iGEM 2023 — Team bnuzh-china

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# Notebook

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* [Notebook](#Description)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:BNUZH-CHINA/Notebook

Week 1-2 (6.26~7.9)

## **Targeting module**

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

## **shRNA silencing module**

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

## **GOx killing module**

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

## **Anaerobic expression module**

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

Week 3-4 (7.10~7.23)

## **Targeting module**

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

## **shRNA silencing module**

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

## **GOx killing module**

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

## **Anaerobic expression module**

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

Week 5-6 (7.24~8.6)

## **Targeting module**

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

## **shRNA silencing module**

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

## **GOx killing module**

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

## **Anaerobic expression module**

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

Week 7-8 (8.7~8.20)

## **Targeting module**

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

## **shRNA silencing module**

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

## **GOx killing module**

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

## **Anaerobic expression module**

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

## **Safety module**

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

Week 9-10 (8.21~9.3)

## **Targeting module**

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

## **shRNA silencing module**

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

## **GOx killing module**

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

## **Safety module**

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

Week 11-12 (9.4~9.17)

## **Targeting module**

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

## **shRNA silencing module**

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

## **GOx killing module**

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

## **Safety module**

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

Week 13-14 (9.18~10.1)

## **shRNA silencing module**

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

## **Safety module**

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

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# Engineering

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Overview

On this page, we will demonstrate the step-by-step improvement of our project FeFighterS. We conducted four rounds of iterations, which followed the "design-build-test-learn" cycle in its entirety. Each full iteration comprised multiple smaller iterations. During each iteration, we extensively referred to the literature and engaged in discussions with experts and other iGEM teams (for more details, refer to [Integrated Human Practice](https://2023.igem.wiki/bnuzh-china/integrated-human-practice)). Additionally, we evaluated the feasibility of the design through dry lab modeling in certain iterations, which provided us with a deeper understanding of the design for further improvement (for more details, refer to [Model](https://2023.igem.wiki/bnuzh-china/model)). These recirculates have led to significant enhancements and matured for our FeFighterS project. Subsequently, we conducted tests to confirm the successful operation of each module within our engineered bacteria.

Targeting

Fenton

shRNA

Safety

Cycle 1 ：Magnetic targeting: conjugating Fe3O4 nanoparticles on the surface of engineered bacteria

## **Design**

Based on the application of ferroptosis in the killing module, we were naturally curious if the engineered
bacteria could be converted to magnetotactic bacteria that can be guided by an external magnetic field.
After reviewing the literature, we found that a research team has successfully linked magnetic Fe3O4
nanoparticles (MNP) covalently to *E. coli*, acting as a catalyst for a Fenton-like reaction[1](#Reference). Taking
inspiration from this study, we designed an amino-modified MNP that can link to N-acetyl muramic acid in
the bacteria’s wall for the purpose of anchoring the MNP to the engineered attenuated *Salmonella* (Figure
1). By altering the strength of the magnetic field *in vitro*, we aimed to enable the engineered bacteria to
specifically target tumor tissue.

![](https://static.igem.wiki/teams/4737/wiki/engineering/scfv/image001.png)

***Figure 1.** Schematic representation of conjugated Fe3O4 nanoparticles on the surface of
engineered bacteria.*

## **Build**

We treated the bacteria solution with magnetic Fe3O4 nanospheres with a diameter of 100 ~ 200 nm for 30
minutes on an ultrasonic vortex apparatus, and then shaked 3 h at 37℃, 220 rpm. After 3 h stir, the
modified bacteria were separated by differential centrifugation (3000 rpm, 5 min) and washed for three
times.

## **Test**

We placed the magnet on the side of the engineered bacteria coupled with Fe3O4 nanoparticles, and observed
that the engineered bacteria moved to the side with the magnet (Figure 2). In other words, engineered
bacteria can directly move under an external magnetic field.

![](https://static.igem.wiki/teams/4737/wiki/engineering/scfv/new/engineer-target-figure-2.png)

***Figure 2.** Engineered bacteria coupled to Fe3O4 nanoparticles were attracted by magnets. a.
Distribution of bacteria when the magnet was first placed. b,c. Bacterial distribution after 24 h of
magnet placement. Red frames outlined in the figure are engineered bacteria attracted by
magnets.*

## **Learn**

Although we successfully conjugated Fe3O4 nanoparticles on the surface of engineered bacteria, we realized
that magnetic targeting lacks specificity and has an imprecise targeting range. Furthermore, in deep tumor
tissue, the effectiveness of engineered bacteria targeting may be compromised. Additionally, through a
literature review, we discovered that attaching Fe3O4 nanoparticles to the surface of engineered bacteria
could potentially impact their invasiveness into cells [2](#Reference).

Cycle 2 ： Antigen-antibody targeting: expressing CEA single chain fragment variable on the outer membrane
of engineered bacteria

## **Design**

When our team communicated with the SCAU-China, they suggested us take advantage of the antigen-antibody
to improve specificity and targeting ability. In order to achieve more precise targeting *in vivo* and
ensure the smooth entry of engineered bacteria into tumor cells, we changed our thought and focused on the
antigen-antibody conjugation, a more precise targeting method. We reviewed the literature, found the
single chain fragment variable (scFv) of carcinoembryonic antigen (CEA). Inspired by the design of the
2018 HZAU-China team and the literature, we decided to conjugate scFv to the lipoprotein (Lpp) and the
outer membrane protein (OmpA) of *E. coli*[3,4](#Reference) (Figure 3). So that the scFv can be fixed on the surface of
VNP20009 and target CEA on the surface of tumor cells.

![](https://static.igem.wiki/teams/4737/wiki/engineering/scfv/image003.png)

***Figure 3.** Schematic representation of the engineered bacteria expressing CEA single chain
fragment variable on the outer membrane.*

## **Build**

To ensure that scFv could be successfully anchored to the cell surface by the Lpp-OmpA expression system,
we conducted molecular modeling of the scFv used in this project. And we employed molecular docking and
corresponding calculations to predict the structure of the antibody and its affinity for CEACAM5 (Figure
4-5).

![](https://static.igem.wiki/teams/4737/wiki/engineering/scfv/new/engineer-target-figure-4.png)

***Figure 4.** Prediction of the transmembrane regions of Lpp-OmpA-scFv.*

Notes: The angle of inclination is defined as the angle between the first
principal axis of the protein and the normal of the membrane. The angle of rotation is the angle
between the second principal axis of the atomic set and the normal of the plane defined by the first
principal axis and the normal of the membrane.

![](https://static.igem.wiki/teams/4737/wiki/engineering/scfv/new/engineer-target-figure-5.png)

***Figure 5.** The docking of antibody molecules with antigen molecules.*

Notes:(a) Results of molecular docking. Lpp-OmpA-scFv is shown in green and
CEACAM5 is shown in orange. (b) Schematic representation of the molecular docking surface. Among them,
amino acids that can form hydrogen bonds are shown.

To verify that the anti-CEA-scFv can actually work, we constructed the *pFPV25.1-[Lpp-OmpA-scFv]-GFP* plasmid and electroporated it into VNP20009.

## **Test**

Western blot was performed to verify whether the scFv could be expressed in the engineered bacteria. Our
data suggested that the scFv successfully expressed in VNP20009 (Figure 6).

![](https://static.igem.wiki/teams/4737/wiki/proof-new-zy/proof-of-concept-figure-5.png)

***Figure 6.** WB analysis of the expression of specific single chain antibody fragments (scFv).*

Notes: Lpp-OmpA-scFv-GFP molecular weight is about 66 kDa and GAPDH is a
reference protein in cells with a molecular weight of 36 kDa.

Next, we chose human gastric cancer cell line NUGC-3 with high-CEA-expression as experimental group, and a
human gastric cancer cell line BGC-823 with low-CEA-expression was used as CEA negative cell line. The
engineered bacteria with GFP tag and the negative control with RFP tag were used to infect the above two
types of cells, and the function of anti-CEA scFv was verified by the infection efficiency of the
bacteria. Unfortunately, during the experiment we found that the green fluorescence of the engineered
bacteria was very weak (Figure 7).

![](https://static.igem.wiki/teams/4737/wiki/engineering/scfv/image007.png)

***Figure 7.** Diagrammatic sketch of engineered bacteria with weak green fluorescence.*

## **Learn**

By reviewing the literature, we found that the VH and VL of scFv are connected by a linker, and the
transient dissociation of VH and VL leads to the instability of scFv and the downstream GFP protein[5](#Reference). This
implies that we need to seek more methods to make green fluorescence stably expressed.

Cycle 3 ： The engineered bacteria with stable green fluorescence were used to verify the function of scFv

## **Design**

In order to ensure that the engineered bacteria could express stable green fluorescence, we selected
VNP20009 competent cells with stable GFP conversion as plasmids receptors.

## **Build**

We prepared VNP20009 competent cells stably transfected with GFP and electroporated the constructed
plasmids into it.

## **Test**

We infected NUGC-3 and BGC-823 with the constructed engineered bacteria and negative control
simultaneously. For NUGC-3 with high-CEA-expression, the infection efficiency of engineered bacteria
(green fluorescence) was higher than that of negative control (red fluorescence), but there was no
significant difference in the infection efficiency of BGC-823 which was with low-CEA-expression. (Figure
8).

![](https://static.igem.wiki/teams/4737/wiki/proof-new-zy/co-infection-bgc-nugc.png)

***Figure 8.** Fluorescent microscopy results of BGC-823 and NUGC-3 cells co-infected by
engineered bacteria, and VNP20009 for 90 minutes. Both the engineered bacteria and
negative control infections were at the MOI of 1:50. Red frames outlined in the figure were engineered bacteria that infected cells.*

## **Learn**

To determine whether scFv really worked, we compared the efficiency of different bacteria in infecting
NUGC-3 and BGC-823. However, we could not exclude the possibility that there was an interaction between
the engineered bacteria and the negative control that affected their infection efficiency, which meant
that further improvement of our experiments was necessary.

In addition, NUGC-3 cells were in a poor state and died in large numbers after a short time of infection,
which resulted in a majority of bacteria being washed off with PBS along with the dead cells during the
infection process.

Cycle 4 ： Cell lines were replaced and scFv function was further verified

## **Design**

In this attempt, we selected the human colorectal cancer cell line LS174T, which also has a high
expression of CEA, as the experimental group.

## **Build**

In this attempt, we added control experiments in which the engineered bacteria and the negative control
infected the cells separately.

## **Test**

By analyzing the results of bacterial infection of LS174T cells and BGC-823 cells, we got the same
conclusions as above. Therefore, the scFv promoted the infection of bacteria no matter when the engineered
bacteria and the negative control infected the cells separately or simultaneously (Figure
9-11).

![](https://static.igem.wiki/teams/4737/wiki/engineering/scfv/image012.png)

***Figure 9.** Microphotographs of LS174T cells with scFv delivery by bacterial infection for 2
hours. Both the engineered bacteria and negative control infections were at the MOI of 1:50.*

![](https://static.igem.wiki/teams/4737/wiki/engineering/scfv/image013.png)

***Figure 10.** Microphotographs of BGC-823 cells with scFv delivery by bacterial infection for 2
hours. Both the engineered bacteria and negative control infections were at the MOI of 1:50.*

![](https://static.igem.wiki/teams/4737/wiki/proof-new-zy/proof-of-concept-figure-6.png)

***Figure 11.** Microphotographs of BGC-823 and LS174T cells co-infected by engineered bacteria
and VNP20009 for 2 hours. Both the engineered bacteria and negative control infections were at the
MOI of 1:50.*

## **Learn**

The experimental results demonstrated that the scFv could be expressed on the surface of VNP20009 and
effectively enhanced the targeting ability of engineered bacteria towards tumor cells. However, since the
bacterial infection of cells occurs within a confined space, it becomes imperative to further enhance the
targeting efficacy of engineered bacteria to meet the need of efficient tumor cell recognition in a
dynamic bloodstream environment.

Cycle 5 ： Site-specific amino acid mutations that may increase antigen-antibody affinity

## **Design**

To enable the engineered bacteria to have stronger tumor cell targeting ability, we tried to find out the
amino acid sites that are involved in antigen binding through modeling. Based on the results of modeling
we hoped to improve the affinity of the scFv by mutating the simulated sites.

## **Build**

We performed interaction-based virtual amino acid mutagenesis of antibody-antigen complexes by Discovery
Studio software. After the structure file from the molecular docking was uploaded, the complex protein was
subjected to a CHARMm force field, and the amino acid residues within 3 Å range around the ligand were
selected. By mutating all of the above amino acids to ALA and ranking the mutational energy results of the
individual mutations, we finally obtained five amino acids that are critical for antibody-antigen
interaction (LEU196, ARG177, ILE193, TYR190, ASN194) (Figure 12).

![](https://static.igem.wiki/teams/4737/wiki/model/model/figure-10.gif)

***Figure 12.** Schematic representation of amino acids critical for antibody affinity.*

Notes: The molecule labeled in yellow represents anti-CEA scFv; The molecule
labeled in blue represents CEACAM5. Amino acids in the antibody that resulted in decreased antibody
affinity after mutation to ALA are highlighted in red, whereas amino acids with increased antibody
affinity after mutation are highlighted in magenta.

## **Test**

In order to gain more detailed information on how mutations enhance antibody affinity, we performed
saturation mutagenesis on the aforementioned five amino acids. From our findings, we identified ASN195 as
a crucial site for improving the affinity of anti-CEA scFv. Substituting ASN195 with TRP, GLN, PHE, GLU or
HIS may significantly enhance the affinity of anti-CEA scFv.

We also calculated mutation energy to evaluate the effect of mutations on protein stability (Figure 13). By
calculation, we found that, except for ASN195 mutated to GLU and GLN, several other mutations with
increased affinity did not affect antibody stability.

![](https://static.igem.wiki/teams/4737/wiki/engineering/scfv/new/engineer-target-figure-13.png)

***Figure 13.** Mutation energy (Binding) analysis in single mutation of key amino acids in
scFv.*

## **Learn**

Based on the above modeling, we can perform site-directed mutagenesis of the scFv and test the affinity of
the modified antigen-antibody complex.

References

1 Feng, L. et al. Magnetic Targeting, Tumor Microenvironment-Responsive Intelligent Nanocatalysts for
Enhanced Tumor Ablation. ACS Nano 12, 11000-11012 (2018). https://doi.org:10.1021/acsnano.8b05042
2 Suh, S. et al. Nanoscale Bacteria-Enabled Autonomous Drug Delivery System (NanoBEADS) Enhances
Intratumoral Transport of Nanomedicine.Adv Sci (Weinh) 6, 1801309 (2019).
https://doi.org:10.1002/advs.201801309
3 Francisco, J. A., Stathopoulos, C., Warren, R. A., Kilburn, D. G. & Georgiou, G. Specific adhesion and
hydrolysis of cellulose by intact Escherichia coli expressing surface anchored cellulase or cellulose
binding domains. Biotechnology (N Y) 11, 491-495 (1993). https://doi.org:10.1038/nbt0493-491
4 Bereta, M. et al. Improving tumor targeting and therapeutic potential of Salmonella VNP20009 by
displaying cell surface CEA-specific antibodies. Vaccine 25, 4183-4192 (2007).
https://doi.org:10.1016/j.vaccine.2007.03.008
5 Wang, J. L. et al. Construction and activity analysis of a recombinant immunotoxin composed of PE38 and
a disulfide stable single-chain antibody. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi 22, 74-77 (2006).

Oxidation-inducing Module:

Inducing the Fenton reaction in tumor cells to regulate iron metabolism thereby inducing ferroptosis in
tumor cells

Cycle 1: Glucose oxidase (GOx) induces the Fenton reaction and
starvation

## **Design**

Through reviewing articles, we noticed that the glucose oxidase (GOx) from *Aspergillus niger* has the
ability to efficiently catalyze O2 to generate H2O2[1](#Reference). H2O2 can be catalyzed by Fe2+ to produce radical
HO• through Fenton reaction. It should be noted that HO•, also known as the hydroxyl radical, is
categorized as a reactive oxygen species (ROS). This particular ROS has the capability to intensify the
lipid peroxidation process of cell membranes. Ultimately, such intensification is responsible for inducing
ferroptosis in tumor cells. Engineered plasmids or proteins carried by recombinant *S. typhimurium* can be
delivered into tumor cells. The unique characteristics of *S. typhimurium* enable targeted protein to be
delivered into tumor cells. We designed to engineer VNP20009 to constitutively express glucose oxidase and
deliver the protein to the cytoplasm after invasion of tumor cells. In the catalysis of GOx, glucose will
be turned into gluconic acid, which can also reduce the energy source of tumor cells, further
strengthening the ferroptosis in tumor cells.

## **Build**

We have constructed a prokaryotic expression plasmid (*pFPV25.1-GOx*) that can be expressed in
*S. typhimurium* (Figure 1-2). We intend to transform the plasmid expressing GOx into VNP20009.

![](https://static.igem.wiki/teams/4737/wiki/engineering/gox/image001.png)

***Figure 1.** The expression of GOx*

![](https://static.igem.wiki/teams/4737/wiki/engineering/gox/image002.png)

***Figure 2.** The map of pFPV25.1-GOx*

## **Learn**

**S. typhimurium** VNP20009 has the ability to survive within host
cells. However, due to its encapsulation in intracellular vesicles, it exhibits limited or minimal
impact on the host cell cytoplasm. By reviewing the literature, we found that the efficiency of
protein delivery after entry into cells by *S. typhimurium* was not appreciable. We need to find a new
mode of delivering functional proteins.

Cycle 2: GOx can be delivered into tumor cells by the type III
secretion system of *S. typhimurium*.

## **Design**

Like many bacterial pathogens of animals, *S. typhimurium* have evolved a specialized protein-secretion
system to deliver bacterial proteins into host cells. *S. typhimurium* has a special secretion system: type
III secretion system (T3SS), which is closely related to bacterial colonization, infection and survival in
host cells[2](#Reference). We designed to use the secretion and translocation
signals of type III secreted protein *Salmonella* outer protein E (SopE) to fuse with GOx. So, engineered VNP20009 can express GOx and inject GOx into the tumor cells.

## **Build**

We integrated the translocation signal of *Salmonella* exoprotein E (SopE), a type III secretory protein,
atthe N-terminus of Gox (Figure 3-4). The gene fragment was synthesized by a commissioned company.
Subsequently, we assembled the fragments into the expression plasmid (*pFPV25.1*) using gibson assembly. We
then transferred the ligation products into DH5α competent cells and extracted plasmids for
identification. The successfully constructed plasmids (*pFPV25.1-SopE-FLAG-GOx*) were transferred into
VNP20009 competent cells. As a result, the engineered VNP20009 (VNP-SopE-GOx) is supposed to utilize T3SS
to facilitate the expression of GOx and deliver it into tumor cells upon contact.

![](https://static.igem.wiki/teams/4737/wiki/engineering/gox/image003.png)

***Figure 3.** The expression of SopE-FLAG-Gox*

![](https://static.igem.wiki/teams/4737/wiki/engineering/gox/image004.png)

***Figure 4.** The map of pFPV25.1-SopE-FLAG-GOx*

## Test

Recombinant **S. typhimurium** (VNP-SopE-GOx) was grown at appropriate
conditions. The proteins from the culture supernatant and bacterial lysates were prepared for an
immunoblotting assay. The results of Western blot showed that VNP20009 electroporated with plasmid
**pFPV25.1-SopE-FLAG-GOx** expressed SopE-FLAG-GOx protein successfully (Figure 5).
![](https://static.igem.wiki/teams/4737/wiki/engineering/gox/image005.png)

***Figure 5.** Western blot analysis shows the expression of gene of interest (SopE-FLAG-GOx) in
VNP-SopE-GOx. The theoretical molecular weight of SopE-FLAG-GOx is about 76 kDa, and the theoretical
molecular weight of GAPDH is about 36 kDa.*

*Salmonella* VNP-SopE-GOx was cultured at 37℃. Engineered bacterial suspensions were prepared using DMEM
medium at a multiplicity of infection (MOI) of 150 bacterial cells per mammalian cell. Additionally, a
control group was established using the unmodified VNP20009 strain. Following infection, cells were
harvested and prepared for SDS-PAGE and Western blot experiments.

However, the results of the Western blot showed no any specific band. The immunoblotting results failed to
meet our expectation.

## **Learn**

At the first time, the temperature to culture the VNP-SopE-GOx is 37℃. From the western blot result of
VNP-SopE-GOx infecting BGC-823, we did not get any specific band. After consulting Dr. Yihao Li, the
postdoctoral of center of biology science and technology, Beijing Normal University at Zhuhai, we found
that at 37℃, inclusion bodies will form in VNP20009. Inclusion bodies are high-density
(1.3 mg/ml) insoluble protein particles encapsulated by membranes when foreign genes are expressed in
prokaryotic cells. According to the advice from Dr. Li, we adjusted the bacteria culture temperature to
30℃.

## **Test**

*Salmonella* VNP-SopE-GOx was cultured at 30℃. Engineered bacterial suspensions were prepared using DMEM
medium at a multiplicity of infection (MOI) of 150 bacterial cells per mammalian cell. Additionally, a
control group was established using the unmodified VNP20009 strain. Following infection, cells were
harvested and prepared for SDS-PAGE and Western blot experiments. The results showed that the recombinant
type III secretion system could effectively present SopE-FLAG-GOx fusion protein to BGC-823 cells, and
that SopE-FLAG-GOx fusion protein could be processed by BGC823 cells, and its secretory related protein
SopE could be further excised[3](#Reference) (Figure 6).

![](https://static.igem.wiki/teams/4737/wiki/engineering/gox/image006.png)

***Figure 6.** Western blot analysis shows the injection of FLAG-GOx into BGC-823 cells by
VNP-SopE-GOx via T3SS. The theoretical molecular weight of FLAG-GOx is about 64 kDa, and the
theoretical molecular weight of GAPDH is about 36 kDa.*

After infection, we used the hydrogen peroxide detection kit (Beyotime) to detect the H2O2 level of the
cell culture supernatant. The results demonstrated that the H2O2 level in the culture supernatant of tumor
cells infected with VNP-SopE-GOx is significantly higher compared to the control group and tumor cells
infected with VNP20009 (Figure 7).

![](https://static.igem.wiki/teams/4737/wiki/engineering/gox/image007.png)

***Figure 7.** H2O2 levels in the cell culture supernatant*

We also used the CCK-8 cell counting kit (Vazyme) to detect the viability of the cells after infection.
The results of the experiment indicated that tumor cells infected with VNP-SopE-GOx exhibited lower
viability compared to non-infected tumor cells and those infected with VNP20009. Additionally, the cell
viability was significantly increased when tumor cells are treated with Fer-1 and infected with
VNP-SopE-Gox. There was no significant difference in cell viability between tumor cells infected with
VNP20009 without Fer-1 treatment and those treated with Fer-1 and infected with VNP20009. Therefore, this
study demonstrated that VNP-SopE-GOx infection can effectively induce ferroptosis in tumor cells (Figure
8).

![](https://static.igem.wiki/teams/4737/wiki/engineering/gox/image008.png)

***Figure 8.** Cell viability of cancer cells treated with bacterial infection and co-treated with
bacterial infection and Fer-1.*

## **Learn**

H2O2 is one of the substrates of aquaporins (AQPs)[4](#Reference). So, the concentration of H2O2 in the culture medium
can reflect the level of H2O2 in the tumor cells. Fer-1 is a selective inhibitor of ferroptosis. The
conclusion we learned was that GOx can catalyze glucose to generate hydrogen peroxide, which can further
promote ferroptosis in tumor cells. To further verify the GOx can induce lipid peroxidation of the tumor
cell membranes, we intended to detect the lipid peroxidation level of the tumor cells infected by the
VNP-SopE-GOx.

To enhance the safety of our project, we need to select the condition that specifically exists in the
tumor tissue to activate the expression of GOx. Tumor microenvironment (TME) often exhibits overall oxygen
deficiency. After communicating with USTC and SCAU-China, we decided to use the specific anaerobic
promoter *pnirB*. The *pnirB* is induced in anaerobic conditions and is up regulated in response to
nitrite and nitrate. Based on the aforementioned considerations, we have selected the *pnirB* to
initiate the expression of GOx.

We intended to use the anaerobic promoter *pnirB* to replace the constitutive promoter *prpsM*. Therefore, the
engineered VNP20009 can drive the expression of GOx in anaerobic conditions.

![](https://static.igem.wiki/teams/4737/wiki/engineering/gox/image009.png)

***Figure 9.** The anaerobic expression of Gox*

![](https://static.igem.wiki/teams/4737/wiki/engineering/gox/image010.png)

***Figure 10.** The map of pFPV25.1-pnirB::SopE-FLAG-GOx*

References

1 Khatami, S. H. et al. Glucose oxidase: Applications, sources, and recombinant production. Biotechnology
and Applied Biochemistry 69, 939-950 (2022). https://doi.org:https://doi.org/10.1002/bab.2165
2 Galán, J. E. & Collmer, A. Type III Secretion Machines: Bacterial Devices for Protein Delivery into Host
Cells. Science 284, 1322-1328 (1999). https://doi.org:10.1126/science.284.5418.1322
3 Kubori, T. & Galán, J. E. Temporal regulation of salmonella virulence effector function by
proteasome-dependent protein degradation. Cell 115, 333-342 (2003).
https://doi.org:10.1016/s0092-8674(03)00849-3
4 Zhang, M. et al. Aquaporin OsPIP2;2 links the H2O2 signal and a membrane-anchored transcription factor
to promote plant defense. Plant Physiol 188, 2325-2341 (2022). https://doi.org:10.1093/plphys/kiab604

Cycle 1: Delivery of shRNA expressing plasmid through attenuated
*S. typhimurium*

## **Design**

Facultative anaerobic strains of *S. typhimurium* can preferentially accumulate in tumor tissues and produce
a potential anti-tumor effect. The progress of RNAi technology for cancer therapy faces the challenge of
how to efficiently deliver RNAi to the cytoplasm of cancer cells. Therefore, we engineered bacteria to
deliver shRNA expressing plasmids into the cytoplasm of tumor cells in order to improve treatment efficacy
and safety. The cystine transporter solute carrier family 7 member 11 (SLC7A11) is highly expressed in many
tumor cells to protect cancer cells from oxidative stress and ferroptosis[1](#Reference). To inhibit the expression of
*SLC7A11*, we engineered *S. typhimurium* to deliver RNAi.

## **Build**

We commissioned companies to synthesize short hairpin RNAs (shRNA) sequences targeting SLC7A11 and ligated into the eukaryotic expression plasmids (*pSilencer 2.1-U6-Neo*). The plasmids were transformed into attenuated *S. typhimurium*.

![](https://static.igem.wiki/teams/4737/wiki/result-new-picture/engineering-shrna-new.png)

***Figure 1.** Construction of pSilencer 2.1-U6-shRNA-SLC7A11-Neo*

## **Test**

We co-cultured the engineered bacteria with mammalian cells for 2 hours, and then performed qRT-PCR experiments to analyze the levels of *SLC7A11* mRNA. However, no significant silencing of target genes was observed.

## **Learn**

The *S. typhimurium* VNP20009 can survive and proliferate within host cells, but as it is encapsulated by intracellular vesicles, it is unable or releases only minimal perturbations into the cytoplasm of host cells. Therefore, VNP20009 is not suitable as a vector for delivering shRNA expressing plasmids to silence genes in mammalian cells2. We need to further engineer *Salmonella* to improve efficiency.

Cycle 2: Silencing *SLC7A11* through trans-kingdom RNAi by engineered bacteria

## **Design**

In addition to utilizing bacteria as the shRNA-expressing plasmid carrier, a trans-kingdom RNAi (tkRNAi) technology was developed based on *E. coli* to deliver RNAi for gene silencing in mammalian cells. This method involves producing high amounts of shRNA and releasing them into the cytoplasm of host cells, both *in vitro* and *in vivo*[3](#Reference). We referred to the tkRNAi system in *E. coli* and aimed to apply it to *Salmonella*. Plasmids were constructed to retain the *HlyA* gene encoding listeriolysin O, which we believed would facilitate product entry into the cytoplasm. Additionly, the invasin gene *Inv* was deleted from the *E. coli* tkRNAi system, as it had no impact on our system due to our inherently invasive chassis. We chose T7 promoter and enhancer which were commonly used in engineered bacteria that mediate trans-kingdom RNAi[4](Reference).

## **Build**

We constructed plasmids named *pSilence-SLC7A11*, which included T7 promoter-driven expression cassette and anaerobically induced the expression of *HlyA*. Oligonucleotides encoding shRNA targeting *SLC7A11* were inserted into the plasmid. Plasmids were transformed into VNP20009 competent cells.

![](https://static.igem.wiki/teams/4737/wiki/engineering/shrna/shrna-engineering-2-new.png)

***Figure 2.** Construction of pSilence-shRNA-SLC7A11*

Test: After 2 hours of co-culturing the engineered bacteria with mammalian cells, we conducted qRT-PCR experiments to confirm the changes in mRNA levels. However, there was no obvious target gene silencing.

## **Learn**

After analysis, we found that the T7 promoter could not function directly in *S. typhimurium*, which is due to the inability of *S. typhimurium* VNP20009 to express T7 RNA polymerase.

Cycle 3: The engineered bacteria were designed to constitutively express T7 RNA polymerase

## **Design**

T7 RNA polymerase is a highly specific RNA polymerase that recognizes the T7 promoter sequence[5](Reference). It can specifically recognize the T7 promoter sequence and activate the transcription of T7 promoter. Consequently, we incorporated the T7 RNA polymerase expression cassette into the plasmid, enabling the proper functioning of the T7 promoter. In order to visually verify the feasibility of this system, we also designed shRNA sequences targeting *GFP*. By observing the intensity of fluorescence, we could determine whether the target gene was silenced.

## **Build**

The T7 RNA polymerase expression cassette was integrated into our plasmid by homologous recombination. By virtue of Gibson Assembly, the shRNA sequence targeting *GFP* was inserted into the plasmid. The plasmids were successfully constructed as confirmed by sequencing. The plasmids were transformed into VNP20009 competent cells.

![](https://static.igem.wiki/teams/4737/wiki/engineering/shrna/shrna-engineering-3-new.png)

***Figure 3.** Design of the plasmid*

## **Test**

We first used engineered bacteria targeting *GFP* to infect human gastric adenocarcinoma cells BGC-823 that transiently transfected with GFP eukaryotic vector. After the engineered bacteria and BGC-823-GFP cells were co-cultured for two hours in serum-free medium, the cells were washed and a fresh complete medium was used. After 24 hours we observed it under a fluorescence microscope.

![](https://static.igem.wiki/teams/4737/wiki/result/shrna-1.png)

***Figure 4.** Results of shGFP delivery by bacterial infection 24 hours later. Both the engineered bacteria and VNP20009 infections were at the MOI of 500. Control was BGC-823 cells that not transfected with the GFP plasmid.*

## **Learn**

The above results proved the feasibility of this system. The engineered bacteria successfully silenced *GFP* by cross-kingdom RNAi delivery of shRNA. We then conducted a follow-up experiment using the system to silence *SLC7A11* and quantitatively analyze its effects.

Cycle 4: Employing the engineered bacteria to silence *SLC7A11* and analyzing the effects

## **Design**

As the engineered bacteria targeting *GFP* successfully silenced the gene and affected its function, we decided to silence *SLC7A11* by this system. The shRNA sequence in the plasmid was replaced from targeting *GFP* to targeting *SLC7A11*. We still chose BGC-823 cells and infected them with bacteria delivered shRNA targeting *SLC7A11*.

## **Build**

By virtue of enzyme-cut and link up, the shRNA sequence targeting *SLC7A11* was inserted into the plasmid. The plasmids were successfully constructed as confirmed by sequencing. Then they were transformed into VNP20009 competent cells.

![](https://static.igem.wiki/teams/4737/wiki/engineering/shrna/image001.png)

***Figure 5.** Construction of pSilence-SLC7A11*

## **Test**

After being exposed to bacteria, cells were washed and replenished with fresh complete medium containing antibiotics. In order to assess the expression level of the *SLC7A11* gene, we collected the samples and conducted quantitative polymerase chain reaction (qPCR). The qPCR results indicated a decrease in the expression of *SLC7A11* in BGC-823 cells post-infection with genetically modified bacteria. To further confirm the impact on cellular function after down-regulating *SLC7A11*, we analyzed the glutathione (GSH) content. The results demonstrated a decrease in GSH content in cells where *SLC7A11* was down-regulated, suggesting that the down-regulation of *SLC7A11* affected the metabolic pathway of the cells. The assessment of cell viability using CCK-8 assay revealed a significant decrease in tumor cell viability when *SLC7A11* expression was down-regulated. These findings collectively indicate that our therapy has effectively produced positive outcomes in BGC-823 cells.

![](https://static.igem.wiki/teams/4737/wiki/engineering/shrna/image005.png)

***Figure 6.** qPCR results after bacterial infection and siRNA-SLC7A11 transfection. a. The SLC7A11 expression level of BGC-823 after siRNA transfection, control was BGC-823 without any treatment, and siRNA-control was siRNA transfected with other genes. Results were significant between siRNA-SLC7A11 and control groups (\*\*\* p<0.001). b. The SLC7A11 expression level of BGC-823 after engineered bacterial infection with different MOI. VNP20009 without functional plasmids at an MOI of 1:5000 was used as control group. The results of VNP-shSLC7A11 (1:3000), VNP-shSLC7A11 (1:5000) and VNP-control groups have significant differences (\* p<0.05, \*\* p<0.01)*

![](https://static.igem.wiki/teams/4737/wiki/result/shrna-2.png)

***Figure 7.** Results of the GSH detection. a. Standard curve of the GSH content varying with the absorbance value. b. The intracellular GSH content in BGC-823 cells after VNP20009 and engineered bacteria infection. Results between VNP-shSLC7A11 (1:500) and VNP20009 (1:500) infection have significant differences (\*\*p< 0.01).*

![](https://static.igem.wiki/teams/4737/wiki/result/shrna-4.png)

***Figure 8.** Results of cell viability test after infection. The siRNA-control is the siRNA transfected with the other genes. The results between siRNA-SLC7A11 and siRNA-control groups have significant differences; and results between engineered bacteria with different MOI and VNP20009 (1:3000) infection have significant differences (\*\*\*\*p < 0.0001).*

## **Learn**

We achieved specific gene silencing of *SLC7A11* by shRNA-expressing *Salmonella* VNP20009-mediated RNAi. The inhibition of *SLC7A11* interfered with lipid homeostasis of tumor cells and significantly inhibited cell growth activity. Next, we hope to further confirm the function of the *SLC7A11* to ensure the applicability of our therapy. We learned from Professor Xiaotian Zhang that different *SLC7A11* expression level dictates differential responses to oxidative stress in cancer cells, which could be complicated. For certain tumor cell lines with high expression of *SLC7A11* and sensitivity to disulfidptosis [6](#Reference), inhibiting the expression of *SLC7A11* does not align with the purpose of our project. Therefore, we will explore other target genes for Xc- system/GSH/GPX4 inhibition in the future.

References

1 Koppula, P., Zhuang, L. & Gan, B. Cystine transporter SLC7A11/xCT in cancer: ferroptosis, nutrient dependency, and cancer therapy. Protein & cell 12, 599-620 (2021).
2 Cabezudo, I., Lobertti, C. A., Véscovi, E. G. & Furlan, R. L. E. Effect-Directed Synthesis of PhoP/PhoQ Inhibitors to Regulate Salmonella Virulence. J Agric Food Chem 70, 6755-6763 (2022). https://doi.org:10.1021/acs.jafc.2c01087
3 Xiang, S., Fruehauf, J. & Li, C. J. Short hairpin RNA-expressing bacteria elicit RNA interference in mammals. Nat Biotechnol 24, 697-702 (2006). https://doi.org:10.1038/nbt1211
4 Guo, H. et al. Targeting tumor gene by shRNA-expressing Salmonella-mediated RNAi. Gene Ther 18, 95-105 (2011). https://doi.org:10.1038/gt.2010.112
5 Borkotoky, S. & Murali, A. The highly efficient T7 RNA polymerase: A wonder macromolecule in biological realm. Int J Biol Macromol 118, 49-56 (2018). https://doi.org:10.1016/j.ijbiomac.2018.05.198
6 Liu, X. et al. Actin cytoskeleton vulnerability to disulfide stress mediates disulfidptosis. Nat Cell Biol 25, 404-414 (2023). https://doi.org:10.1038/s41556-023-01091-2

Cycle 1: Devised the first generation of "logical suicide circuit" to maintain two functional plasmids

## **Design**

Initially, we planned to utilize the conventional toxin-antitoxin system to sustain two operative plasmids in our project. This involves incorporating the toxin gene into plasmid *pFenton*, while introducing the antitoxin gene into plasmid *pSilence*. Consequently, if *pSilence* is no longer present, the expression of toxin in plasmid *pFenton* will result in the eradication of the modified bacteria. Nevertheless, if plasmid *pFenton* is lost, the TA system will become nonfunctional. As a result, we devised the first generation of "logical suicide circuit". Our intention was to engineer specific components of the circuit within each of the two plasmids, so that in the event of one plasmid being lost, the modified strain would initiate suicide.

## **Build**

The "logical suicide circuit" was introduced in two plasmids (Figure 1). The corresponding gene fragments were amplified by PCR and introduced into the plasmid by homologous recombination to construct plasmids that can be used to verify the logic suicide circuit. Plasmid *pSilence* constitutively expressed LacI and toxin Hok. The expression of tetR was regulated by the repressible promoter *pLacO*, which is repressed by the LacI protein. Similarly, the expression of *Sok* was regulated by the repressible promoter *pLtetO*, which is repressed by the tetR protein. If *pSilence* is lost, LacI protein levels will gradually degrade, resulting in cessation of its production. Subsequently, *pLacO* will promote the expression of tetR, leading to repression of *Sok* transcription. As *Sok* degrades, *Hok* mRNA will translate into the toxin protein and eliminate the engineered bacteria. If *pFenton* is lost, the Hok-Sok system will induce bacterial death. (see in [design](https://2023.igem.wiki/bnuzh-china/design)).

![](https://static.igem.wiki/teams/4737/wiki/proof-new-zy/engineer-safty-figure-1.png)

***Figure 1.** Schematic representation of the first generation of "logical suicide circuit"*

## **Test**

The "logical suicide circuit" was introduced to experts and other teams for their advice and gain a lot from them, especially in the communication in 10th Conference of China iGEMer Community (CCiC) . It was suggested that we should pay more attention to the induction of the suicide of engineered bacteria after the end of the mission. Otherwise, the engineered bacteria might harm the normal cells.

## **Learn**

We realized that it was necessary for artificially manipulating the initiation of engineered bacterial suicide to strengthen the safety of our project. Especially in the later stage of treatment, if the volume of the tumor tissue decreases and the tumor regress, the engineered bacteria are likely to invade the normal tissue cells. Then it came to the second generation of "logical suicide circuit".

Cycle 2: Enable the engineered bacteria to be induced to suicide with Dox

## **Design**

The second generation of the "logical suicide circuit" was improved based on the initial generation (refer to Figure 2). In order to enable the engineered bacteria to be induced after treatment with doxycycline (Dox), we altered the arrangement of the LacI and tetR expression cassettes in the circuit. Doxycycline is a tetR acycline analogue that can bind to tetR, consequently preventing tetR from suppressing the *pLtetO* promoter. As a result, the *pLtetO* can express the LacI protein normally, which then inhibits the transcription of the antitoxin *Sok*, ultimately leading to the death of the engineered bacteria caused by the toxin Hok.

![](https://static.igem.wiki/teams/4737/wiki/engineering/safety/safety2.png)

***Figure 2.** Schematic representation of the second generation of "logical suicide circuit"*

## **Build**

To validate the second generation of "logical suicide circuit", *pET-T7::Hok-Sok-LacI* and *pGEN-prpsm::tetR* plasmids were re-constructed by restriction enzyme ligation and homologous recombination to validate the circuit.

![](https://static.igem.wiki/teams/4737/wiki/result-new-picture/safety-result-4.png)

***Figure 3.** Construction of plasmid pET-T7::Hok-Sok-LacI and pGEN-prpsM::tetR to validate the third generation of "logical suicide circuit"*

## **Test**

Firstly, the system was modeled to predict its functionality. The modeling results predicted that the expression of toxin Hok would surpass that of antitoxin *Sok*, which is logically impossible (Figure 4). Additionally, the *pET-T7::Hok-Sok-LacI* and *pGEN-prpsM::tetR* were introduced into BL21 (DE3) competent cells simultaneously, and they were plated on LB-agar plates containing ampicillin, kanamycin, and IPTG. The presence of IPTG enables the binding with LacI, thus preventing LacI from repressing the expression of *Sok* and ensuring the survival of the engineered bacteria. However, it was observed that the engineered bacteria did not proliferate on the LB-agar plates supplemented with IPTG (Figure 5).

![](https://static.igem.wiki/teams/4737/wiki/engineering/safety/safety4.png)

***Figure 4.** Schematic representation of the concentration of each substance over time. The black curve is the concentration of Hok, which is significantly higher than Sok*

![](https://static.igem.wiki/teams/4737/wiki/engineering/safety/safety5.png)

***Figure 5.** The plasmid pET-T7::Hok-Sok-LacI and pGEN-prpsM::tetR were transformed into BL21 (DE3) competent cells, which was plated on the LB-agar plates with ampicillin, kanamycin and IPTG. No colonies grow up after more than 36 hours culture at 37℃*

## **Learn**

The model was highly significant in predicting the performance of our system. We conducted model validation and experiments, which revealed that the strength of toxin protein expression driven by the T7 promoter was excessively high compared to the expression of antitoxin. Consequently, we determined the need to employ a weaker promoter to drive toxin protein expression.

Cycle 3: Decrease the expression of toxin with a weaker promoter

## **Design**

Therefore, it comes to the third generation of "logical suicide circuit". The T7 promoter that drives toxin protein expression becomes a weaker *trp* promoter than the T7 promoter.

![](https://static.igem.wiki/teams/4737/wiki/engineering/safety/safety6.png)

***Figure 6.** Schematic representation of the third generation of "logical suicide circuit"*

## **Build**

The plasmid *pET-trp::Hok-Sok-LacI* was constructed with *pET-T7::Hok-Sok-LacI*. We removed T7 promoter from *pET-T7::Hok-Sok-LacI* by reverse PCR. The *trp* promoter gene fragments were amplified by PCR and introduced into the plasmid by homologous recombination. However, later we found that BL21 (DE3) competent cells will endogenous express LacI protein. As a result, we failed to validate "logical suicide circuit" in BL21 (DE3).

![](https://static.igem.wiki/teams/4737/wiki/engineering/safety/safety7.png)

***Figure 7.** Construction of plasmid pET-trp::Hok-Sok-LacI to validate the third generation of "logical suicide circuit"*

## **Test**

The model initially tested the third generation of the "logical suicide circuit" and predicted that it would function as intended (see in [model](https://2023.igem.wiki/bnuzh-china/model)). However, it is a pity that we don’t have enough time to validate complete "logical suicide circuit" in our chassis strain. We only tested the functionality of *pLacO* and *pLtetO*, and evaluated *pET-Hok-Sok-lacI* separately in BL21 (DE3). The results confirmed the functionality of *pLacO* and *pLtetO* as expected, as well as the successful performance of the Hok-Sok system (see in [results](https://2023.igem.wiki/bnuzh-china/results)).

## **Learn**

By studying the background of the *E. coli* BL21 (DE3) strain, we found that the strain carried the DE3λ prophage on the chromosome, which in turn carried the *lacI* and *sam7I* genes and the T7 RNA polymerase gene controlled by the *lacUV5* promoter. Therefore, *E. coli* BL21 (DE3) will endogenous express LacI protein, we need to validate complete "logical suicide circuit" in a new bacteria strain.

We designed the "logical suicide circuit" according to our needs and built step by step. It was tested with different and effective methods. During these processes, we learned from problems that we met and then improve the system. We finally designed the "logical suicide circuit" that meets the requirements through “DBTL”.

{% endblock %}

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{% block title %}Experiments {% endblock %}
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![](https://static.igem.wiki/teams/4737/wiki/shoutu/experiment-new.jpg)

# Experiments

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**Experiments**

The Preparation of Electroporation Competent Bacteria

1. Take the attenuated **Salmonella typhimurium** VNP20009 preserving in glycerol to inoculate
in 1 mL
LB
liquid medium and culture it overnight at 37°C.

2. Inoculate the obtained bacterial solution in antibiotic-free LB liquid medium at 1%, in sure that the
final volume is 100 mL. Culture the bacteria to OD600=0.4~0.5 (37°C, 180 rpm), and collect for
later
use.

3. Pre-cool the bacterial solution for 10 min and sterilized 10% sterile glycerol in advance by ice bath.
Centrifuge the bacterial solution at 4°C and 5000 rpm for 5 min, and collect the bacteria
precipitation.

4. Wash the precipitate 2 or 3 times with 10% glycerol solution at 1/10 volume of bacteria solution,
centrifuge at 4°C and 5000 rpm for 5 min to collect the precipitation.

5. Resuspend the precipitation with 10% glycerol at 1/100 volume of bacteria solution, and divide into 50
μL per tube.

Electroporation

1. Take 2-3 μL of plasmid (the concentration of plasmid higher than 150 ng/μL), gently mix with 50 μL of
ice-cold competent bacteria suspension, add it to a pre-cooled electroporation cup with a distance of 0.1
cm, put it on ice and stand for 5 min, the setting conditions of the electroporator are 1800 V, 200 Ω, 25
μF; Or add to a pre-cooled electroporation cup with a spacing of 0.2 cm, put it on ice and stand for 5
min, set the conditions to 2500~3000 V, 200 Ω, 25 μF.

2. After the electroporation, quickly suck the liquid in the electroporation cup into the centrifuge tube,
add 800 μL of LB liquid medium, culture the bacteria for 1.5 h (37°C, 180 rpm), centrifuge at 25°C and
5000 rpm for 5 min, discard the excess supernatant, remain 100 μL supernatant to resuspend the bacteria
precipitation, coat on LB solid medium with Amp antibiotic, incubate overnight at 37°C, and screen
positive monoclonals.

3. After the experiment, the electroporation cup needs to be cleaned: first rinse with ddH2O,
then rinse with 75% sprinkles, and finally soak in 75% alcohol. Usually, the electroporation cup is soaked
in 75% alcohol. Before using, the electroporation cup needs ultraviolet irradiation for 15~30 minutes in
clean bench.

Infection of Tumor Cells with the Engineered Bacteria VNP20009

1. Observe the degree of convergence of tumor cells under a microscope, digest the cells in the biosafety
cabinet and count the cells under the inverted microscope.

2. Inoculate the tumor cells in 24-well plates (5×104 cells/well), culture them in 500 μL DMEM
medium
containing 10% fetal bovine serum without penicillin and streptomycin, and observe the cell apposition on
the following day.

3. Take 10 μL of VNP20009 bacteria from glycerol preservation, and add to 5 mL LB liquid medium for
expansion, culture at 37°C, 220 rpm for 12 h.

4. Transfer the above bacterial solution to 50 mL LB liquid medium and expand to OD600=0.5.

5. Centrifuge the bacterial solution at 4000 rpm for 5 min, and carefully remove the supernatant.

6. Resuspend the precipitate with 500 μL PBS, centrifuge at 4000 rpm for 5 min, and carefully remove the
supernatant.

7. Resuspend the precipitate with 500 μL DMEM medium containing 10% fetal bovine serum without penicillin
and streptomycin, centrifuge at 4000 rpm for 5 min, and remove the supernatant carefully.

8. Resuspend the bacteria with DMEM medium containing 10% fetal bovine serum without penicillin and
streptomycin, and then divided into four tubes for dilution, inoculated in 24-well plates according to the
ratio of cells/bacteria = 1:30/1:150/1:300 (to find the appropriate multiplicity of infection, MOI), and
co-cultured for about 5 h. Observe the state of the cells under the microscope.

9. Cells are washed twice with PBS, replaced with complete medium, and continued to incubate for 36 h.

10. If observing with fluorescence microscope, there is no need to wait for protein expression.

scFv Experiments: The Experiments of scFv Surface Presentation Technique

Prepare Cells for Infection:

1. Place CEA-positive NUGC-3 cells and CEA-negative BGC-823 cells in a 37°C, 5% CO2 incubator for
culture
(use company-supplied medium for culturing NUGC-3 cells, and DMEM complete medium for culturing BGC-823
cells. RPMI-1640 medium for culturing NUGC-3 cells does not need to prepare antibiotic cell culture medium
in advance).

2. Perform cell counting, inoculate these two kinds of cells in different wells of 24-well plate at 5×104
cells per well, and set up a control group. Culture the cells overnight.

Prepare Bacteria for Infection:

1. Take 10 μL of engineered VNP20009 (express Lpp-OmpA-scFv) from the glycerol preservation, and add to 1 mL
LB liquid medium for expansion (37°C, 200 rpm, 12 h).

2. Transfer 50 μL of bacterial solution into 5 mL of LB liquid medium containing Amoxicillin antibiotic
(1:100) in a 12 mL bacteria-culturing tube, and culture at 37℃ until OD600 =0.5
(OD600=0.5 equals to
approximately 1× 108 CFU/mL).

3. Wash the bacteria prepared to infect BGC-823 cells once with 1×PBS, then wash twice with antibiotic-free
DMEM medium containing 10% FBS, and finally resuspend with 5 mL of antibiotic-free DMEM medium containing
10% FBS; Wash the bacteria prepared to infect NUGC-3 cells once with 1×PBS, then wash with antibiotic-free
RPMI-1640 medium containing 10% FBS, and finally resuspend with 5 mL of antibiotic-free RPMI-1640 medium
containing 10% FBS.

Infection:

1. Replace the complete medium containing antibiotics with antibiotic-free cell culture medium containing
10% FBS (DMEM medium for BGC-823 cells and RPMI-1640 medium for NUGC-3 cells) and culture the cells at 37°C
and 5% CO2 for 30 min.

2. Inoculate the cells with bacteria (Multiplicity of infection, MOI=1:100) by adding the treated bacterial
solution to the cell culture medium.

3. Incubate the cells at 37°C and 5% CO2 for 2 hours.

4. Aspirate the culture medium and wash twice with 1× PBS.

5. Add fresh medium containing 100 μg/mL gentamicin and incubate at 37°C and 5% CO2 for 2
hours.

6. Replace the medium containing 100 μg/mL of gentamicin with fresh medium containing 20 μg/mL of gentamicin
for 1 hour.

7. Add 16 μg/mL of tetratoxum and continue to incubate the cells.

8. Observe the results of bacterial targeting under the fluorescence microscope after incubating the cells
for 5 min, 1 h and 2 h.

GOx Experiments: The Experiment of Generation of H2O2 to Promote Fenton Reaction

Prokaryotic Expression Verification Experiments

1. Separately take 10 μL of VNP20009 bacteria and 10 μL of SopE-FLAG-GOx engineered VNP20009 bacteria from
the glycerol preservation, and add to 5 mL LB liquid medium for expansion (30°C, 200 rpm, 12 h).

2. Take out 100 μL of bacterial solution from the overnight culture bacteria solution, centrifuge the rest
of the bacterial solution at 4000 rpm for 5 min. Discard the supernatant, precipitate and break, and carry
out Western Blot experiment.

Type III secretion system
(T3SS) Functional Verification Experiments

1. Culture BGC-823 cells in 24-well plates, 5×105 cells per well.

2. Separately take 10 μL of VNP20009 bacteria and 10 μL of SopE-FLAG-GOx engineered VNP20009 bacteria from
glycerol preservation, and add to 5 mL LB liquid medium for expansion (30°C, 200 rpm, 12 h).

3. Transfer the above bacterial solution to 50 mL LB liquid medium and expand to
OD600=0.5.

4. After the cells are passsaged for 12 h, the VNP20009 and SopE-FLAG-GOx engineered VNP20009 bacteria
are
added to the cell culture system at a MOI ratio of 1:150, and coculture 5 h. And then wash the cells 3
times
with sterile PBS, complete DMEM medium containing 5% fetal bovine serum and 50 μg/mL gentamicin was added
to
continue the culture for 24 h.

5. Aspirate the supernatant of the medium, break the cells, and perform Western Blot experiments.

Function Verification
Experiments

1. Measure Hydrogen Peroxide Level:

Bacterial infection is carried out according to the above bacterial infection experimental protocol (T3SS
Work verification experiment step 1~4), and then the medium and cells are taken separately, and hydrogen
peroxide detection is carried out separately using the hydrogen peroxide detection kit.

2. Measurement of Cell Viability:

According to the above bacterial infection protocol (T3SS Work verification experiment steps 1~4), a CCK-8
kit is used to detect cell viability. The absorbance of A450 nm is determined under a microplate reader
by
incubating with cells for 2 h under a co-incubation condition under a light shield.

3. Measure Lipid Peroxidation Level:

According to the above bacterial infection experimental protocol (T3SS Work verification experiment steps
1~4), bacterial infection is performed, the medium is removed, cells are taken, and lipid peroxidation
detection is performed using the lipid peroxidation detection kit.

4. Measurement of Cell Viability treated with Fer-1:

According to the above bacterial infection protocol (T3SS Work verification experiment steps 1~4), a CCK-8
kit is used to detect cell viability. The cells are treated with Fer-1 in this experiment. The absorbance
of A450 nm is determined under a microplate reader by incubating with cells for 2 h under a co-incubation
condition under a light shield.

shRNA Experiments: The Experiments on Engineered Bacteria Mediated Gene Silencing

Bacterial Infection:

1. Inoculate cancer cells in a culture dish and culture for a period of time to achieve appropriate state
and density.

2. Take 10 μL of VNP20009 bacteria from glycerol preservation, and add to 5 mL LB liquid medium for
expansion (37°C, 220 rpm, 12 h).

3. Transfer the above bacterial solution to 50 mL LB liquid medium and expand to OD600=0.5
(OD600=0.5 equals 1.6×108 bacterial cells/mL).

4. At least 30 minutes before co-incubation of bacteria, replace the original culture medium of cancer
cells
with DMEM basic culture medium with 10% FBS without antibiotics.

5. Take an appropriate amount of bacterial solution (the amount of bacterial solution depends on the MOI
value), centrifuge and wash the bacteria twice with 1× PBS, and resuspend with DMEM without antibiotics or
serum.

6. Add diluted cell culture medium containing corresponding engineered bacteria to cancer cells at the
required MOI, and co-incubate the bacteria and cancer cells at 37°C for 2 hours.

7. After co-incubation for 2 hours, wash the cancer cells twice with 1× PBS, and then wash them once with
DMEM culture medium added with antibiotics.

8. Continue to culture cancer cells in an atmosphere saturated with 37°C, 5% CO2, and water
vapor for 24 hours.

9. The effect of bacterial treatment can be observed through fluorescence microscopy, measured by
quantitative real-time RT-PCR for mRNA levels, and displayed by cell proliferation assay.

RNA Extraction from Adherent Cells:

1. Discard the culture medium and clean once with PBS.

2. Add 1 mL RNA isolator to every 10 cm2 of cultured cells to fully cover the cell surface, and blow the
cells off using a pipette.

3. Transfer the lysate into a 1.5 mL centrifuge tube, blow repeatedly with a pipette until it is fully
lysed, and let it stand on ice for 5 minutes.

4. Add 1/5 volume to the above cracking solution (200 μL) chloroform, shake vigorously to form an
emulsion,
and let stand at 4℃ for 5 minutes.

5. Centrifuged at 11200 rpm and 4 ℃ for 15 min.

6. Carefully remove the centrifuge tube, and the solution is divided into three layers: the upper aqueous
phase, the middle white layer, and the lower red organic phase. Carefully suck the upper layer into a new
centrifuge tube.

7. Add equal volume of pre-cooled isopropanol, invert and mix well. Allow to stand at 4 ℃ for 10
minutes.

8. 11200 rpm, centrifuged at 4 ℃ for 10 minutes, white precipitates appeared.

9. Carefully discard the supernatant and add 1 ml of 75% ethanol (prepared with RNase-free ddH2O). Gently flick the bottom of the tube to suspend the sediment, inverting it several times. Allow to
stand at room temperature for 3-5 minutes.

10. Centrifuge at 11200 rpm and 4 ℃ for 5 minutes, discard the supernatant.

11.Dry and precipitate at room temperature for 2-5 minutes in a clean environment (be careful not to
excessively dry, otherwise RNA will be difficult to dissolve).

12. Add an appropriate amount of RNase free ddH2O to dissolve the precipitate, gently blow with
a
pipette, take a small amount for testing after complete dissolution, and store the rest at -80℃ or use for
the next reverse transcriptional reaction.

Reverse Transcription Total RNA:

![](https://static.igem.wiki/teams/4737/wiki/experiment/pic-1.png)

Gently blow 10 times with a pipette until thoroughly mixed.Procedure of reaction: 50℃ for 15 minutes; 85℃,
5
sec.

Quantitative Real-time PCR:

![](https://static.igem.wiki/teams/4737/wiki/experiment/pic-2.png)

Procedures of reaction:

![](https://static.igem.wiki/teams/4737/wiki/experiment/pic-4.png)

Anaerobism Experiments: The Experiments of Validation of Anaerobic Promoter *pnirB*

1. Add 5 mL LB liquid medium and 50 μL bacteria solution to 12 mL culture cube, culture 5 h at 37℃, 220
rpm.

2. Pretreatment to create low-oxygen conditions.

(i) Prepare 100 g/L Na2SO3 solution using sterilized water. Sterile filtration using
0.2 μm filter
membranes.

(ii) Prepare 5 mL LB liquid medium with 1 g/L Na2SO3. In the meanwhile, the culture
medium should be
pre-reduced by placing it in an anaerobic tank containing anaerobic bags at room temperature over 10 hours
in advance, which can absorb dissolved oxygen in the culture medium thoroughly.

3. Centrifuge the bacteria at 3000×g at room temperate for 5 min. Discard the supernatant.

4. Resuspend bacteria and transfer it to the medium treated with hypoxia as soon as possible.

5. Place the 12 mL culture cube in an anaerobic tank containing an anaerobic bag. Shake overnight at
37℃.

6. Take 100 μL bacterial solution for Western blotting.

TA System Experiments: The Experiments of Validation of Toxin-antitoxin System

Measurement of Plasmid Stability:

1. BL21 (DE3) transformed with *pET-RFP-lacI* and *pGEN* plasmids were grown on LB-agar plates supplemented with kanamycin and ampicillin.

2. Pick a single colony into 250 mL LB liquid medium without antibiotics, which was cultured at 37℃, 220 rpm.

3. Every 12 hours, 10 μL of bacteria solution was taken and transferred to another 20 mL of fresh LB liquid medium, which was still cultured at 37℃, 220 rpm for the next 12-hour growing cycle.

4. Repeat the above operations. When the accumulated time reached 48 h and 72 h, the appropriate amount of the culture was plated on the non-antibiotic LB-agar plates.

5. After single colonies grew on non-antibiotic LB-agar plates, count the white colonies and red colonies on each plate separately. The number of white colonies on each plate was divided by the total number of colonies on the plate to obtain the loss rate of plasmid containing *pET-RFP-lacI* in 48 hours and 72 hours.

6. A certain number of single colonies were randomly picked from non-antibiotic LB-agar plates to LB liquid medium containing ampicillin and grown in a 37℃ incubator. The loss rate of *pGEN* plasmid in the corresponding culture time was obtained by dividing the number of colonies ungrown by the total number of colonies which were picked from non-antibiotic LB-agar plates.

Measurement of BL21(DE3) Specific Growth Rates:

1. Separately take 10 μL of BL21 (DE3) bacteria and 10 μL of engineered BL21 (DE3) bacteria containing *pET-RFP-lacI* and *pGEN* from the glycerol preservation, and add to 1 mL LB liquid medium for expansion (37°C, 220 rpm, 2.5 h).

2. Take out 100 μL of BL21 (DE3) bacterial solution from the culture add to 5 mL LB liquid medium and 100 μL of BL21 (DE3) bacterial solution from the culture add to 5 mL LB liquid medium with kanamycin, shaking at 37°C, 220 rpm.

3. Measure the OD600 of the samples at 15 minutes intervals after record the 0-time absorbance.

Validation of T7-lac operator and *pLtetO*:

1. Separately take 10 μL of BL21 (DE3) bacteria containing the *pET-GFP-lacI* plasmid and 10 μL of original BL21 (DE3) bacteria from the glycerol preservation and add into 2 mL LB liquid medium for expansion (37°C, 200 rpm, 12 h).

2. Transfer 20 μL of BL21 (DE3) *pET-GFP-lacI*-containing bacteria solution into 2 mL of LB liquid medium containing kanamycin antibiotic (1:100) in six 12 mL bacteria-culturing tubes and 20 μL of BL21 (DE3) bacteria solution into 2 mL of LB liquid medium in three 12 mL bacteria-culturing tubes, culture at 37℃ until OD600=0.5 (OD600=0.5 equals to approximately 1× 108 CFU/mL).

3. Add 0.1 mM IPTG into three of the BL21 (DE3) *pET-GFP-lacI*-containing bacteria solution, culture for 6 h at 37℃, 220 rpm.

4. Measure the GFP fluorescence of nine bacteria solutions.

5. Validation of *pLtetO* with *pJKR-L-tetR* engineerd BL21 (DE3) bacteria in the same way with doxycycline.

References

1 Yu C, Zhai CK, Liao CS, et al. Construction and characterization of type III secretion system of
attenuated Salmonella typhimurium. Chin J Biotech, 2016, 32(12): 1664–1675.
2 Liu Z, Liu X, Cao W, Hua ZC. Tumor-specifically hypoxia-induced therapy of SPRY1/2 displayed differential
therapeutic efficacy for melanoma. Am J Cancer Res. 2015 Jan 15;5(2):792-801. PMID: 25973316; PMCID:
PMC4396039.

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![](https://static.igem.wiki/teams/4737/wiki/shoutu/result-new.jpg)

# Results

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Infection

Targeting

Fenton

shRNA

Anaerobism

Safety

Validation of VNP20009 infecting tumor cells

1. VNP20009 Engineering

We utilized the plasmid *pFPV25.1-GFP* to transform the attenuated *Salmonella typhimurium*
VNP20009, aiming to verify its efficient invasion of cancer cells and explore the optimal multiplicity of
infection (MOI) ratio more intuitively.

![](https://static.igem.wiki/teams/4737/wiki/result-new-picture/infection-result-1.png)

***Figure 1.** Schematic diagram of the *pFPV25.1-GFP* plasmid*

We transformed this plasmid into *E. coli* DH5α clonally competent cells for expansion. The cloned
plasmids were then transformed into VNP20009 by electroporation. We collected the transformed bacteria for
bacterial fluid PCR identification, then successful transformation of *pFPV25.1-GFP* into VNP20009
was demonstrated by UV light.

![](https://static.igem.wiki/teams/4737/wiki/result/10-8/junqinran/infection-result-figure-2.png)

***Figure 2.** Comparison of fluorescence intensity of VNP-GFP (A) and VNP-scFv (B) under UV
light irradiation. VNP-GFP: engineered VNP20009 with pFPV25.1-GFP. VNP-scFv: engineered VNP20009
with pFPV25.1-Lpp-OmpA-scFv*

2. Bacterial infection: explore the optimal ratio of MOI and
infection time

BGC-823 cells were infected by VNP-GFP for 5 h and observed under the fluorescence microscope
(Olympus).

![](https://static.igem.wiki/teams/4737/wiki/result/10-8/junqinran/infection-result-figure-3.png)

***Figure 3.** VNP-GFP infected BGC-823 cells for 5 h. Bright field, fluorescent field and superimposed graphs of different MOIs as well as BGC-823 cells without bacterial infestation (Control) were observed under fluorescence microscope (Olympus).*

BGC-823 cells were infected by VNP-GFP for 5 h, then washed twice with PBS, replaced with complete medium,
and continued to incubate for 36 h and finally observed under the fluorescence microscope
(Olympus).

![](https://static.igem.wiki/teams/4737/wiki/result/10-8/junqinran/infection-result-figure-4.png)

***Figure 4.** VNP-GFP infected BGC-823 cells for 36 h. Bright field, fluorescence field, and superimposed graphs of different MOIs as well as BGC-823 cells without bacterial infestation (Control) were observed under fluorescence microscope (Olympus).*

Based on the results of the aforementioned experiments, it was discovered that the engineered bacteria
successfully infiltrated BGC-823 cells within 5 hours of infection with VNP-GFP. The invasion of cells by
the bacteria was successfully accomplished at various MOI ratios. Particularly, when MOI=1:150, a
significant number of tumor cells were infected with the engineered bacteria, and the tumor cells remained
in good condition. Therefore, it is recommended that a MOI ratio of 1:150 is utilized for bacterial
infection.

Validation of Targeting

1. Plasmids construction

We utilized the expression of high-affinity carcinoembryonic antigen (CEA)-specific single chain antibody
fragments (scFv) on the surface of bacteria in order to enhance the tumor cell targeting ability of
engineered bacteria. To test that scFv can actually work, we constructed the
*pFPV25.1-[Lpp-OmpA-scFv]-GFP* plasmid (Figure 1).

![](https://static.igem.wiki/teams/4737/wiki/result/10-8/scfv/result-target-figure-1.png)

***Figure 1.** The pFPV25.1-[Lpp-OmpA-scFv]-GFP plasmid that was constructed for verifying the
function of scFv.*

We used the linearized vector the same as *nirB* promotor validation module and amplified the target
fragment *Lpp-OmpA-scFv* synthesized by the company (Figure 2). Next, we ligated target fragment to
the plasmids through homologous recombination to obtain positive recombinant clones. Then we carried out
colony PCR (Figure 3) and sanger sequencing to verify whether the target fragment was ligated into the
plasmid. Finally, we transform the constructed plasmids into *E. coli*, followed by using alkaline
lysis methods to extract recombinant plasmids DNA and then transform them into VNP20009 with
electroporation.

![](https://static.igem.wiki/teams/4737/wiki/result/10-8/scfv/result-target-figure-2.png)

***Figure 2.** Agarose gel electrophoresis analysis of Lpp-OmpA-scFv fragments (1119bp).*

![](https://static.igem.wiki/teams/4737/wiki/result/10-8/scfv/result-target-figure-3.png)

***Figure 3.** Colony PCR were used to confirmed whether Lpp-OmpA-scFv was successfully ligated
with the vector.*

2. Expression of anti-CEA single chain antibody fragments (scFv) in
VNP20009

Western blotting proved that Lpp-OmpA-scFv-GFP fusion protein could be expressed in VNP20009 (Figure 4). A
GFP tag was added to the C-terminus of *Lpp-OmpA-scFv* on the plasmid, in order to characterize the
expression of anti-CEA scFv.

![](https://static.igem.wiki/teams/4737/wiki/proof-new-zy/proof-of-concept-figure-5.png)

***Figure 4.** WB analysis of the expression of specific single chain antibody fragments (scFv).
Lpp-OmpA-scFv-GFP molecular weight is about 66 kDa and GAPDH is a reference protein in cells with a
molecular weight of 36 kDa. The upper band shows the expression of Lpp-OmpA-scFv-GFP fusion protein,
and the lower band shows the expression of GAPDH in VNP20009.*

3. Functional verification of the high-affinity carcinoembryonic
antigen (CEA)-specific single chain antibody fragments (scFv)

We chose human gastric cancer cell line NUGC-3 with high-CEA-expression as experimental group, and a human
gastric cancer cell line BGC-823 with low-CEA-expression as CEA negative cell lines. The engineered
bacteria with GFP tag and the negative control with RFP tag were used to infect the above two types of
cells, and the function of anti-CEA scFv was verified by the infection efficiency of the bacteria.

**PRACTICE 1**

Firstly, we infected cells by engineered bacteria with *pFPV25.1-[Lpp-OmpA-scFv]-GFP* vector and
found that the green fluorescence of the engineered bacteria was very weak.

![](https://static.igem.wiki/teams/4737/wiki/result/10-8/scfv/result-target-figure-5.png)

***Figure 5.** Diagrammatic sketch of Practice 1*

**DEBUG 1**

By reviewing the literature, we found that the VH and VL of scFv are connected
by a linker, and the transient dissociation of VH and VL leads to the instability of
scFv and the downstream GFP protein[1](#Reference).

**PRACTICE 2**

In the subsequent iteration, we removed the GFP tag downstream of scFv by enzyme digestion, followed
by the electroporation of the modified vector into competent cells stably transfected with GFP. Then we
infected NUGC-3 and BGC-823 with the constructed engineered bacteria and negative control simultaneously.
For NUGC-3 with high-CEA-expression, the infection efficiency of engineered bacteria (green fluorescence)
was higher than that of negative control (red fluorescence), but there was no significant difference in
the infection efficiency of BGC-823 which was with low-CEA-expression (Figure 6).

![](https://static.igem.wiki/teams/4737/wiki/proof-new-zy/co-infection-bgc-nugc.png)

***Figure 6.** Fluorescent microscopy results of BGC-823 and NUGC-3 cells co-infected by
engineered bacteria and VNP2009 for 90 minutes Both the engineered bacteria and negative control
infections were at the MOI of 1:50. Red frames outlined in the figure were Engineered bacteria that
infected cells.*

**DEBUG 2**

To determine whether scFv really worked, compared the efficiency of different bacteria in infecting
NUGC-3 and BGC-823. However, we could not exclude the possibility that there was an interaction between
the engineered bacteria and the negative control that affected their infection efficiency, which meant
that further improvement of our experiments was necessary.

**PRACTICE 3**

In this attempt, we added control experiments in which the engineered bacteria and the negative
control infected the cells separately while infecting the cells with the two bacteria simultaneously.
However, NUGC-3 grew slowly, was in poor condition, and died in large numbers after a short time of
infection with engineered bacteria. This resulted in a large number of engineered bacteria being washed
off with PBS along with the dead cells during the infection process, which was very inconvenient for our
observation. Therefore, we selected the human colorectal cancer cell line LS174T, which also has a high
expression of CEA, as the experimental group. The morphology of LS174T cells changed for the long-time
infection, but by analyzing the number of engineering bacteria under the fluorescence field, we found that
the efficiency of engineering bacteria to infect LS174T cells was higher than that of other groups,
whether the engineering bacteria and negative control infection at the same time or separately (Figure
7~9).

![](https://static.igem.wiki/teams/4737/wiki/result/10-8/scfv/result-target-figure-7.png)

***Figure 7.** Microphotographs of LS174T cells with scFv delivery by bacterial infection for 2
hours. Both the engineered bacteria and negative control infections were at the MOI of 1:50.*

![](https://static.igem.wiki/teams/4737/wiki/result/10-8/scfv/result-target-figure-8.png)

***Figure 8.** Microphotographs of BGC-823 cells with scFv delivery by bacterial infection for 2
hours. Both the engineered bacteria and negative control infections were at the MOI of 1:50.*

![](https://static.igem.wiki/teams/4737/wiki/proof-new-zy/proof-of-concept-figure-6.png)

***Figure 9.** Microphotographs of BGC-823 and LS174T cells co-infected by engineered bacteria
and VNP2009 for 2 hours. Both the engineered bacteria and negative control infections were at the
MOI of 1:50.*

What’s more, we used imageJ software to calculate the fluorescence intensity in the images, and the data showed that the infection efficiency of the engineered bacteria was significantly higher than that of the negative control, regardless of whether it was co-cultured or separately infected（Figure 10）.

![](https://static.igem.wiki/teams/4737/wiki/result/target10.png)

***Figure 10.** Quantitative data of both bacteria infect cells at the same time and separately.*

To conclude, anti-CEA single chain antibody fragments (scFv) can be expressed on the surface of
VNP20009 and effectively enhance the targeting of tumor cells by engineered bacteria.

Reference

1 Wang, J. L. et al. Construction and activity analysis of a recombinant immunotoxin composed of PE38 and
a disulfide stable single-chain antibody. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi 22, 74-77 (2006).

Expression of glucose oxidase and mediating the Fenton response in
tumor cells

1. Plasmids construction

We designed the GOx killing module to enable the engineered bacteria VNP20009 to express the glucose
oxidase (GOx) and inject the protein into tumor cells through the type III secretion system (T3SS), which
induce lipid peroxidation in tumor cells and promote ferroptosis. Based on the *pFPV25.1* plasmid
backbone, we successfully constructed the plasmid *pFPV25.1-SopE-FLAG-GOx* (Figure 1).

![](https://static.igem.wiki/teams/4737/wiki/parts/gox-1.png)

***Figure 1.** Schematic diagram of the construction of plasmid for expression of SopE-Flag-GOx
in VNP20009.*

We obtained a linearized plasmid by double digestion of vector *pFPV25.1* using restriction
endonuclease *Xba*I and *Mfe*I (Figure 2). Simultaneously, we amplified the target gene
*SopE-FLAG-Gox* from *pUC19-SopE-FLAG-Gox* (synthesized by BGI company) (Figure 3). The target
fragment is assembled to the linearized *pFPV25.1* plasmid with Gibson Assembly®. PCR detection was
used to screen positive recombinants. The positive recombinants were then sequenced (Sangon Biotech) to
verify that *SopE-FLAG-GOx* was successfully ligated to the plasmid *pFPV25.1.*

![](https://static.igem.wiki/teams/4737/wiki/parts/gox-2.png)

***Figure 2.** Linearization plasmid *pFPV25.1* was isolated by Agarose gel
electrophoresis.*

![](https://static.igem.wiki/teams/4737/wiki/parts/gox-3.png)

***Figure 3.** *prpsM::SopE-FLAG-GOx* was isolated by Agarose gel electrophoresis.*

![](https://static.igem.wiki/teams/4737/wiki/parts/gox-4.png)

***Figure 4.** PCR detection was used to verify that *SopE-FLAG-GOx* was successfully
ligated to the vector *pFPV25.1*.*

2. Validation of the expression of the SopE-Flag-GOx fusion protein
by western blot

To confirm the efficient expression of the functional plasmid *pFPV25.1-SopE-FLAG-GOx* in VNP20009,
we utilized electroporation to transfer *pFPV25.1-SopE-FLAG-GOx* into VNP20009 competent cells.
Subsequently, we conducted screening to identify the positive recombinants (VNP-SopE-GOx) and proceeded to
culture the bacteria overnight at 37°C, 220 rpm. Simultaneously, we follow the same conditions to amplify
the non-engineered VNP20009 bacteria. Upon completion of the expansion, we collected the bacterial
solution, bacterial precipitate and medium supernatant separately. The bacterial solution and the
bacterial precipitate were disrupted, and the protein was collected. To test the samples, SDS-PAGE and
Western blot experiments were performed (Figure 5).

According to the Western blot results (Figure 5), VNP20009 electroporated with plasmid
*pFPV25.1-SopE-FLAG-GOx* successfully expressed the SopE-FLAG-GOx protein, indicating the successful
transfer and expression of our plasmid into VNP20009 when compared to the control.

![](https://static.igem.wiki/teams/4737/wiki/parts/gox-wb-bac.png)

***Figure 5.** Western blot analysis shows the expression of gene of interest (SopE-FLAG-GOx) in
VNP-SopE-GOx. The theoretical molecular weight of SopE-FLAG-GOx is about 76 kDa, and the theoretical
molecular weight of GAPDH is about 36 kDa.*

3. Validation of VNP-SopE-GOx injects FLAG-GOx into tumor cells via
the type III secretion system (T3SS) by Western blot

We chose the BGC-823 cell line, which is a common gastric cancer cell line, for conducting bacterial
infection experiments. We used the engineered bacteria VNP-SopE-GOx to infect BGC-823, while BGC-823 cells
without bacterial infection were set up as a blank control and VNP20009 infected BGC-823 cells were used
as a negative control. Following the bacterial infection experiment, we collected both the cell culture
medium supernatant and the cell precipitate separately. We disrupted the cell precipitate, collected the
protein. All the samples were performed SDS-PAGE and Western blot experiments (Figure 6). Previous
research has shown that the recombinant type III secretion expression system can efficiently deliver
fusion proteins to mammalian cells, and the fusion protein will undergo cellular processing, with the
secretion-related protein SopE being further cleaved[1](#Reference). According to
our Western blot results (Figure 6), after bacterial infection experiment, only the BGC-823 cells infected
by VNP-SopE-GOx had been injected FLAG-GOx, which means that the engineered bacteria VNP-SopE-GOx
successfully injected FLAG-GOx into tumor cells through T3SS.

![](https://static.igem.wiki/teams/4737/wiki/parts/gox-wb-cell.png)

***Figure 6.** Western blot analysis shows the injection of FLAG-GOx into BGC-823 cells by
VNP-SopE-GOx via T3SS. The theoretical molecular weight of FLAG-GOx is about 64 kDa, and the
theoretical molecular weight of GAPDH is about 36 kDa. The results showed that the recombinant type
III secretion system could effectively present SopE-FLAG-GOx fusion protein to BGC-823 cells, and
that SopE-FLAG-GOx fusion protein could be processed by BGC823 cells, and its secretory related
protein SopE could be further excised1.*

4. Functional validation of glucose oxidase delivered by
engineered bacteria

**(i) Detection of Hydrogen peroxide (H2O2) level**

We inoculated BGC-823 cells into 24-well plates with a density of 2.5×105 cells per well.
VNP20009 and VNP-SopE-GOx were used to infect BGC-823 cells for 5 h, following the protocol for bacterial
infection. The control group cells were cultured for 5 hours in DMEM medium without antibiotics and with
5% FBS. DMEM complete medium containing 50 μg/mL gentamicin was then used to culture the BGC-823 cells for
24 h.
Because previous study indicated that hydrogen peroxide produced in cells can be transported to the
outside of cells through aquaporins (AQPs), resulting in increased hydrogen peroxide content in the
microenvironment1. After 24 h of incubation, we utilized the hydrogen peroxide detection kit
(Beyotime) to measure the H2O2 level of the cell culture medium supernatant. The
experiment clearly demonstrate that the H2O2 level in the culture medium supernatant
from tumor cell infected by VNP-SopE-GOx is significantly higher compared to that of the control group and
tumor cell infected by VNP20009. Our experimental results provided evidence that the successful expression
and injection of FLAG-GOx into tumor cells by T3SS enables glucose and oxygen consumption in the medium to
produce H2O2.

![](https://static.igem.wiki/teams/4737/wiki/result-new-picture/gox-result-7.jpg)

***Figure 7.** H2O2 levels in the cell culture medium supernatant*

**(ii) Detection of cell viability with cell counting kit-8 (CCK-8)**

Ferrostatin-1 (Fer-1), a potent and selective inhibitor of ferroptosis, effectively suppresses
Erastin-induced ferroptosis in tumor cells. Specifically, Fer-1 acts as a synthetic antioxidant that
mitigate damage to membrane lipids through a reductive mechanism, thereby inhibits cell death. Glucose
oxidase (GOx) consumes glucose and oxygen in tumor cells, resulting in the production of gluconic acid and
H2O2. Under the catalysis of Fe2+, H2O2 generates
hydroxyl radicals that cause lipid peroxidation of tumor cell membranes, ultimately leading to ferroptosis
in tumor cells.

BGC-823 cells were inoculated in 96-well plates at a density of 5×104 cells per well. In the
group without Fer-1 treatment, VNP20009 and VNP-SopE-GOx were employed to infect BGC-823 cells for 5 h
following the bacterial infection protocol. Meanwhile, the control group cells were cultured with DMEM
medium containing 5% FBS and 20 μM Fer-1, and no antibiotics for 5 h. Subsequently, the medium was
switched to DMEM complete medium supplemented 50 μg/mL gentamicin to culture the BGC-823 cells for 24 h.
In the group with Fer-1 treatment, the cells were pretreated with DMEM without antibiotics and with 5% FBS
and 20 μM Fer-1 for 30 minutes. VNP20009 and VNP-SopE-GOx were used to infect BGC-823 cells for 5 h
according to the bacterial infection protocol, while using DMEM medium without antibiotics and with 5% FBS
and 20 μM Fer-1 to culture the control group cells for 5 h. Following this, the medium was switched to
DMEM complete medium containing 50 μg/mL gentamicin and 20 μM Fer-1 to culture the BGC-823 cells for 24
h.

After 24 hours of culture, we used the CCK-8 cell counting kit (Vazyme) to assess the viability of the
cells. The experimental results indicate that the viability of tumor cells infected with VNP-SopE-GOx is
significantly lower compared to both uninfected tumor cells and tumor cells infected with VNP20009.
Moreover, when comparing tumor cells treated with Fer-1 and infected with VNP-SopE-GOx to those only
infected with VNP-SopE-GOx, there was a significant increase in cell viability. No significant difference
in cell viability was observed between tumor cells infected with VNP20009 without Fer-1 treatment and
tumor cells treated with Fer-1 and infected with VNP20009. In conclusion, we have successfully
demonstrated that the viability of tumor cells infected with VNP-SopE-GOx is significantly reduced, and
ferroptosis serves as the primary mechanism of tumor cell death. Thus, our findings support the
significant induction of ferroptosis in tumor cells through VNP-SopE-GOx infection.

![](https://static.igem.wiki/teams/4737/wiki/result-new-picture/gox-result-8.jpg)

***Figure 8.** Cell viability of cells treated with bacterial infection and co-treated with
bacterial infection and Fer-1*

Reference

1. Kubori, T. & Galán, J. E. Temporal regulation of salmonella virulence effector function by
proteasome-dependent protein degradation. Cell 115, 333-342 (2003).
https://doi.org:10.1016/s0092-8674(03)00849-3.

Expression of shRNA to induce *SLC7A11* gene silencing

1. Plasmids construction

In order to silence the *SLC7A11* gene in tumor cells and induce ferroptosis, a functional plasmid
*pSilence-SLC7A11* was constructed. This plasmid included a short hairpin RNA (shRNA) sequence to
silence the target gene *SLC7A11*, and the *HlyA* gene encoding Listeriolysin-O protein was also
added to the plasmid. Based on literature review, we learned that T7 promoter is often used to initiate
shRNA transcription in the prokaryote to realize cross-kingdom RNAi, however the deficiency of T7 RNA
polymerase in our chassis bacteria VNP20009 will be a problem. Therefore, we used a constitutive promoter
to initiate T7 RNA polymerase expression in the engineered bacteria.

At the same time, in order to qualitatively verify whether the engineered bacteria can release shRNA after
invading tumor cells to silence the expression of the corresponding genes in tumor cells, we constructed
the plasmid *pVB230-shRNA-GFP*, which consists of a shRNA sequence for silencing green fluorescent
protein (GFP).

The above constructed plasmids were transferred into attenuated *Salmonella typhimurium* VNP20009 by
electric shock transformation method to obtain engineered bacteria capable of down-regulating gene
expression in tumor cells.

![](https://static.igem.wiki/teams/4737/wiki/result/10-8/shrna/result-shrna-1.png)

***Figure 1.** Constructed pSilence-SLC7A11 plasmid to deliver shSLC7A11 to tumor cells*

![](https://static.igem.wiki/teams/4737/wiki/result/10-8/shrna/result-shrna-2.png)

***Figure 2.** Constructed pVB230-shRNA-GFP plasmid to deliver shGFP to tumor cells*

2. Western blot analysis

To verify the engineered bacteria can achieve the expression of *HlyA*, bacterial lysates were
prepared from *S. typhimurium* strain VNP20009 carrying appropriate expression vectors for
shRNA-SLC7A11 and subjected to immunoblotting analysis. *HlyA* expression detected by Western
blotting indicates that our plasmids were successfully transferred into VNP20009 and effectively expressed
(Figure 3).

![](https://static.igem.wiki/teams/4737/wiki/result/10-8/shrna/result-shrna-3.png)

***Figure 3.** Western blot analysis of Listeriolysin O. a. Listeriolysin-O is a pore-forming
protein (59kDa) encoded by gene HlyA; b. GAPGH is a reference protein in cells with a molecular
weight of 36 kDa.*

3. Delivery of shRNA to tumor cells by bacteria infection

**3.1 Delivery of *shRNA-GFP* to silence the expression of GFP**

We selected human gastric adenocarcinoma cells BGC-823 and transiently transfected with the GFP
eukaryotic expression vector. After the fluorescent protein was effectively expressed in BGC-823 24 h
after transfection, the cells were co-cultured with the engineered bacteria expressing shRNA-GFP.

We chose the multiplicity of infection (MOI) of cells and engineered bacteria (VNP20009-shGFP) as 1:500,
at the same time, we also used VNP20009 without *shRNA-GFP* at an MOI of 1:500 to infect tumor cells
as negative control results. We observed the results under fluorescence microscope after 24 hours (Figure.
4). Compared with the VNP20009 control, the tumor cells’ fluorescence was significantly weakened after
VNP20009-shGFP infection, indicating that the engineered bacteria actually delivered *shRNA-GFP*
after entering the tumor cells, and effectively down-regulated the gene expression.

![](https://static.igem.wiki/teams/4737/wiki/result/shrna-1.png)

***Figure 4.** Results of shGFP delivery by bacterial infection 24 hours later. Both the
engineered bacteria and VNP20009 infections were at the MOI of 1:500. Control was BGC-823 not
transfected with the GFP transient plasmid.*

**3.2 Delivery of *shRNA-SLC7A11* to silence tumor cells’ expression of SLC7A11**

After verifying the feasibility of engineered bacteria to deliver *shRNA-SLC7A11*, we used
engineered bacteria (VNP20009-SLC7A11) to infect BGC-823. Bacteria in early log phase were washed, diluted
in DMEM and added at the desired MOI from 1:1500 to 1:5000. We used VNP20009 without functional plasmid to
infect cells as the negative control (MOI=1:5000). Meanwhile, we also used siRNA transfection with BGC-823
as a positive control to further verify the efficiency of the delivery of shRNA by the engineered
bacteria.

After 24 h of co-culture infection and siRNA transfection, we quantified gene expression level to verify
VNP20009-shSLC7A11 mediates *SLC7A11* gene silencing in BGC-823 gastric adenocarcinoma cells. Total
RNA from infected cells was extracted and cDNA was synthesized using random primers. qRT-PCR was performed
with AceQ® Universal SYBR Green qPCR Master Mix (Vazyme, Nanjing, China) in a QuantStudio(TM) 6 Flex
System (Applied Biosystems, America) to determined mRNA level. For both siRNA transfection (Figure 5a) and
engineered bacteria carrying *shRNA-SLC7A11* (Figure 5b), *SLC7A11* gene expression in tumor
cells was significantly down-regulated compared with control cells. This demonstrates the feasibility of
using engineered bacteria to conduct gene silence by delivering shRNA, and this system operates
efficiently in our project.

![](https://static.igem.wiki/teams/4737/wiki/result/10-8/shrna/result-shrna-5.png)

***Figure 5.** qPCR results after bacterial infection and siRNA-SLC7A11 transfection. a. The
SLC7A11 expression level of BGC-823 after siRNA transfection, control was BGC-823 without any
treatment, and siRNA-control was siRNA transfected with other genes. Results were significant
between siRNA-SLC7A11 and control groups (\*\*\* p < 0.001). b. The SLC7A11 expression level of BGC-823
after engineered bacterial infection with different MOI. VNP20009 without functional plasmids at
an MOI of 1:5000 was used as control group. The results of VNP-shSLC7A11 (1:3000), VNP-shSLC7A11
(1:5000) and VNP-control groups have significant differences (\* p < 0.05, \*\* p < 0.01)*

4. Verification the functions of SLC7A11 protein in the induction
of ferroptosis

Based on the above experiments, the results have shown that the cross-kingdom RNAi delivered from
VNP20009-shSLC7A11 have been successfully achieved. Next, we will verify that SLC7A11 inhibition promotes
lipid peroxidation in tumor cells and accelerate the occurrence of ferroptosis, including the viability of
tumor cells and the content of Glutathione (GSH) in tumor cells after bacterial infection. The results of
the above several modules could be used as indicators to determine whether the down-regulation of
*SLC7A11* could promote the ferroptosis of tumor cells.

**4.1 Cell viability detection**

Cell viability was measured using the CCK-8 reagent after bacterial infection. BGC-823 cells were
cultured in 96-well plates, and engineered bacteria were added to it with MOI at 1:500, 1:1500 and 1:3000
respectively. Bacteria and BGC-823 cells were co-cultured at MOI of 1:3000 as negative control, and siRNA
transfection as positive control. We used microplate reader to measure light absorption values at 450 nm,
and then calculated the percentage of surviving cells in each well. The results are shown that after
siRNA-SLC7A11 transfection and engineered bacterial infection, the proportion of surviving cells were
below 50% compared to the control cells, indicating that the down-regulation of *SLC7A11* gene could
significantly inhibit the cellular activity of tumors (Figure 6).

![](https://static.igem.wiki/teams/4737/wiki/result/shrna-4.png)

***Figure 6.** Results of cell viability test after bacteria infection. The siRNA-control is the
siRNA transfected with the other genes. The results between siRNA-SLC7A11 and siRNA-control groups
have significant differences; and results between engineered bacteria with different MOI and
VNP20009 (1:3000) infection have significant differences (\*\*\*\*p< 0.0001).*

**4.2 Detection of intracellular GSH content in tumor cells**
To verify whether the down-regulation of *SLC7A11* gene has an effect on the Xc-
system/GSH/GPX4 pathway in tumor cells, we detected the intracellular GSH content after bacterial
infection. To achieve this goal, we firstly established a standard curve of GSH (Figure 7a). According to
this standard curve, we calculated the GSH content in the supernatant after lysis of the experimental and
control cells by combining the absorptive value of the treated samples at 412 nm. Compared with the
control group infected with VNP20009 without functional plasmid, the GSH content within BGC-823 after
VNP20009-shSLC7A11 infection was significantly reduced (Figure 7b). This indicated that the metabolic
pathway of cysteine and other substances in tumor cells were effectively inhibited by the down-regulation
of *SLC7A11* gene expression.

![](https://static.igem.wiki/teams/4737/wiki/result/shrna-2.png)

***Figure 7.** Results of the GSH detection. a.Standard curve of the GSH content varying with the
absorbance value. b.The intracellular GSH content in BGC-823 cells after VNP20009 and engineered
bacteria infections. Results between VNP-shSLC7A11 (1:500) and VNP20009 (1:500) infection have
significant differences (\*\*p< 0.01).*

The above results all indicate that the engineered VNP20009 carrying functional plasmids can invade tumor
cells and deliver shRNA into tumor cells to silence the expression of *SLC7A11* gene. Down-regulation
of *SLC7A11* gene will inhibit Xc- system/GSH/GPX4 pathway, leading to the accumulation of
intracellular lipid peroxides, and ultimately promote ferroptosis of tumor cells. This suggests that the
use of engineered bacteria-mediated RNAi is an effective way to silence intracellular gene expression,
which will provide a new approach for cancer treatment.

Verification of Anaerobic Expression

1. Plasmids construction

To enhance verification of the functioning of *nirB* promoter (*pnirB*) in VNP20009, we
constructed the *pnirB::HlyA-6×His plasmid* (Figure 1).

![](https://static.igem.wiki/teams/4737/wiki/result/10-8/pnirb/result-nirb-figure-1.png)

***Figure 1.** The *pnirB::HlyA-6×His* vector that was constructed for verifying the
function of nirB promoter.*

![](https://static.igem.wiki/teams/4737/wiki/result/10-8/pnirb/result-nirb-figure-2.png)

***Figure 2.** Agarose gel electrophoresis analysis of linearized vector pFPV25.1.*

![](https://static.igem.wiki/teams/4737/wiki/result/10-8/pnirb/result-nirb-figure-3.png)

***Figure 3.** Agarose gel electrophoresis analysis of pnirB (407 bp) and HlyA-6×His (1608 bp).
Red frames outlined in the figure are target fragments. a. HlyA-6×His fragment; b. pnirB
fragment.*

We obtained the linearized vector by double digestion of vector *pFPV25.1* with endonucleases
*Xba*I and *Mfe*I (Figure 2). We amplified the target fragment *pnirB* from *E. coli*
K12 genome (Figure 3a) and the *HlyA-6×His* fragment synthesized by the company by PCR (Figure 3b),
after which we connected above target fragments to the linearized vector through homologous recombination
to obtain positive clone recombinants. Then we carried out colony PCR (Figure 4) and Sanger sequencing
(Sangon Biotech) to verify whether the *HlyA-6×His* was ligated into the vector, which meant
*pnirB::HlyA-6×His* plasmid was constructed successfully.

![](https://static.igem.wiki/teams/4737/wiki/result/10-8/pnirb/result-nirb-figure-4.png)

***Figure 4.** Colony PCR was used to confirm whether pnirB::HlyA-6×His was successfully ligated
with the vector.*

We transform the constructed plasmids into *E. coli* for incubating, then use the lysis method to get
new plasmids and transform them into VNP20009 respectively with electroporation.

2. Expression of effector proteins in *S. typhimurium* driven
by *pnirB*.

Western blotting proved that engineered bacteria can be induced to produce Listeriolysin-O under hypoxic
conditions (Figure 5). We added a 6×His tag to the C-terminus of Listeriolysin-O on the vector, and the
expression of Listeriolysin-O was characterized through it.

![](https://static.igem.wiki/teams/4737/wiki/result/10-8/pnirb/result-nirb-figure-5.png)

***Figure 5.** SDS-PAGE analysis of the expression of Listeriolysin-O. 6×His tag molecular weight
is about 59.5 kDa and GAPDH is a reference protein in cells with a molecular weight of 36 kDa.*

Based on the above results, the engineered bacteria we constructed successfully expressed target protein
under hypoxic conditions.

Verification of logical suicide circuit system

In the safety module we have designed “logical suicide circuit” to maintain two plasmids in the project
and to induce engineered bacteria to commit suicide through intake of doxycycline after the end of the
mission (more details see in [Design](https://2023.igem.wiki/bnuzh-china/design)). The “logical
suicide circuit” involves two strong and tightly repressible promoters *pLacO* and *pLtetO*. To
validate the “logical suicide circuit”, we first tested the functionality of the *pLacO* and
*pLtetO* promoters.

![](https://static.igem.wiki/teams/4737/wiki/design/design-figure-10.png)

***Figure 1.** Schematic representation of the "logical suicide circuit"*

1. Validation of *pLacO* and *pLtetO*

Aiming to test the functionality of the *pLacO* and *pLtetO* promoters, we constructed the
*pET-GFP-LacI* and *pJKR-L-tetR* plasmids. The *pET-GFP-LacI* plasmid utilized the
constitutive promoter *prpsM* to express the LacI protein, which effectively represses the
*pLacO* promoter. Meanwhile, *pLacO* regulates the expression of GFP. Similarly, the
*pJKR-L-tetR* plasmid contains a tetR repressor protein that efficiently represses the *pLtetO*
promoter, with *pLacO* governing the expression of GFP. We separately transformed the
*pET-GFP-LacI* and *pJKR-L-tetR* plasmids into BL21 (DE3) cells to validate a portion of our
logical circuit (called BL21-pET-GFP-LacI and BL21-pJKR-L-GFP-tetR).

![](https://static.igem.wiki/teams/4737/wiki/result-new-picture/safety-result-2.png)

***Figure 2.** Construction of plasmid pET-GFP-LacI and pJKR-L-tetR plasmids to test pLacO and
pLtetO*

The engineered bacteria BL21-pET-GFP-LacI was cultured at 37°C, 220 rpm until OD600=0.5, and
original BL21 as a blank control operate accordingly. Subsequently, 0.1 mM IPTG was added to the solution
of BL21-pET-GFP-LacI bacteria, followed by culturing for 6 h at 37℃, 220 rpm. A negative control
consisting of BL21-pET-GFP-LacI (IPTG-free) was also cultured under the same condition. Then we measured
the GFP fluorescence intensity of these cultures. The fluorescence intensity of BL21-pET-GFP-LacI+IPTG was
found to be significantly higher than that of BL21-pET-GFP-LacI and blank control, indicating that
*pLacO* functions effectively (Figure 3).

Similarly, BL21-pJKR-L-GFP-tetR was cultured at 37°C, 220 rpm until OD600=0.5, and original
BL21 as a blank control operate accordingly. Subsequently, we added 0.01 μg/mL, 0.1 μg/mL, 0.2 μg/mL, 0.3
μg/mL doxycycline (Dox) into BL21-pJKR-L-GFP-tetR separately and culture for 6 h at 37℃, 220 rpm.
BL21-pJKR-L-GFP-tetR (Dox-free) was cultured accordingly as a negative control. Measure the GFP
fluorescence intensity of these cultures. The fluorescence intensity of BL21-pJKR-L-GFP-tetR+Dox are
significantly higher than BL21-pJKR-L-GFP-tetR and blank control (especially BL21-pJKR-L-GFP-tetR+0.1
μg/mL Dox) which means *pLtetO* functions effectively and the best concentration of Dox is about 0.1
μg/mL (Figure 3).

![](https://static.igem.wiki/teams/4737/wiki/result-new-picture/safety-figure-prism-new-new.png)

***Figure 3.** The fluorescence intensity of BL21-pET-GFP-lacI with/without IPTG and
BL21-pJKR-L-GFP-tetR with different concentration of Dox*

2. Validation of Hok-Sok

In order to further validate "logical suicide circuit" and test the function of Hok-Sok, we constructed
two plasmids, *pET-Hok-Sok-LacI* and *pGEN-prpsM::tetR*. These two plasmids were then
transformed into BL21 (DE3) competent cells together. The *pGEN-prpsM::tetR* plasmid utilized the
constitutive promoter *prpsM* to express the tetR protein. Within the *pET-Hok-Sok-LacI*
plasmid, the *pLtetO* regulates the expression of LacI repressor, the promoter *pLacO* regulates
the expression of antitoxin *Sok* and the *trp* promoter regulates the toxin Hok. However, it
was later discovered that BL21 (DE3) endogenously express LacI protein making it difficult to validate
"logical suicide circuit" in this strain.

![](https://static.igem.wiki/teams/4737/wiki/result-new-picture/safety-result-4.png)

***Figure 4.** Construction of plasmid pET-Hok-Sok-LacI and pGEN-prpsM::tetR*

Therefore, we tested *pET-Hok-Sok-LacI* plasmid alone. When *pET-Hok-Sok-LacI* works alone,
toxin Hok and LacI repressor will be constitutively expressed. LacI will repress the expression of
antitoxin *Sok* and leads to the death of engineered bacteria. The addition of IPTG, which can bind
with LacI and inhibit its repression *Sok* production, preventing engineered bacteria from being
killed by Hok.

The *pET-Hok-Sok-LacI* plasmid was transformed into BL21 (DE3) (BL21-Hok/Sok). BL21-Hok/Sok can grow
in the LB-agar plates with IPTG as expected. We selected nine colonies and individually transferred them
to 10 μL LB liquid medium. Subsequently, the bacteria solution was transferred to the LB-agar plates
without IPTG. They were cultured at 37℃ for 36 h. It was found that all of the colonies can’t grow up
which means *pET-Hok-Sok-LacI* can work well as we designed.

However, it is a pity that we don’t have enough time to validate complete "logical suicide circuit" in our
chassis strain.

![](https://static.igem.wiki/teams/4737/wiki/proof-of-concept/proof-of-concept-figure-7.png)

***Figure 5.** Bacterial BL21-Hok/Sok can grow in the LB-agar plates with IPTG (left), but cannot
live in the LB-agar plates without IPTG (right).*

3. Plasmid Stability Test

To assess the stability of plasmids without selection pressure, we constructed the *pET-RFP-lacI-Kan*
plasmid. Afterwards, the *pET-RFP-LacI-Kan* plasmid was transformed into BL21 (DE3) along with the
*pGEN-Amp* plasmid, which is the skeleton of functional plasmid *pGEN-prpsM::tetR*. The
*pET-RFP-LacI-Kan* is the skeleton of functional plasmid *pET-Hok-Sok-LacI* with a reporter gene
*RFP*.

![](https://static.igem.wiki/teams/4737/wiki/result-new-picture/safety-result-6.png)

***Figure 6.** Plasmid construction of pET-RFP-LacI-Kan and pGEN-Amp*

The engineered bacteria (BL21-Kan/Amp) were cultured in the LB liquid medium without antibiotics for 48 h
and 72 h. And then we found that 5.1% of BL21-Kan/Amp lost *pET-RFP-LacI-Kan* while none of
BL21-Kan/Amp lost *pGEN-Amp* in 48h. And 15.4% of the BL21-Kan/Amp lost *pET-RFP-LacI-Kan* while
1.07% of BL21-Kan/Amp lost *pGEN-Amp* in 72 h (specific methods see in [Experiments](https://2023.igem.wiki/bnuzh-china/experiments)). These data have been provided to
model.

![](https://static.igem.wiki/teams/4737/wiki/result-new-picture/safety-result-7.png)

***Figure 7. a**, BL21-Kan/Amp colonies of 48 h and 72 h in LB-agar plates without antibiotic.
Red colonies are engineered bacteria containing pET-RFP-lacI-Kan while white lost pET-RFP-LacI-Kan.
**b**, A certain number of single colonies were randomly picked from non-antibiotic LB-agar
plates (a) to LB liquid medium containing ampicillin and grown in a 37℃ incubator. H10, H11 and H12
are LB liquid medium without colonies as blank control. Measure the OD600 of these
culture and compare to blank control to find out the lost rates of pGEN-Amp.*

To calculate the total number of generations of engineered bacteria by culturing time, we measured the
specific growth rates of original BL21 (DE3) and BL21-Kan/Amp (specific methods see in [Experiments](https://2023.igem.wiki/bnuzh-china/experiments)). It revealed that engineered
bacteria grow slower than original bacteria due to the burden of the exogenous plasmids. The maximum
specific growth rates of engineered bacteria decrease 15.9% to original bacteria. These data have been
provided to model.

![](https://static.igem.wiki/teams/4737/wiki/result-new-picture/safety-result-8.png)

***Figure 8.** The specific growth rates of original BL21 (DE3) (left) and BL21-Kan/Amp (right).
We measured the specific growth rates by measuring the OD600 of the samples at 15 minutes
intervals.
The maximum specific growth rates were measured in the exponential growth time.*

{% endblock %}