Florecent
Salmonella
Results
“
Our project explores the groundbreaking potential of genetically engineered Salmonella in cancer therapy. By selectively targeting tumor cells and inducing apoptosis, we aim to create a more effective and less invasive treatment option.
Engineered Salmonella Beat Cancer
Verification of VNP20009 strain
Figure 1.1 16s Assay Result of <em>VNP20009</em>
Figure 1.1 16s Assay Result of VNP20009
After doing 16s assay on the strain we received from BNUZH-China, 2023 iGEM team, we did BLAST on National Library of Medicine. The result is shown in figure, which indicates that the strain we got is Salmonella enterica serovar Typhimurium strain LT2. Moreover, we did PCR about the deleted gene (msb, purL) of VNP20009, with wild type strain as positive control. The gel image below shows that the strain we got didn’t have msb or purL gene, and was probably VNP20009.
Fig 1.2 1.5% Agarose Gel of Gene Deletion Verification of <em>VNP20009</em>
Fig 1.2 1.5% Agarose Gel of Gene Deletion Verification of VNP20009
Recognition System
Plasmid Construction
To verify the recognition of HER2 scFv to HER2 antigen, we synthesized the gene sequence of Lpp-OmpA-1-HER2_scFv and insert it into BPK764 vector, where Lpp-OmpA-1 is the surface display system derived from Francisco, J A, et al.’s work (Francisco et al., 1992). Moreover, we synthesized two other surface display systems, one is from iGEM Standard Biological Parts, Lpp-OmpA-2, and the other is Non-OmpA from Jeiranikhameneh et al.’s work (Jeiranikhameneh et al., 2017), in order to compare their display efficacy. And we insert the synthesized genes to BPK764 as well.
Figure 2.1 Construction of Lpp/Non-OmpA-scFv or scFv Plasmids for Function Verification
Figure 2.1 Construction of Lpp/Non-OmpA-scFv or scFv Plasmids for Function Verification
Considering that it is difficult to purify membrane protein from E.coli, we amplified HER2 scFv from BPK764 and insert it into pET28(a)+. Poly his sequence is added at 3’-terminal of HER2 scFv for following purification.
HER2scFv Purification
With online calculator, we got the size of HER2 scFv, which is 25.85 kDa.
Before Ni-NTA beads are added, we did SDS-PAGE and Western Blot to determine whether it could be expressed in SHuffle® E. coli, which is a genetically engineered chemically competent K12 cells of E. coli, can express proteins containing disulfide bonds in the cytoplasm.
Figure 2.2.1 SDS-PAGE and Western Blot Result of Samples from Steps during Purification
Figure 2.2.1 SDS-PAGE and Western Blot Result of Samples from Steps during Purification
From left to right (same for the two pictures): a) Marker: ColorMixed Protein Marker 180 (10-180 kDa) (RM19001). b) culture: LB culture from the overnight culture of SHuffle. c) supernatant of culture: the supernatant after centrifuging the overnight LB culture of SHuffle. d) precipitation of culture: the precipitation after centrifuging the overnight LB culture of SHuffle. e) mixture after sonication: the mixture after sonicating the SHuffle precipitation. f) supernatant after sonication: the supernatant after centrifuging the sonication mixture. g) precipitation after sonication: the precipitation after centrifuging the sonication mixture.
Direct antibody: Mouse monoclonal antibody against His label (LABLEAD H1104) LABLEAD
The left figure in Figure 2.2.1 shows that the antibody can be successfully expressed, although it was not so clear in the gel bands. There are always many other proteins in E.coli and it may show very dirty on the SDS-PAGE gel.
After the purification described in the protocol, we got the protein chromatography result. We also collected samples of each step during purification, followed by doing SDS-PAGE and Western Blot.
Figure 2.2.2 SDS-PAGE and Western Blot Result of Samples from Purification by AKTA
Figure 2.2.2 SDS-PAGE and Western Blot Result of Samples from Purification by AKTA
(a) Marker: ColorMixed Protein Marker 180 (10-180 kDa) (RM19001).
(b) a: Flow through step.
(c) b: Wash step.
(d) c: Elute B1.
(e) d: Elute B4.
(f) e: Elute B5.
(g) f: Elute C4.
(h) g: Elute C5.
(i) h: Elute C6.
(j) i: Elute C7.
(k) j: Elute C8.
(l) k: Elute C9.
Direct antibody: Mouse monoclonal antibody against His label (LABLEAD H1104) LABLEAD.
There are two peaks from chromatography, and the bands in SDS-PAGE are relatively messy. We suppose the highest peak is polymer, but the result of Western Blot shows that there is only one band of scFv. So we used the samples from B4 and B5 to do the following experiments since they have higher purity.
Enzyme Linked Immunosorbent Assay (ELISA)
Figure 2.3 The Results of ELISA
Figure 2.3 The Results of ELISA We did ELISA of the B5 hole of protein purified by AKTA.
It shows that the HER2 scFv we purified can efficiently recognize HER2 scFv. The results of 25.00 ng/mL, 50.000 ng/mL and 100.00 ng/mL are even above the detection limit of the ELISA kit. From the picture that is taken 20 min after adding TMB substrate, we can even see some precipitation in the left three holes. Hence, the binding efficiency of HER2 scFv used in our project is enough to recognize HER2 antigen on cancer cells efficiently.
Fluorescence Microscopy
Figure 2.4.1 Co-culturing of MCF10A and Lpp-2 <em>VNP20009</em>
Figure 2.4.1 Co-culturing of MCF10A and Lpp-2 VNP20009
(a): Bright field (b): The Cy5 channel of Far Red (c): Merged image of (a) and (b)
Figure 2.4.2 Co-culturing of SK-BR-3 and Lpp-2 <em>VNP20009</em>
Figure 2.4.2 Co-culturing of SK-BR-3 and Lpp-2 VNP20009
(a): Bright field (b): The Cy5 channel of Far Red (c): Merged image of (a) and (b)
An obvious colocalization can be observed in Figure 2.4.1: all the fluorescent spots located in the cells whose apoptosis process had begun. A similar phenomenon can also be observed in Figure 2.4.2. However, due to the high infection efficiency towards SK-BR-3 and resulting great apoptosis rate (proven by subsequent Flow Cytometry), the colocalization is relatively weak. Overall, it shows that VNP20009 has the ability to invade the cells SK-BR-3 and MCF10A.
Flow Cytometry
Figure 2.5.1 GFP Channel in Co-culture FACS
Figure 2.5.1 GFP Channel in Co-culture FACS
(a): SK-BR-3 without bacteria (b): SK-BR-3 with Lpp-1 VNP20009 (c): SK-BR-3 with Lpp-2 VNP20009 (d): SK-BR-3 with Lpp-2 E.coli (e): MCF10A with Lpp-1 VNP20009 (f): MCF10A with Lpp-2 VNP20009 SK-BR-3 is a HER2 positive breast cancer cell line. MCF10A is a HER2 negative non-tumorigenic epithelial cell line.
Figure 2.5.2 DAPI Channel in Co-culture FACS
Figure 2.5.2 DAPI Channel in Co-culture FACS
(a): MCF10A without bacteria (b): MCF10A with Lpp-2 VNP20009 (c): SK-BR-3 without bacteria (d): SK-BR-3 with Lpp-2 VNP20009 SK-BR-3 is a HER2 positive breast cancer cell line. MCF10A is a HER2 negative non-tumorigenic epithelial cell line.
In this flow cytometry, all the green fluorescence protein was from bacteria, and only the cell infected with bacteria would show positive signal in GFP channels. In Figure 2.5.1, only the SK-BR-3 co-cultured with Lpp1/Lpp-2 VNP20009 showed strong positive signal (more than 60%) in the GFP channel, and only a few (15% on average) cells were invaded by VNP20009 for MCF10A. That's a strong statement that our recognition system has an excellent specific targeting ability.
Moreover, Figure 2.5.2 showed the apoptosis rate change with bacteria: MCF10A was unaffected by co-culturing while SK-BR-3 showed significant decrease in survival rate, which proved the engineered bacteria is safe for normal cells.
Delivery System
Plasmid Construction
To verify that PsseJ promoter is only activated when Salmonella is in human cells, we constructed two plasmids from pYX00004. The original pYX00004 (pBAD-mCherry-2aa) vector includes araBAD promoter and mCherry. We firstly replaced the araBAD promoter and araC with PsseJ promoter, and got pYX00004 (PsseJ-mCherry-2aa). Then we replaced the mCherry with lyseE gene from φ174X.
Figure 3.1 Plasmid Construction Based on pYX00004 Vector for <em>PsseJ</em> Function Verification
Figure 3.1 Plasmid Construction Based on pYX00004 Vector for PsseJ Function Verification
Extracellular Verification
Figure 3.2 Fluorescence Comparison of the Activation Strength of pBAD and PsseJ
Figure 3.2 Fluorescence Comparison of the Activation Strength of pBAD and PsseJ
From left to right: 0.02% arabinose, pBAD-mCherry; 0.00% arabinose, pBAD-mCherry; 0.00% arabinose, PsseJ-mCherry.
We cultured 3 groups of VNP20009 transformed with pYX00004 (PsseJ-mCherry) and pYX00004 (pBAD-mCherry) to LB Broth, and add 0.02% arabinose to one group of pYX00004 (pBAD-mCherry). After 24 hours incubation, we washed the bacteria culture with PBS and observed them under fluorescence microscopy. The fluorescence strength of PsseJ-mCherry group is similar to pBAD-mCherry group cultured in 0% arabinose, and is much weaker than pBAD-mCherry group cultured in 0.02% arabinose. It shows that PsseJ is not activated when Salmonella is not in human cells.
Flow Cytometry
Figure 3.3.1 mCherry Channel in co-culturing FACS
Figure 3.3.1 mCherry Channel in co-culturing FACS
(a): SK-BR-3 without bacteria (b): SK-BR-3 without bacteria (c): MCF10A with PsseJ-mCherry VNP20009 (d): SK-BR-3 with PsseJ-mCherry VNP20009 SK-BR-3 is a HER2 positive breast cancer cell line. MCF10A is a HER2 negative non-tumorigenic epithelial cell line.
The flow cytometry result also supports that mCherry fluorescence would be only expressed when PsseJ-mCherry VNP20009 co-cultured with SK-BR-3, verifying the function of PsseJ .
Verification of the function of BAX gene in B16-F10 cells
Plasmid Construction
To verify the function of BAX gene, specifically its ability to induce programmed cell apoptosis, we first used the PCR method to amplify and cut the BAX sequence from pSL886 PMusAFP-MusBax-(MS2)24-HHR-pA (Shao et al., 2024) and inserted the sequence into CD513B-Bax vector. The constructed plasmids were transfected into DH5α competent cells and verified through sequencing, which confirmed the correct sequence of the BAX gene.
Figure 4.1 Construction of CD513B-Bax Plasmid for BAX Function Verification
Figure 4.1 Construction of CD513B-Bax Plasmid for BAX Function Verification
Cells and Fluorescence Signal Observation
Next, to examine the function of BAX inside the cancer cells, we extracted the plasmid using an EndoFree DNA purification kit for subsequent cell transformation, then transfected the purified CD513B-Bax plasmid into the B16-F10 cell line. To verify its ability to inhibit cell growth and induce apoptosis, we used an inverted microscope for initial observations and flow cytometry with Annexin V-FITC and PI double staining to measure cell death and the death rate. After that, fluorescence microscopy and Western blot analysis were employed to confirm the expression of BAX gene inside the cell line, thereby demonstrating the function of BAX gene.
Figure 4.2 B16-F10 Cell Images after 24h Transfection Under 10x Microscope
Figure 4.2 B16-F10 Cell Images after 24h Transfection Under 10x Microscope
(a) B16-F10 cells before transfection (b) Untreated B16-F10 cells after 24h transfection in bright field (c) B16-F10 cells with Liposomal Transfection Reagent added after 24h transfection in bright field (d) B16-F10 cells transfected with CD513B-SV40 plasmid by Liposomal Transfection Reagent after 24h transfection in bright field (e) B16-F10 cells transfected with CD513B-Bax plasmid by Liposomal Transfection Reagent after 24h transfection in bright field (f) Merged image of B16-F10 cells transfected with CD513B-Bax plasmid by Liposomal Transfection Reagent after 24h transfection in bright field and in green field
Table 2. Cell Number of Each Group after 24h Transfection
Note:
cell: untreated B16-F10 cells
lipo: B16-F10 cells with Liposomal Transfection Reagent added
CD513B: B16-F10 cells transfected with CD513B-SV40 plasmid by Liposomal Transfection Reagent
BAX: B16-F10 cells transfected with CD513B-Bax plasmid by Liposomal Transfection Reagent
It is observed that the untreated cells, cells mixed with the transfection reagent, and cells transfected with the control plasmid (CD513B-SV40) continued to grow under experimental conditions over 24 hours. Most cells maintained their normal shape, and the seeding density increased from about 70% to above 90%. In contrast, cells transfected with the CD513B-Bax plasmid failed to increase in seeding density and exhibited clusters of dead cells. Referring to the cell numbers in Table 1, the BAX-transfected group had a cell count that was only about one-third of the other groups.
When the bright field and green field images were merged as in Figure 2(f), it was noted that the fluorescence signal was present in approximately 60% of the cells, indicating successful expression of the BAX gene in B16-F10 cells. Moreover, a relatively higher fluorescence intensity was observed in dead cells, which is consistent with the known function of the BAX gene.
Flow Cytometry
Figure 4.3 Plots of different treatment groups of B16-F10 cells double-stained by Annexin-FITC and PI after 12h and 24h transfection
Figure 4.3 Plots of different treatment groups of B16-F10 cells double-stained by Annexin-FITC and PI after 12h and 24h transfection
(a) Plots of untreated B16-F10 cells after 12h transfection (b) Plots B16-F10 cells with Liposomal Transfection Reagent added after 12h transfection (c) Plots B16-F10 cells transfected with CD513B-SV40 plasmid by Liposomal Transfection Reagent after 12h transfection (d) Plots B16-F10 cells transfected with CD513B-Bax plasmid by Liposomal Transfection Reagent after 12h (e) Plots of untreated B16-F10 cells after 24h transfection (f) Plots B16-F10 cells with Liposomal Transfection Reagent added after 24h transfection (g) Plots B16-F10 cells transfected with CD513B-SV40 plasmid by Liposomal Transfection Reagent after 24h transfection (h) Plots B16-F10 cells transfected with CD513B-Bax plasmid by Liposomal Transfection Reagent after 24h
In the flow cytometry test, positive results in the FITC channel, derived from Annexin V-FITC staining, indicate cells in the early apoptosis stage. Positive results in the PerCP channel, from PI staining, indicate cells in the late apoptosis stage. In the double-staining condition, double-positive results are specifically regarded as dead cells.
When comparing the different treatment groups, after 12 hours of transfection, the death rate in all four groups was relatively low, with more than 85% of the cells still in the early apoptosis stage. However, after 24 hours of transfection, the CD513B-Bax transfected group showed a death rate three times higher than the other three groups, confirming the BAX gene's function in inducing cell apoptosis.
Western Blot
Table 3. Western Blot SDS-PAGE Layout for BAX Protein Detection (Experiment Date: 9/30)
(a) BAX(ab32503): Rabbit Anti-Bax antibody [E63].
(b) Marker: Thermo #26619.
(c) NC: Negative control, representing the protein extracted from cells that were not transfected with the BAX plasmid.
(d) BAX: Protein extracted from cells that were transfected with the BAX plasmid.
(e) Bio-switch on: Protein extracted from cells transfected with the bio-switch plasmid, in the "on" state.
(f) Bio-switch off: Protein extracted from cells transfected with the bio-switch plasmid, in the "off" state.
Primary antibody: Rabbit Anti-Bax antibody [E63] (ab32053) 21kDa abcam 1:1000 dilution
Secondary antibody: Goat Anti-Rabbit IgG H&L (HRP) (ab205718) abcam 1:2000 dilution
Marker: Thermo #26619
Western Blot Image Chemiluminescence Detection
Figure 4.4 Chemiluminescence Detection of BAX Protein in BAX and Negative Control Samples
(a) BAX (above the image): Protein extracted from cells transfected with the BAX plasmid.
(b) NC: Negative control, representing protein extracted from cells that were not transfected with the BAX plasmid.
(c) 21 kDa: The expected and observed molecular weight of the BAX protein band, visible at 21 kDa.
(d) BAX (beside the image): The primary antibody used was anti-BAX, targeting and detecting the BAX protein.
In the primary antibody analysis of the BAX gel, the BAX group showed a significantly stronger protein signal compared to the negative control. This indicated that the BAX plasmid was successfully transfected into the B16-F10 cells and that the protein was expressed effectively. Furthermore, the relatively high expression level of BAX suggested that it is responsible for inducing apoptosis in the cells. When comparing the protein extracted on September 25 to that extracted on September 30, the signal from the latter was notably stronger, indicating protein deduction due to storage.
Verification of the bio-switch in B16-F10 cells
Cells and Fluorescence Signal Observation
Merged Image
Figure 5.1. Merged Image of B16-F10 Cell Images after 24h Transfection of pSL816 and SB100 Under 10x Microscope in Bright Field and Green Field
Cell Images
Figure 5.2 B16-F10 Cell Images after 24h Transfection of Bio-switch Plasmids Under 10x Microscope
(a) B16-F10 cells transfected with bio-switch plasmids after 24h transfection in bright field
(b) B16-F10 cells transfected with both target protein plasmids and bio-switch plasmids after 24h transfection in bright field
Western Blot
To verify the function of the bio-switch, we first transfected the B16-F10 cell line with pSL816 (Shao et al., 2024) plasmid containing the target protein NS3a and EGFP to express NS3a within the cells. After 24 hours, intense green fluorescence was observed, indicating a transfection efficiency of over 70% and confirming the expression of NS3a.
Subsequently, we co-transfected the three bio-switch plasmids (pSL886, pSL582, and pSL776) (Shao et al., 2024) into the B16-F10 cells. Under inverted microscope observations after 24 hours, cells without NS3a continued to grow normally with typical cell shapes. In contrast, cells co-transfected with NS3a and the bio-switch plasmids exhibited cell clustering, indicating the activation of the bio-switch. This activation upregulates the expression of the killer gene BAX in the presence of the target protein and remains inactive in its absence.
Table 4. Western Blot SDS-PAGE Layout (Experiment Date: 9/30)
(a) BAX(ab32503): Rabbit Anti-Bax antibody [E63].
(b) Marker: Thermo #26619.
(c) NC: Negative control, representing the protein extracted from cells that were not transfected with the BAX plasmid.
(d) BAX: Protein extracted from cells that were transfected with the BAX plasmid.
(e) Bio-switch on: Protein extracted from cells transfected with the bio-switch plasmid, in the "on" state.
(f) Bio-switch off: Protein extracted from cells transfected with the bio-switch plasmid, in the "off" state.
Primary antibody: Rabbit Anti-Bax antibody [E63] (ab32053) 21kDa abcam 1:1000 dilution
Secondary antibody: Goat Anti-Rabbit IgG H&L (HRP) (ab205718) abcam 1:2000 dilution
Marker: Thermo #26619
Chemiluminescence Detection of BAX
Figure 5.2 Chemiluminescence Detection of BAX Protein in Bio-Switch "On" and "Off" States
(a) Bio-switch on: Protein extracted from cells transfected with the bio-switch plasmid, in the "on" state.
(b) Bio-switch off: Protein extracted from cells transfected with the bio-switch plasmid, in the "off" state.
(c) 21 kDa: The expected and observed molecular weight of the BAX protein band, visible at 21 kDa.
(d) BAX (beside the image): The primary antibody used was anti-BAX, targeting and detecting the BAX protein.
In the primary antibody analysis of the BAX gel, the bio-switch "on" group displayed a significantly stronger protein signal compared to the bio-switch "off" group. This suggested that the bio-switch plasmid was successfully transferred into the B16-F10 cells, leading to effective protein expression and successful control of BAX expression. Additionally, the relatively high level of BAX expression in the bio-switch "on" group indicated that the bio-switch can regulate apoptosis in the cells. However, when comparing the protein extracted on September 25 to that extracted on September 30, the signal from the former did not exhibit any expected trend.
Supplementary information of Western Blot experiment
Table 5. Western Blot SDS-PAGE Layout for β-ACTIN Protein Detection
(a) β-ACTIN(643802): Purified anti-β-actin Antibody
(b) Marker: Thermo #26619
(c) NC: Negative control, representing the protein extracted from cells that were not transfected with the BAX plasmid.
(d) BAX: Protein extracted from cells that were transfected with the BAX plasmid.
Primary antibody: Purified anti-β-actin Antibody(643802) 43kDa BioLegend 1:500 dilution
Secondary antibody: HRP-conjugated Goat Anti-Mouse IgG(H+L) (SA00001-1) proteintech 1:2000 dilution
Marker: Thermo #26619
Chemiluminescence Detection of β-actin
Figure 6.1 Chemiluminescence Detection of β-actin Protein in BAX and Negative Control Samples
(a) BAX (above the image): Protein extracted from cells transfected with the BAX plasmid.
(b) NC: Negative control, representing protein extracted from cells that were not transfected with the BAX plasmid.
(c) 43 kDa: The expected and observed molecular weight of the β-actin protein band, visible at 43 kDa.
(d) β-actin: The primary antibody used was anti-β-actin, targeting and detecting the β-actin protein.
In the primary antibody analysis of the β-actin gel, which served as the internal reference, the negative control group exhibited a slightly stronger signal than the BAX group, and the bio-switch "off" group showed a marginally stronger signal than the bio-switch "on" group. This trend suggested that the cell count in the BAX group was lower than in the negative control group, and the cell count in the bio-switch "on" group was lower than in the bio-switch "off" group, likely due to cell apoptosis (we collected cells of the same volume for each group). We guessed that the BAX plasmid was successfully transfected into the cells, leading to relatively high levels of BAX protein expression, which induced cell apoptosis, as did the bio-switch plasmids.
Complete Chemiluminescence Detection of BAX Protein
Figure 6.2 The Complete Chemiluminescence Detection of BAX Protein (Experiment Date: 9/30)
(a) Marker: Thermo #26619.
(b) NC: Negative control, representing the protein extracted from cells that were not transfected with the BAX plasmid.
(c) BAX: Protein extracted from cells that were transfected with the BAX plasmid.
(d) Bio-switch on: Protein extracted from cells transfected with the bio-switch plasmid, in the "on" state.
(e) Bio-switch off: Protein extracted from cells transfected with the bio-switch plasmid, in the "off" state.
(f) 21 kDa: The expected and observed molecular weight of the BAX protein band, visible at 21 kDa.
Complete Chemiluminescence Detection of β-actin Protein
Figure 6.3 The Complete Chemiluminescence Detection of β-actin Protein
(a) Marker: Thermo #26619.
(b) NC: Negative control, representing the protein extracted from cells that were not transfected with the BAX plasmid.
(c) BAX: Protein extracted from cells that were transfected with the BAX plasmid.
(d) Bio-switch on: Protein extracted from cells transfected with the bio-switch plasmid, in the "on" state.
(e) Bio-switch off: Protein extracted from cells transfected with the bio-switch plasmid, in the "off" state.
(f) 43 kDa: The expected and observed molecular weight of the β-actin protein band, visible at 43 kDa.
References:
  1. Francisco, J. A., Earhart, C. F., & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A, 89(7), 2713-2717. https://doi.org/10.1073/pnas.89.7.2713
  2. Jeiranikhameneh, M., Razavi, M. R., Irani, S., Siadat, S. D., & Oloomi, M. (2017). Designing novel construction for cell surface display of protein E on Escherichia coli using non-classical pathway based on Lpp-OmpA. AMB Express, 7(1), 53. https://doi.org/10.1186/s13568-017-0350-0
  3. Shao, J., Li, S., Qiu, X., Jiang, J., Zhang, L., Wang, P., Si, Y., Wu, Y., He, M., Xiong, Q., Zhao, L., Li, Y., Fan, Y., Viviani, M., Fu, Y., Wu, C., Gao, T., Zhu, L., Fussenegger, M., . . . Xie, M. (2024). Engineered poly(A)-surrogates for translational regulation and therapeutic biocomputation in mammalian cells. Cell Research, 34(1), 31-46. https://doi.org/10.1038/s41422-023-00896-y