Plasmid

Part:BBa_K4841018

Designed by: Qiu Zexuan   Group: iGEM23_TSBC-SZ   (2023-08-12)


pET28a-GOX1

BBa_K4841018 (pET28a-Gox1) Documentation

BBa_K4841018 (pET28a-Gox1) Documentation

Construction Design

Figure 1
Figure 1: The plasmid map of pET28a-Gox1

BBa_K4841018 (pET28a-Gox1) is mainly composed of BBa_K2934000/K4841000 (Gox1) and K3521004 (pET-28a backbone). Gox1 is used to code for the glucose oxidase enzyme to digest glucose which we designed to promote TNBC ferroptosis. pET-28a backbone commonly used fusion protein type prokaryotic expression vector for host E. coli DH5α and BL21(DE3).

Engineering Principle

Figure 2
Figure 2: Project Engineering Schematic Diagram

Ferroptosis is a new type of programmed cell death, which is different from apoptosis, cell necrosis, and autophagy. The main mechanism is that under the action of iron or ester oxygenase, the highly expressed unsaturated fatty acids on the cell membrane are stimulated, and lipid overoxidation occurs, thus inducing cell death. The pathway of ferroptosis has two independent molecular mechanisms, namely glutathione peroxidase 4 (GPX4) and ferroptosis suppressor protein 1 (FSP1). GPX4 converts cytotoxic lipid peroxides to non-toxic lipid alcohols by consuming glutathione (GSH), while FSP1 catalyzes Coenzyme Q10 cycling to Coenzyme Q10H2 to trap lipid peroxides free radicals.

As shown in Figure 2, 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.

Experimental Approach and Construction Result

Figure 3
Figure 3: Experiment design flowchart

By restriction enzyme double digestion and ligation, we inserted the Gox1 gene into the pET-28a plasmid. Then, we transformed it into the DH5α E.coli, which is also used to store the plasmid, and transformed it into BL21 E.coli to express protein, respectively.

Figure 4
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

Characterization/Measurement

Figure 5
Figure 5: SDS-PAGE gel of pET28a-Gox1 protein

Through IPTG induction, protein purification experiments, we successfully expressed and purified the protein, as shown in Figure 5. The molecular weight of the target protein Gox1 is 65kDa.

Figure 6
Figure 6: Gox1 deregulates 4T1 cancer cells by glucose oxidase.

From Fig.6A and 6B 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.6C which present the result of CCK-8, we can observe that the area of light pink color with higher concentrations of GOX1 proteins means fewer 4T1 cells. 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.6D. 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
Figure 7: Gox1 deregulates 4T1 cancer cells’ GSH activity.

By detecting the same sample as CCK-8, in Fig.7C 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.7D 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.

References

  1. Conrad, Marcus et al. “Targeting Ferroptosis: New Hope for As-Yet-Incurable Diseases.” Trends in molecular medicine vol. 27,2 (2021): 113-122. doi:10.1016/j.molmed.2020.08.010
  2. Ke Li, Chuanchuan Lin, Menghuan Li, et al. Multienzyme-like Reactivity Cooperatively Impairs Glutathione Peroxidase 4 and Ferroptosis Suppressor Protein 1 Pathways in Triple-Negative Breast Cancer for Sensitized Ferroptosis Therapy. ACS Nano 2022, 16, 2, 2381–2398.
  3. Youjing Yang, Yu Ma, Qianmin Li, et al. STAT6 inhibits ferroptosis and alleviates acute lung injury via regulating P53/SLC7A11 pathway. Cell Death Dis. 2022 Jun; 13(6): 530.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 6220
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 4440
    Illegal NgoMIV site found at 4600
    Illegal NgoMIV site found at 6188
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 30
    Illegal BsaI site found at 361
    Illegal BsaI.rc site found at 307
    Illegal BsaI.rc site found at 1132


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