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Parts Code |
Parts Name |
Contribution Type |
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Gox1 |
New experimental data to an existing Part |
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pet28a-backbone |
New experimental data to an existing Part |
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pET28a-GOX1 |
New composite part |
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pCOLA-PesaS-RBS-RFP-rrnbT1-CI857-PR-SRRz-rrnB backbone |
New composite part |
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pCOLA-PesaS-RBS-RFP-rrnbT1-CI857-PR-SRRz-rrnB-Gox1 |
New composite part |
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pACYC-pJ23117-esaR(I70V)-rrnBT -LSPT-esaI-BCD22-P8 |
New composite part |
1. Add new experimental data to existing parts: BBa_K3521004 and BBa_K2934000 (BBa_K4841000) and Create a new part: BBa_K4841018
In order to obtain the GOX1 protein through E. coli, we designed to use pET28a-backbone (BBa_K3521004) as the vector and ligated the synthetic GOX1 (BBa_K2934000 / BBa_K4841000) to it so as to construct the recombinant plasmid pET28a-GOX1 (BBa_K4841018) and following we transformed the plasmid into E. coli DH5α and BL21(DE3), respectively.
① Plasmid construction
The target gene Gox1 was synthesized by the company Genscript and the vector pET28a we used is provided by SubCat. Firstly we amplified Gox1 and pET28a through PCR and then plasmid pET28a-Gox1 was constructed by T4 ligation. The construction results were shown below where the gels indicate correct bands of GOX1 (Fig 1).
Figure 1. Construction of pET28a-Gox1 plasmid. Fig.1A shows the gel electrophoresis result of PCR for Gox1; Fig.1B represents a double enzyme gel cleavage diagram. Fig.1C is PCR validation gel map. Fig.1D is monoclone plate.
② Sequencing and protein expression
From Fig.2A, the red arrow shows that the sequencing results match correctly. Subsequently, the IPTG induction expression and protein purification were conducted with pET28a-Gox1 E. coli BL21. As shown in Fig.2B, SDS-PAGE gel result indicates that the molecular weight of the target protein GOX1 is 65kDa.
Figure 2. Gene sequencing results and protein expression of pET28a-Gox1 plasmid. Fig.2A shows the result of the gene sequencing. Fig.2B shows the target protein Gox1 at 65 kDa.
③ Function Tests: Glucose Oxidase Activity; CCK-8; GSH Activity
- Glucose Oxidase Activity; CCK-8;
From Fig.3A and 3B 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 and we can infer that the higher the glucose oxidase activity the easier the protein promotes iron death.
In Fig.3C 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.3D. 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 3. GOX1 protein glucose oxidase activity and CCK-8 Test with 4T1 cells. Fig.3A is 4T1 cells (mouse breast cancer cells). Fig.3B is glucose oxidase activity values of different concentrations of the protein. Fig.3C CCK-8 samples on a 96 well plate. Fig.3D CCK-8 activity values.
- GSH Activity
Fig.4A and Fig.4B is a standard curve of glutathione content served as a reference. By detecting the same sample as CCK-8, in Fig.4C 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.4D shows that 64μg/mL of Gox1 protein and RSL (1μM) can achieve about the same level of cancer inhibition which could possess a higher potential to promote iron death of TNBC cells.
Figure 4. Gox1 GSH Activity test on 4T1 cells. Fig.4A shows a 96 well plate diagram of the GSH standard samples. Fig.4B is the GSH activity standard curve. Fig.4C shows the 96 well plate diagram of GSH experimental samples. Fig.4D shows the relative GSH activity values of experimental samples.
2. Create new parts: BBa_K4841017, BBa_K4841019, BBa_K4841020
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. This mainly consists of two parts:
pACYC-pJ23117-esaR(I70V)-rrnBT-LSPT-esaI-BCD22-P8 plasmid is the sensor plasmid of the quorum sensing system. The esal in the plasmid expresses the AHL factor, and the esaR (I70V) promoter is responsible for controlling the expression of the AHL factor. The expression of the AHL factor can induce the PesaS promoter to express a red fluorescent protein. pCOLA-PesaS-RBS-RFP-rrnbT1-CI857-PR-SRRz-rrnB backbone is the protein expression vector in this system. The red fluorescent protein mentioned above is expressed here. The CI857/PR (T41C) promoter is the heat shock promoter. , the maximum heat shock temperature can reach 380 degrees. The SRRz lysis cassette is extracted from phage and used for autolysis. pCOLA-PesaS-RBS-RFP-rrnbT1-CI857-PR-SRRz-rrnB-Gox1 is the autolysis vector with added GOX1.
① Plasmid construction
Figure 5. electrophoresis gel results of pCOLA-CI857-SRRz-Gox1 plasmid construction
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 5 shows the construction results of pCOLA-CI857-SRRz-Gox1 plasmid and its quorum sensing vector PaCYC-QS. Figure 5A shows the pCOLA-CI857-SRRz-Gox1 vector constructed after Gibson assembly. Figure 5B 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 5C is the colony PCR identification result of the quorum sensing vector PaCYC-QS. Figure 5D is colony PCR identification of the Gox1 gene in the pCOLA-CI857-SRRz-Gox1 plasmid.
② Protein expression and activity evaluation
- Protein expression, cell lysis and SDS-PAGE
Figure 6 Lysis result of heat-induced autolysis plasmid (pCOLA-CI857-SRRz-GOX1 and PaCYC-QS)
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 6 prove that the cells were fully lysed, and the protein coded by Gox1 gene will be purified following.
Figure 7 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 7, the same of that as Figure 2B.
- Glucose Oxidase Activity; CCK-8
Figure 8 glucose oxidase activity and CCK-8 activity of the GOX1 protein purified after heat-induced autolysis
For the GOX1 protein purified after heat-induced autolysis, we also conducted glucose oxidase activity assay and CCK-8 test. Figure 8A 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 8B also backs up this inference.
Figure 9 GSH Activity test on 4T1 cells of the GOX1 protein purified after heat-induced autolysis
GSH Activity Assay was also conducted with the GOX1 protein purified after heat-induced autolysis. In Figure 9, 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.