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.

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.

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).

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).

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).

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.

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

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.

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.

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 protein1.

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).

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).

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.

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.

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）.

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).

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 XbaI and MfeI (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.

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

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

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.

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 cleaved1. 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.

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.

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.

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.

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

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).

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.

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.

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).

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.

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).

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

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

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 XbaI and MfeI (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.

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.

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). 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.

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).

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).

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.

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.

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.

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). These data have been provided to model.

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). 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.

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.