Ferroptosis is a natural way for cells to die. There are two independent molecular mechanisms for iron death, namely glutathione peroxidase 4 (GPX4) and ferroptosis suppressor protein 1 (FSP1)1,2. One of these ways is using glutathione. Therefore, we would inhibit glutathione to make ferroptosis happen3,4. To be more specific, ferroptosis needs lipid peroxidation to intrigue cells to die. Generally, cells would consume glutathione to convert cytotoxic lipid peroxides to non-toxic lipid alcohols so as to inhibit ferroptosis. In our project, we aimed at the Gox1 gene. TNBC cells have a high metabolic rate, so they need sufficient nutrition such as glucose.
As shown in the Figure 1, what we do is to use the Gox1 encodes glucose oxidase to inhibits GPX4/GSH and FSP1/CoQ10H2, the two iron death inhibition pathways, and interferes with lipid metabolism or REDOX balance, which can lead to iron death in various cancers and inhibit tumor cell proliferation.
Figure 1. Project Engineering Schematic Diagram 3
Design:
a. Obtain the synthetic gene Gox1
b. Design primers
c. Clone the target gene and ligate it to the vector pET-28a
d. E. Coli transformation (DH5αand BL21)
e. Protein expression and purification: SDS-PAGE
f. Protein activity test: glucose oxidation; CCK-8; GSH detection
Figure 2. Experiment design flowchart
Build:
We first multiplied the Gox1 gene. Meanwhile, we used a restriction enzyme to digest to cut a part of the pET-28a plasmid. The pET-28a plasmid is a common plasmid in E.coli, so it is suitable to use it as a carrier. By using ligation, we inserted Gox1 gene into pET-28a plasmid. Then we transformed it into the DH5α E.coli which is also used to store plasmid and transformed it into BL21 E.coli to express protein, respectively.
We use PCR to amplify the Gox1 gene, as shown in Fig.4A. Then, the linear plasmid pET-28a obtained by restriction enzyme double digestion is shown in Fig.4B. From Fig.4C and Fig.4D, it can be seen that the recombinant plasmid was successfully constructed and successfully transformed to Escherichia coli DH5α and BL21(DE3).
The sequencing results in Figure 5 also demonstrate that our plasmid was successfully constructed and no mutations occurred.
Figure 4. Electrophoresis gel results of pET28a-Gox1: A shows the gel electrophoresis result of PCR for Gox1; B represents a double enzyme gel cleavage diagram. C is PCR validation gel map. D is monoclone plate.
Figure 5. Gene sequencing results of pET28a-Gox1 plasmid
Test:
- SDS-PAGE
IPTG induction expression and protein purification were conducted with pET28a-Gox1 E. coli BL21. As shown in Figure 6. The molecular weight of the target protein GOX1 is 65kDa as expected.
Figure 6. SDS-PAGE map of pET28a-Gox1 protein
- Glucose Oxidase Activity; CCK-8
After the target protein GOX1 is successfully expressed, its function is verified through a series of experiments in order to measure its glucose oxidase activity and inhibition ability against 4T1 cells.
From Fig.7A and 7B which present the glucose oxidase activity of the protein GOX1. As the protein concentration increases, the glucose oxidase activity also increases. This trend confirmed the positive glucose oxidase activity of protein GOX1.
In Fig.7C which present the result of CCK-8, we can observe that the area of light pink colour with higher concentrations of GOX1 proteins means fewer 4T1cells. After measuring the activity by a microplate reader, we can see that there is a declining trend of 4T1 cells against GOX1 protein concentrations in Fig.7D. Therefore, we can conclude that GOX1 proteins possess the inhibition ability against 4T1 cells and higher concentrations of the protein, better suppressed the 4T1 cells under given setting.
Figure 7. Gox1 deregulars 4T1 cancer cells by glucose oxidase.
- GSH Activity
By detecting the same sample as CCK-8, in Fig.8C we could draw the similar conclusion that higher concentrations of the protein Gox1 possessed higher glutathione digestion ability.
After being measured by a microplate reader, Fig.8D shows that 64 μg/mL of Gox1 protein and RSL (1 μM) can achieve about the same level of cancer inhibition which could possess a higher potential to promote iron death of TNBC cells.
Figure 8. Gox1 deregulars 4T1 cancer cells’ GSH activity.
Learn:
Throughout the whole cycle, we could confirm the great potential of the protein GOX1 in TNBC treatment based on ferroptosis theory. The protein possesses good enzyme activity to digest glucose and inhibit the growth of 4T1 cells in experiments. All these evidences support the conclusion that this new protein therapy on TNBC has a promising future.
Besides, during this research, we learned many experiment skills and techniques. But we also notice that there is still improvement required, such as protein purification and ferroptosis theory. This experience inspired us to continue the pursuit of a biological career in the future.
DBTL
Cycle of Part
BBa_K4841020
(COLA-PesaS-RBS-RFP-rrnbT1-CI857-PR-SSRz-rrnB-Gox1)
Design
To enable possible in vivo lysis progression and increase lysis efficiency and protein yield, a heat-induced autolysis pathway was designed and monitored with a quorum sensing system.
Figure 9. Scientific concept of heat-induced autolysis system
The heat-induced lysis system, its functional test has more cleavage efficiency tests than the ordinary pET28a vector. The quorum sensing system will express whether it is lysed through the brightness of red fluorescence. The plasmid vector used in this system is pCOLA. The procedure shown on below：
Figure 10. shows the experimental design of heat-induced lysis system.
Build:
Figure 11 electrophoresisgel results of pCOLA-CI857-SRRz-Gox1 plasmid construction
Fig.11A: gel map of the vector after Gibson assembly
Fig.10B: gel map of the Gox1 gene fragment
Fig.11C: PCR identification gel map of PaCYC-QS quorum sensing vector by colony PCR.
Fig.11D and 10E: the PCR identification gel map of pCOLA-CI857-SRRz-Gox1 vector by colony PCR.
After amplifying Gox1, we used the Gibson assembly to complete the construction of the pCOLA-CI857-SRRz-GOX1 vector. And colony PCR identification was performed to validate the successful construction. Figure 11 shows the construction results of pCOLA-CI857-SRRz-Gox1 plasmid and its quorum sensing vector PaCYC-QS. Figure 11A shows the pCOLA-CI857-SRRz-Gox1 vector constructed after Gibson assembly. Figure 11B shows the gel map of the Gox1 gene which encodes Gox1 cassette. After assembly, we transformed the plasmids into DH10β competent cells (PaCYC-QS vector has been synthesized by the company). After the transformation and cultivation, we conducted colony PCR identification. Figure 11C is the colony PCR identification result of the quorum sensing vector PaCYC-QS. Figure 11D is colony PCR identification of the Gox1 gene in the pCOLA-CI857-SRRz-Gox1 plasmid.
Test:
- Protein expression, cell lysis and SDS-PAGE
Figure 12 Lysis result of heat-induced autolysis plasmid (pCOLA-CI857-SRRz-GOX1 and PaCYC-QS).Figures 12A and 12B are two replicates.
After overnight incubation of the pCOLA-CI857-SRRz-Gox1 BL21 E. coli, lysis was measured by the microplate reader and monitored to ensure enough lysis. The results in Figure 11 prove that the cells were fully lysed, and the protein coded by Gox1 gene will be purified following.
Figure 13 SDS-PAGE of pCOLA-CI857-SRRz-Gox1 plasmid protein expression after heat-induced autolysis.
After purifying samples from the lysis of pCOLA-CI857-SRRz-Gox1 BL21 E. coli, we performed SDS-PAGE to verify the expression of the target protein. The size of the target protein, Gox1 is 65kDa, as shown in Figure 13, the same of that as Figure 6
- Glucose Oxidase Activity; CCK-8
Figure 14 glucose oxidase activity and CCK-8 activity of the GOX1 protein purified after heat-induced autolysis
Figure 14A glucose oxidase activity of the autolysis experimental group.
Figure 14B relative CCK-8 activity of the autolysis experimental group.
For the GOX1 protein purified after heat-induced autolysis, we also conducted glucose oxidase activity assay and CCK-8 test. Figure 14A shows that when the protein concentration increases, the glucose concentration increases in a positive trend, confirming that the protein is able to promote ferroptosis by consuming glutathione. The CCK-8 test in Figure 14B also backs up this inference.
Figure 15 GSH Activity test on 4T1 cells of the GOX1 protein purified after heat-induced autolysis
Figure 15A GSH activity standard curve of the autolysis experimental group
Figure 15B relative GSH activity of the autolysis experimental group.
GSH Activity Assay was also conducted with the GOX1 protein purified after heat-induced autolysis. In Figure 15, compared with the control group, as the protein concentration increases, the GSH content decreases. In other words, higher protein concentration possesses higher glutathione digestion performance, which also means a better effect on promoting 4T1 cell ferroptosis at the given range.
Learn:
Through the 2nd cycle, we successfully fulfilled the in vivo lysis progression through the heat-induced autolysis pathway with a quorun sensing system. We also evaluate the protein activity of Gox1 and its potential in curing TNBC through ferroptosis. In the future, the efficiency of in vivo lysis still requires further investigation and measurement which will be the next move of this sensing system.
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2. Yang, Youjing et al. “STAT6 inhibits ferroptosis and alleviates acute lung injury via regulating P53/SLC7A11 pathway.” Cell death & disease vol. 13,6 530. 6 Jun. 2022, doi:10.1038/s41419-022-04971-x
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4. Yang, Fan et al. “Ferroptosis heterogeneity in triple-negative breast cancer reveals an innovative immunotherapy combination strategy.” Cell metabolism vol. 35,1 (2023): 84-100.e8. doi:10.1016/j.cmet.2022.09.021