, usually requiring more than 10 minutes at 37°C.
  + The cell comes from an epithelial cell, tending to form large pieces of tissue. The cell density is easily underestimated.
  + The medium component of the cell is relatively complex, some components might influence the measurement and experiment results.
  + The cells will die out soon under low cell density. Control the subculturing ratio.

Thawing:

1. Gentle agitation in 37°C water bath in 2 min to thaw.
2. Remove from the water bath as soon as thawed.
3. Decontaminate with 75% ethanol.

   *Operation starting from here should be under strict aseptic conditions.*
4. Centrifuge with 5.0 mL medium, 500g, 3-5 min.
5. Discard the supernatant.
6. Resuspend in medium and dispense into a 10 cm² dish.
7. Incubation.

Subculturing:

1. Remove the medium.
2. Rinse the cell layer with PBS to wash off the trypsin inhibitor.
3. Add 1.0 mL 0.25% Trypsin-0.53 mM EDTA. Wait until dispersed (usually 2 min).

   **Notice:** Do not shake when digesting or there will be clumping.
4. Add 1.0 mL serum-contained medium or soybean trypsin inhibitors to stop digesting.
5. (Optional) Centrifuge at an appropriate speed and discard the supernatant, then resuspend in the appropriate volume of the medium.
6. Transfer cells into new culture vessels in the appropriate ratio.
7. Incubation.

Cryopreservation:

1. Prepare the cryoprotectant, reference recipe: 60% Complete growth medium + 30% FBS + 10% DMSO.
2. Do Steps 1-5 in Subculturing, resuspend in 2 mL appropriate cryoprotectant instead of medium in step 5.
3. Put the cryopreservation tube in a cell-freezing container, then put the container at -80°C.

Lipo3000 Protocol

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Hieff Trans® Liposomal Transfection Reagent

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Equipment

![NanoPhotometer® NP80](https://static.igem.wiki/teams/5363/facilities/image1.png)

NanoPhotometer® NP80 from IMPLEN

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Amersham ImageQuant™ 800 from cytiva

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#### Westlake IGEM 2024

The Westlake iGEM team is a dedicated group of undergraduate researchers focused on advancing synthetic biology. Our project employs genetically engineered Salmonella for targeted cancer therapies. Committed to innovation and collaboration, we strive to make a meaningful impact in the fight against cancer while inspiring our community through research and education.

#### Links

* [Description](./description)
* [Model](./model)
* [Engineering](./engineering)
* [Education](./education)
* [integrated Human Practice](./human-practices)

#### Contact

No. 600 Dunyu Road,
XihuDistrict, Hangzhou,
Zhejiang, 310030, P.R. China

(+86)15058007607

gudingqi@westlake.edu.cn

---

© 2024 - Content on this site is licensed under a [Creative Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).

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![Florecent](https://static.igem.wiki/teams/5363/xcl/2-10-24-5-25.jpeg)

![Salmonella](https://static.igem.wiki/teams/5363/xcl/2-10-24-5-26.jpeg)

Results

“

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

Verification of *VNP20009* strain

![Figure 1.1 16s Assay Result of <em>VNP20009</em>](https://static.igem.wiki/teams/5363/results/1-1.png)

Figure 1.1 16s Assay Result of *VNP20009*

After doing 16s assay on the strain we received from BNUZH-China, 2023 iGEM team, we did BLAST on National Library of Medicine. The result is shown in figure, which indicates that the strain we got is *Salmonella* *enterica serovar Typhimurium* strain LT2. Moreover, we did PCR about the deleted gene (*msb*, *purL*) of *VNP20009*, with wild type strain as positive control. The gel image below shows that the strain we got didn’t have *msb* or *purL* gene, and was probably *VNP20009*.

![Fig 1.2 1.5% Agarose Gel of Gene Deletion Verification of <em>VNP20009</em>](https://static.igem.wiki/teams/5363/results/1-2.png)

Fig 1.2 1.5% Agarose Gel of Gene Deletion Verification of *VNP20009*

Recognition System

Plasmid Construction

To verify the recognition of HER2 scFv to HER2 antigen, we synthesized the gene sequence of Lpp-OmpA-1-HER2\_scFv and insert it into BPK764 vector, where Lpp-OmpA-1 is the surface display system derived from Francisco, J A, et al.’s work (Francisco et al., 1992). Moreover, we synthesized two other surface display systems, one is from iGEM Standard Biological Parts, Lpp-OmpA-2, and the other is Non-OmpA from Jeiranikhameneh et al.’s work (Jeiranikhameneh et al., 2017), in order to compare their display efficacy. And we insert the synthesized genes to BPK764 as well.

![Figure 2.1 Construction of Lpp/Non-OmpA-scFv or scFv Plasmids for Function Verification](https://static.igem.wiki/teams/5363/results/2-1.png)

Figure 2.1 Construction of Lpp/Non-OmpA-scFv or scFv Plasmids for Function Verification

Considering that it is difficult to purify membrane protein from E.coli, we amplified HER2 scFv from BPK764 and insert it into pET28(a)+. Poly his sequence is added at 3’-terminal of HER2 scFv for following purification.

HER2scFv Purification

With online calculator, we got the size of HER2 scFv, which is 25.85 kDa.

Before Ni-NTA beads are added, we did SDS-PAGE and Western Blot to determine whether it could be expressed in **SHuffle*®* *E. coli*, which is a genetically engineered chemically competent K12 cells of *E. coli*, can express proteins containing disulfide bonds in the cytoplasm.

![Figure 2.2.1 SDS-PAGE and Western Blot Result of Samples from Steps during Purification](https://static.igem.wiki/teams/5363/results/2-2-1.png)

Figure 2.2.1 SDS-PAGE and Western Blot Result of Samples from Steps during Purification

From left to right (same for the two pictures): a) Marker: ColorMixed Protein Marker 180 (10-180 kDa) (RM19001). b) culture: LB culture from the overnight culture of *SHuffle*. c) supernatant of culture: the supernatant after centrifuging the overnight LB culture of *SHuffle*. d) precipitation of culture: the precipitation after centrifuging the overnight LB culture of *SHuffle*. e) mixture after sonication: the mixture after sonicating the *SHuffle* precipitation. f) supernatant after sonication: the supernatant after centrifuging the sonication mixture. g) precipitation after sonication: the precipitation after centrifuging the sonication mixture.

Direct antibody: Mouse monoclonal antibody against His label (LABLEAD H1104) LABLEAD

The left figure in Figure 2.2.1 shows that the antibody can be successfully expressed, although it was not so clear in the gel bands. There are always many other proteins in E.coli and it may show very dirty on the SDS-PAGE gel.

After the purification described in the protocol, we got the protein chromatography result. We also collected samples of each step during purification, followed by doing SDS-PAGE and Western Blot.

![Figure 2.2.2 SDS-PAGE and Western Blot Result of Samples from Purification by AKTA](https://static.igem.wiki/teams/5363/results/2-2-2.png)

Figure 2.2.2 SDS-PAGE and Western Blot Result of Samples from Purification by AKTA

(a) Marker: ColorMixed Protein Marker 180 (10-180 kDa) (RM19001).
(b) a: Flow through step.
(c) b: Wash step.
(d) c: Elute B1.
(e) d: Elute B4.
(f) e: Elute B5.
(g) f: Elute C4.
(h) g: Elute C5.
(i) h: Elute C6.
(j) i: Elute C7.
(k) j: Elute C8.
(l) k: Elute C9.

Direct antibody: Mouse monoclonal antibody against His label (LABLEAD H1104) LABLEAD.

There are two peaks from chromatography, and the bands in SDS-PAGE are relatively messy. We suppose the highest peak is polymer, but the result of Western Blot shows that there is only one band of scFv. So we used the samples from B4 and B5 to do the following experiments since they have higher purity.

Enzyme Linked Immunosorbent Assay (ELISA)

![Figure 2.3 The Results of ELISA](https://static.igem.wiki/teams/5363/results/2-3.png)

Figure 2.3 The Results of ELISA We did ELISA of the B5 hole of protein purified by AKTA.

It shows that the HER2 scFv we purified can efficiently recognize HER2 scFv. The results of 25.00 ng/mL, 50.000 ng/mL and 100.00 ng/mL are even above the detection limit of the ELISA kit. From the picture that is taken 20 min after adding TMB substrate, we can even see some precipitation in the left three holes. Hence, the binding efficiency of HER2 scFv used in our project is enough to recognize HER2 antigen on cancer cells efficiently.

Fluorescence Microscopy

![Figure 2.4.1 Co-culturing of MCF10A and Lpp-2 <em>VNP20009</em>](https://static.igem.wiki/teams/5363/results/f08ef233-9da1-4213-bebf-9f72d55ee66e.png)

Figure 2.4.1 Co-culturing of MCF10A and Lpp-2 *VNP20009*

(a): Bright field (b): The Cy5 channel of Far Red (c): Merged image of (a) and (b)

![Figure 2.4.2 Co-culturing of SK-BR-3 and Lpp-2 <em>VNP20009</em>](https://static.igem.wiki/teams/5363/results/2-4-2.png)

Figure 2.4.2 Co-culturing of SK-BR-3 and Lpp-2 *VNP20009*

(a): Bright field (b): The Cy5 channel of Far Red (c): Merged image of (a) and (b)

An obvious colocalization can be observed in Figure 2.4.1: all the fluorescent spots located in the cells whose apoptosis process had begun. A similar phenomenon can also be observed in Figure 2.4.2. However, due to the high infection efficiency towards SK-BR-3 and resulting great apoptosis rate (proven by subsequent Flow Cytometry), the colocalization is relatively weak. Overall, it shows that *VNP20009* has the ability to invade the cells SK-BR-3 and MCF10A.

Flow Cytometry

![Figure 2.5.1 GFP Channel in Co-culture FACS](https://static.igem.wiki/teams/5363/results/2-5-1.png)

Figure 2.5.1 GFP Channel in Co-culture FACS

(a): SK-BR-3 without bacteria (b): SK-BR-3 with Lpp-1 *VNP20009* (c): SK-BR-3 with Lpp-2 *VNP20009* (d): SK-BR-3 with Lpp-2 E.coli (e): MCF10A with Lpp-1 *VNP20009* (f): MCF10A with Lpp-2 *VNP20009* SK-BR-3 is a HER2 positive breast cancer cell line. MCF10A is a HER2 negative non-tumorigenic epithelial cell line.

![Figure 2.5.2 DAPI Channel in Co-culture FACS](https://static.igem.wiki/teams/5363/results/2-5-2.png)

Figure 2.5.2 DAPI Channel in Co-culture FACS

(a): MCF10A without bacteria (b): MCF10A with Lpp-2 *VNP20009* (c): SK-BR-3 without bacteria (d): SK-BR-3 with Lpp-2 *VNP20009* SK-BR-3 is a HER2 positive breast cancer cell line. MCF10A is a HER2 negative non-tumorigenic epithelial cell line.

In this flow cytometry, all the green fluorescence protein was from bacteria, and only the cell infected with bacteria would show positive signal in GFP channels. In Figure 2.5.1, only the SK-BR-3 co-cultured with Lpp1/Lpp-2 *VNP20009* showed strong positive signal (more than 60%) in the GFP channel, and only a few (15% on average) cells were invaded by *VNP20009* for MCF10A. That's a strong statement that our recognition system has an excellent specific targeting ability.

Moreover, Figure 2.5.2 showed the apoptosis rate change with bacteria: MCF10A was unaffected by co-culturing while SK-BR-3 showed significant decrease in survival rate, which proved the engineered bacteria is safe for normal cells.

Delivery System

Plasmid Construction

To verify that *PsseJ* promoter is only activated when *Salmonella* is in human cells, we constructed two plasmids from pYX00004. The original pYX00004 (pBAD-mCherry-2aa) vector includes *araBAD* promoter and mCherry. We firstly replaced the *araBAD* promoter and *araC* with *PsseJ* promoter, and got pYX00004 (PsseJ-mCherry-2aa). Then we replaced the mCherry with *lyseE* gene from *φ*174X.

![Figure 3.1 Plasmid Construction Based on pYX00004 Vector for <em>PsseJ</em> Function Verification](https://static.igem.wiki/teams/5363/results/3-1.png)

Figure 3.1 Plasmid Construction Based on pYX00004 Vector for PsseJ Function Verification

Extracellular Verification

![Figure 3.2 Fluorescence Comparison of the Activation Strength of pBAD and PsseJ](https://static.igem.wiki/teams/5363/results/3-20.png)

Figure 3.2 Fluorescence Comparison of the Activation Strength of *pBAD* and *PsseJ*

From left to right: 0.02% arabinose, pBAD-mCherry; 0.00% arabinose, pBAD-mCherry; 0.00% arabinose, PsseJ-mCherry.

We cultured 3 groups of *VNP20009* transformed with pYX00004 (PsseJ-mCherry) and pYX00004 (pBAD-mCherry) to LB Broth, and add 0.02% arabinose to one group of pYX00004 (pBAD-mCherry). After 24 hours incubation, we washed the bacteria culture with PBS and observed them under fluorescence microscopy. The fluorescence strength of PsseJ-mCherry group is similar to pBAD-mCherry group cultured in 0% arabinose, and is much weaker than pBAD-mCherry group cultured in 0.02% arabinose. It shows that *PsseJ* is not activated when *Salmonella* is not in human cells.

Flow Cytometry

![Figure 3.3.1 mCherry Channel in co-culturing FACS](https://static.igem.wiki/teams/5363/results/3-3-1.png)

Figure 3.3.1 mCherry Channel in co-culturing FACS

(a): SK-BR-3 without bacteria (b): SK-BR-3 without bacteria (c): MCF10A with PsseJ-mCherry *VNP20009* (d): SK-BR-3 with PsseJ-mCherry *VNP20009* SK-BR-3 is a HER2 positive breast cancer cell line. MCF10A is a HER2 negative non-tumorigenic epithelial cell line.

The flow cytometry result also supports that mCherry fluorescence would be only expressed when PsseJ-mCherry *VNP20009* co-cultured with SK-BR-3, verifying the function of *PsseJ* .

Verification of the function of *BAX* gene in B16-F10 cells

Plasmid Construction

To verify the function of *BAX* gene, specifically its ability to induce programmed cell apoptosis, we first used the PCR method to amplify and cut the BAX sequence from pSL886 PMusAFP-MusBax-(MS2)24-HHR-pA (Shao et al., 2024) and inserted the sequence into CD513B-Bax vector. The constructed plasmids were transfected into DH5α competent cells and verified through sequencing, which confirmed the correct sequence of the *BAX* gene.

![Figure 4.1 Construction of CD513B-Bax Plasmid for BAX Function Verification](https://static.igem.wiki/teams/5363/results/4-1.png)

Figure 4.1 Construction of CD513B-Bax Plasmid for BAX Function Verification

Cells and Fluorescence Signal Observation

Next, to examine the function of BAX inside the cancer cells, we extracted the plasmid using an EndoFree DNA purification kit for subsequent cell transformation, then transfected the purified CD513B-Bax plasmid into the B16-F10 cell line. To verify its ability to inhibit cell growth and induce apoptosis, we used an inverted microscope for initial observations and flow cytometry with Annexin V-FITC and PI double staining to measure cell death and the death rate. After that, fluorescence microscopy and Western blot analysis were employed to confirm the expression of *BAX* gene inside the cell line, thereby demonstrating the function of *BAX* gene.

![Figure 4.2 B16-F10 Cell Images after 24h Transfection Under 10x Microscope](https://static.igem.wiki/teams/5363/results/4-2.png)

Figure 4.2 B16-F10 Cell Images after 24h Transfection Under 10x Microscope

(a) B16-F10 cells before transfection (b) Untreated B16-F10 cells after 24h transfection in bright field (c) B16-F10 cells with Liposomal Transfection Reagent added after 24h transfection in bright field (d) B16-F10 cells transfected with CD513B-SV40 plasmid by Liposomal Transfection Reagent after 24h transfection in bright field (e) B16-F10 cells transfected with CD513B-Bax plasmid by Liposomal Transfection Reagent after 24h transfection in bright field (f) Merged image of B16-F10 cells transfected with CD513B-Bax plasmid by Liposomal Transfection Reagent after 24h transfection in bright field and in green field

![Table 2. Cell Number of Each Group after 24h Transfection](https://static.igem.wiki/teams/5363/results/2024-10-02-165529.png)

Note:

cell: untreated B16-F10 cells

lipo: B16-F10 cells with Liposomal Transfection Reagent added

CD513B: B16-F10 cells transfected with CD513B-SV40 plasmid by Liposomal Transfection Reagent

BAX: B16-F10 cells transfected with CD513B-Bax plasmid by Liposomal Transfection Reagent

It is observed that the untreated cells, cells mixed with the transfection reagent, and cells transfected with the control plasmid (CD513B-SV40) continued to grow under experimental conditions over 24 hours. Most cells maintained their normal shape, and the seeding density increased from about 70% to above 90%. In contrast, cells transfected with the CD513B-Bax plasmid failed to increase in seeding density and exhibited clusters of dead cells. Referring to the cell numbers in Table 1, the BAX-transfected group had a cell count that was only about one-third of the other groups.

When the bright field and green field images were merged as in Figure 2(f), it was noted that the fluorescence signal was present in approximately 60% of the cells, indicating successful expression of the *BAX* gene in B16-F10 cells. Moreover, a relatively higher fluorescence intensity was observed in dead cells, which is consistent with the known function of the *BAX* gene.

Flow Cytometry

![Figure 4.3 Plots of different treatment groups of B16-F10 cells double-stained by Annexin-FITC and PI after 12h and 24h transfection](https://static.igem.wiki/teams/5363/results/4-3.png)

Figure 4.3 Plots of different treatment groups of B16-F10 cells double-stained by Annexin-FITC and PI after 12h and 24h transfection

(a) Plots of untreated B16-F10 cells after 12h transfection (b) Plots B16-F10 cells with Liposomal Transfection Reagent added after 12h transfection (c) Plots B16-F10 cells transfected with CD513B-SV40 plasmid by Liposomal Transfection Reagent after 12h transfection (d) Plots B16-F10 cells transfected with CD513B-Bax plasmid by Liposomal Transfection Reagent after 12h (e) Plots of untreated B16-F10 cells after 24h transfection (f) Plots B16-F10 cells with Liposomal Transfection Reagent added after 24h transfection (g) Plots B16-F10 cells transfected with CD513B-SV40 plasmid by Liposomal Transfection Reagent after 24h transfection (h) Plots B16-F10 cells transfected with CD513B-Bax plasmid by Liposomal Transfection Reagent after 24h

In the flow cytometry test, positive results in the FITC channel, derived from Annexin V-FITC staining, indicate cells in the early apoptosis stage. Positive results in the PerCP channel, from PI staining, indicate cells in the late apoptosis stage. In the double-staining condition, double-positive results are specifically regarded as dead cells.

When comparing the different treatment groups, after 12 hours of transfection, the death rate in all four groups was relatively low, with more than 85% of the cells still in the early apoptosis stage. However, after 24 hours of transfection, the CD513B-Bax transfected group showed a death rate three times higher than the other three groups, confirming the *BAX* gene's function in inducing cell apoptosis.

Western Blot

![Table 3. Western Blot SDS-PAGE Layout for BAX Protein Detection (Experiment Date: 9/30)](https://static.igem.wiki/teams/5363/results/2024-10-02-165538.png)

(a) BAX(ab32503): Rabbit Anti-Bax antibody [E63].

(b) Marker: Thermo #26619.

(c) NC: Negative control, representing the protein extracted from cells that were not transfected with the BAX plasmid.

(d) BAX: Protein extracted from cells that were transfected with the BAX plasmid.

(e) Bio-switch on: Protein extracted from cells transfected with the bio-switch plasmid, in the "on" state.

(f) Bio-switch off: Protein extracted from cells transfected with the bio-switch plasmid, in the "off" state.

Primary antibody: Rabbit Anti-Bax antibody [E63] (ab32053) 21kDa abcam 1:1000 dilution

Secondary antibody: Goat Anti-Rabbit IgG H&L (HRP) (ab205718) abcam 1:2000 dilution

Marker: Thermo #26619

![Western Blot Image](https://static.igem.wiki/teams/5363/results/2024-10-02-154224.png)
![Chemiluminescence Detection](https://static.igem.wiki/teams/5363/results/2024-10-01-164045.png)

Figure 4.4 Chemiluminescence Detection of BAX Protein in BAX and Negative Control Samples

(a) BAX (above the image): Protein extracted from cells transfected with the BAX plasmid.

(b) NC: Negative control, representing protein extracted from cells that were not transfected with the BAX plasmid.

(c) 21 kDa: The expected and observed molecular weight of the BAX protein band, visible at 21 kDa.

(d) BAX (beside the image): The primary antibody used was anti-BAX, targeting and detecting the BAX protein.

In the primary antibody analysis of the BAX gel, the BAX group showed a significantly stronger protein signal compared to the negative control. This indicated that the BAX plasmid was successfully transfected into the B16-F10 cells and that the protein was expressed effectively. Furthermore, the relatively high expression level of BAX suggested that it is responsible for inducing apoptosis in the cells. When comparing the protein extracted on September 25 to that extracted on September 30, the signal from the latter was notably stronger, indicating protein deduction due to storage.

Verification of the bio-switch in B16-F10 cells

Cells and Fluorescence Signal Observation

![Merged Image](https://static.igem.wiki/teams/5363/results/5-1.png)

Figure 5.1. Merged Image of B16-F10 Cell Images after 24h Transfection of pSL816 and SB100 Under 10x Microscope in Bright Field and Green Field

![Cell Images](https://static.igem.wiki/teams/5363/results/5-2.png)

Figure 5.2 B16-F10 Cell Images after 24h Transfection of Bio-switch Plasmids Under 10x Microscope

(a) B16-F10 cells transfected with bio-switch plasmids after 24h transfection in bright field

(b) B16-F10 cells transfected with both target protein plasmids and bio-switch plasmids after 24h transfection in bright field

Western Blot

To verify the function of the bio-switch, we first transfected the B16-F10 cell line with pSL816 (Shao et al., 2024) plasmid containing the target protein NS3a and EGFP to express NS3a within the cells. After 24 hours, intense green fluorescence was observed, indicating a transfection efficiency of over 70% and confirming the expression of NS3a.

Subsequently, we co-transfected the three bio-switch plasmids (pSL886, pSL582, and pSL776) (Shao et al., 2024) into the B16-F10 cells. Under inverted microscope observations after 24 hours, cells without NS3a continued to grow normally with typical cell shapes. In contrast, cells co-transfected with NS3a and the bio-switch plasmids exhibited cell clustering, indicating the activation of the bio-switch. This activation upregulates the expression of the killer gene BAX in the presence of the target protein and remains inactive in its absence.

![Table 4. Western Blot SDS-PAGE Layout (Experiment Date: 9/30)](https://static.igem.wiki/teams/5363/results/2024-10-02-165552.png)

(a) BAX(ab32503): Rabbit Anti-Bax antibody [E63].

(b) Marker: Thermo #26619.

(c) NC: Negative control, representing the protein extracted from cells that were not transfected with the BAX plasmid.

(d) BAX: Protein extracted from cells that were transfected with the BAX plasmid.

(e) Bio-switch on: Protein extracted from cells transfected with the bio-switch plasmid, in the "on" state.

(f) Bio-switch off: Protein extracted from cells transfected with the bio-switch plasmid, in the "off" state.

Primary antibody: Rabbit Anti-Bax antibody [E63] (ab32053) 21kDa abcam 1:1000 dilution

Secondary antibody: Goat Anti-Rabbit IgG H&L (HRP) (ab205718) abcam 1:2000 dilution

Marker: Thermo #26619

![Chemiluminescence Detection of BAX](https://static.igem.wiki/teams/5363/results/2024-10-01-164048.png)

Figure 5.2 Chemiluminescence Detection of BAX Protein in Bio-Switch "On" and "Off" States

(a) Bio-switch on: Protein extracted from cells transfected with the bio-switch plasmid, in the "on" state.

(b) Bio-switch off: Protein extracted from cells transfected with the bio-switch plasmid, in the "off" state.

(c) 21 kDa: The expected and observed molecular weight of the BAX protein band, visible at 21 kDa.

(d) BAX (beside the image): The primary antibody used was anti-BAX, targeting and detecting the BAX protein.

In the primary antibody analysis of the BAX gel, the bio-switch "on" group displayed a significantly stronger protein signal compared to the bio-switch "off" group. This suggested that the bio-switch plasmid was successfully transferred into the B16-F10 cells, leading to effective protein expression and successful control of BAX expression. Additionally, the relatively high level of BAX expression in the bio-switch "on" group indicated that the bio-switch can regulate apoptosis in the cells. However, when comparing the protein extracted on September 25 to that extracted on September 30, the signal from the former did not exhibit any expected trend.

Supplementary information of Western Blot experiment

![Table 5. Western Blot SDS-PAGE Layout for β-ACTIN Protein Detection](https://static.igem.wiki/teams/5363/results/2024-10-02-165600.png)

(a) β-ACTIN(643802): Purified anti-β-actin Antibody

(b) Marker: Thermo #26619

(c) NC: Negative control, representing the protein extracted from cells that were not transfected with the BAX plasmid.

(d) BAX: Protein extracted from cells that were transfected with the BAX plasmid.

Primary antibody: Purified anti-β-actin Antibody(643802) 43kDa BioLegend 1:500 dilution

Secondary antibody: HRP-conjugated Goat Anti-Mouse IgG(H+L) (SA00001-1) proteintech 1:2000 dilution

Marker: Thermo #26619

![Chemiluminescence Detection of β-actin](https://static.igem.wiki/teams/5363/results/2024-10-01-184955.png)

Figure 6.1 Chemiluminescence Detection of β-actin Protein in BAX and Negative Control Samples

(a) BAX (above the image): Protein extracted from cells transfected with the BAX plasmid.
(b) NC: Negative control, representing protein extracted from cells that were not transfected with the BAX plasmid.
(c) 43 kDa: The expected and observed molecular weight of the β-actin protein band, visible at 43 kDa.
(d) β-actin: The primary antibody used was anti-β-actin, targeting and detecting the β-actin protein.

In the primary antibody analysis of the β-actin gel, which served as the internal reference, the negative control group exhibited a slightly stronger signal than the BAX group, and the bio-switch "off" group showed a marginally stronger signal than the bio-switch "on" group. This trend suggested that the cell count in the BAX group was lower than in the negative control group, and the cell count in the bio-switch "on" group was lower than in the bio-switch "off" group, likely due to cell apoptosis (we collected cells of the same volume for each group). We guessed that the BAX plasmid was successfully transfected into the cells, leading to relatively high levels of BAX protein expression, which induced cell apoptosis, as did the bio-switch plasmids.

![Complete Chemiluminescence Detection of BAX Protein](https://static.igem.wiki/teams/5363/results/thumbnail-0930-baxwin-20240930-220347-03-ch-chemi-marker-1.png)

Figure 6.2 The Complete Chemiluminescence Detection of BAX Protein (Experiment Date: 9/30)

(a) Marker: Thermo #26619.
(b) NC: Negative control, representing the protein extracted from cells that were not transfected with the BAX plasmid.
(c) BAX: Protein extracted from cells that were transfected with the BAX plasmid.
(d) Bio-switch on: Protein extracted from cells transfected with the bio-switch plasmid, in the "on" state.
(e) Bio-switch off: Protein extracted from cells transfected with the bio-switch plasmid, in the "off" state.
(f) 21 kDa: The expected and observed molecular weight of the BAX protein band, visible at 21 kDa.

![Complete Chemiluminescence Detection of β-actin Protein](https://static.igem.wiki/teams/5363/results/beta-actin-0925-20240926-193341-08-ch-chemi-marker-1.png)

Figure 6.3 The Complete Chemiluminescence Detection of β-actin Protein

(a) Marker: Thermo #26619.
(b) NC: Negative control, representing the protein extracted from cells that were not transfected with the BAX plasmid.
(c) BAX: Protein extracted from cells that were transfected with the BAX plasmid.
(d) Bio-switch on: Protein extracted from cells transfected with the bio-switch plasmid, in the "on" state.
(e) Bio-switch off: Protein extracted from cells transfected with the bio-switch plasmid, in the "off" state.
(f) 43 kDa: The expected and observed molecular weight of the β-actin protein band, visible at 43 kDa.

References:

1. Francisco, J. A., Earhart, C. F., & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. *Proc Natl Acad Sci U S A, 89*(7), 2713-2717. https://doi.org/10.1073/pnas.89.7.2713
2. Jeiranikhameneh, M., Razavi, M. R., Irani, S., Siadat, S. D., & Oloomi, M. (2017). Designing novel construction for cell surface display of protein E on Escherichia coli using non-classical pathway based on Lpp-OmpA. *AMB Express, 7*(1), 53. https://doi.org/10.1186/s13568-017-0350-0
3. Shao, J., Li, S., Qiu, X., Jiang, J., Zhang, L., Wang, P., Si, Y., Wu, Y., He, M., Xiong, Q., Zhao, L., Li, Y., Fan, Y., Viviani, M., Fu, Y., Wu, C., Gao, T., Zhu, L., Fussenegger, M., . . . Xie, M. (2024). Engineered poly(A)-surrogates for translational regulation and therapeutic biocomputation in mammalian cells. *Cell Research, 34*(1), 31-46. https://doi.org/10.1038/s41422-023-00896-y

#### Westlake IGEM 2024

The Westlake iGEM team is a dedicated group of undergraduate researchers focused on advancing synthetic biology. Our project employs genetically engineered Salmonella for targeted cancer therapies. Committed to innovation and collaboration, we strive to make a meaningful impact in the fight against cancer while inspiring our community through research and education.

#### Links

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#### Contact

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