Group: TSBC-SZ iGEM 2023

Summary

Gox1 gene codes for the glucose oxidase enzyme to digest glucose. In this project, we used this protein for proceeding with TNBC ferroptosis. We used the synthetic Gox1 (BBa_K2934000) gene fragment to connect to the pET28a vector to construct a recombinant plasmid pET28a-Gox1 (BBa_K4841018). For this new composite part, we have tested the glucose oxidase activity, GSH activity, and TNBC inhibition activity of the Gox1 protein.

Documentation

a. Usage and Biology

Glucose oxidase is an enzyme that has widespread applications in industry and biotechnology. Due to this, a deep understanding of its structure and function are warranted. Glucose degradation is the most universal metabolic process. In addition to its breakdown in glycolysis, glucose can also be directly oxidized to glucono-δ-lactone by a number of enzymes.[1] GOx is a member of the glucose-methanol-choline oxidoreductase (GMC oxidoreductase) superfamily. The members of this family are all FAD-dependent oxidoreductases that share a common fold [2,3]. They consist of two functional domains, an N-terminal FAD-binding domain, which contains a strictly conserved βαβ mononucleotide-binding motif and a more variable substrate binding-domain. As the name suggests, the members of this family oxidize a variety of substrates containing hydroxyl functional groups, including mono and di-saccharides, alcohols, cholesterol, and choline.[1]

Figure 1: Gox1 was successfully attached to the vector per the sequencing result, and Gox1 protein was found at 65 Kda by SDS-PAGE

Characterization/Measurement

We amplified Gox1 and pET28a through PCR and then plasmid pET28a-Gox1 was constructed by T4 ligation. The sequencing result validates correct construction (Figure 1).

Figure 2: Gox1 protein glucose oxidase activity and CCK-8 Test with 4T1 cells. Fig. 2A is 4T1 cells (mouse breast cancer cells). Fig. 2B is glucose oxidase activity values of different concentrations of the protein. Fig. 2C CCK-8 samples on a 96 well plate. Fig. 2D CCK-8 activity values.

From Figures 2A and 2B, as the protein concentration increases, the glucose oxidase activity also increases. We can be seen that the higher the glucose oxidase activity, the easier it is to promote iron death. Fig 2C and D are both results of CCK-8, and the part which color is pink is the part where the cells are better suppressed. We can be seen that the higher the glucose oxidase activity, the easier it is to promote iron death.

Figure 3: Gox1 GSH Activity test on 4T1 cells. Fig. 3A shows a 96 well plate diagram of the GSH standard samples. Fig. 3B is the GSH activity standard curve. Fig. 3C shows the 96 well plate diagram of GSH experimental samples. Fig. 3D shows the relative GSH activity values of experimental samples.

Fig. 3A and Fig. 3B are a linear function with different concentrations of GSH for reference. And Fig. 3C is the result of CCK8, and transparent parts indicate low GSH activity. Fig. 3C is able to visualize the difference in color, Fig. 3D shows that 64 μg/mL of Gox1 protein and RSL (1 μM) can achieve about the same level of cancer inhibition. Fig. 4D shows that RSL (1 μM) and 64 μg/mL of Gox1 protein inhibit GSH activity relatively well and could promote iron death.

References

Bauer JA, Zámocká M, Majtán J, Bauerová-Hlinková V. Glucose Oxidase, an Enzyme "Ferrari": Its Structure, Function, Production and Properties in the Light of Various Industrial and Biotechnological Applications. Biomolecules. 2022 Mar 19;12(3):472.
Cavener D.R. GMC oxidoreductases: A newly defined family of homologous proteins with diverse catalytic activities. J. Mol. Biol. 1992;223:811–814.
Sützl L., Foley G., Gillam E.M.J., Bodén M., Haltrich D. The GMC superfamily of oxidoreductases revisited: Analysis and evolution of fungal GMC oxidoreductases. Biotechnol. Biofuels. 2019;12:118.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
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