iGEM 2024 — Team yiye-china

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{% block title %}Notebook{% endblock %}
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# Notebook

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{% block title %}Engineering Success{% endblock %}
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# Engineering Success

1. Upregulated G3BP1 mRNA level in GC cells

Ras-GTPase-activating protein SH3 domain binding protein 1 (G3BP1) is a protein overexpressed in gastric
cancer (GC) tumor cells [1]. Meanwhile, G3BP1 proteins have the activity to degenerate highly structured
3’UTR (HSU) structures in mRNA molecules [2].

To verify that G3BP1 is overexpressed in GC cells, we conducted a qRT-PCR test to measure the mRNA level of
G3BP1 in healthy stomach mucosal cell line GES-1 and GC cell lines AGS and MGC-803. We found that G3BP1 mRNA
levels were significantly higher in GC cell lines as compared to healthy stomach mucosal cell lines. The
results are shown below in Figure 1.

![](https://static.igem.wiki/teams/5082/engineering-img-1.png)

Figure 1. Relative G3BP1 mRNA expression in normal and GCs., \*\*P < 0.01, \*\*\*P<
0.001,
two-tailed t-test.

2. Plasmid preparation

2.1 Plasmid construction

In this project, we constructed two plasmids containing different reporter genes with HSU regions at 3’UTR,
and their control counterparts without HSU regions. The plasmid maps for the above four plasmids are shown
in Figure 2.

![](https://static.igem.wiki/teams/5082/engineering-img-2.png)
![](https://static.igem.wiki/teams/5082/engineering-img-3.png)

Figure 2. Plasmid maps. (A) pCMV-EGFP-EIF3B-HSU. (B) pMIR-EIF3B-HSU. (C) pCMV-EGFP-EIF3B-MUT. (D)
pMIR-EIF3B-MUT.

2.2 Plasmid transformation

To amplify the plasmids, we transformed them into DH5α bacteria. We successfully grew bacterial colonies on
the ampicillin plates, proving that the bacterial cells contained Ampicillin resistance genes and that our
plasmid transfection was successful. The bacteria cultures are shown in Figure 3.

![](https://static.igem.wiki/teams/5082/engineering-img-4.png)

Figure 3. Solid plates. (A) pCMV-EGFP-EIF3B-HSU. (B) pMIR-EIF3B-HSU. (C) pCMV-EGFP-EIF3B-MUT. (D) and
pMIR-EIF3B-MUT.

2.3 Plasmid sequencing

To verify that the plasmid was successfully constructed, we picked out single bacterial colonies and
conducted DNA sequencing. The sequencing results were shown in Figure 4.

![](https://static.igem.wiki/teams/5082/engineering-img-5.png)

Figure 4. Sequencing of plasmids. (A) pCMV-EGFP-EIF3B-HSU. (B) pCMV-EGFP-EIF3B-MUT.

3.The effect of pCMV-EGFP-EIF3B-HSU sensor in gastric cancer cells

The expression of G3BP1 is highly associated with the early stage of GC [3]. So, we constructed plasmids
[pCMV-EGFP-EIF3B-HSU (kkb website)] and tried to test the possibility of detecting tumor cells by using the
plasmid. The pCMV-EGFP-EIF3B-MUT (kkb website) without HSU structure and the plasmid backbone pCMV-EGFP (kkb
website) have been used as negative controls. The pCMV-EGFP-EIF3B-HSU plasmid was transfected into the three
cell lines: normal gastric cell line-GES-1, gastric cancer cell line-AGS, and gastric cancer cell
line-MGC-803 in 24-well plates with 0.8 ug of plasmid for each well. As negative controls, the
pCMV-EGFP-EIF3B-MUT and pCMV-EGFP plasmids were also transfected into all three cell lines at the same
concentration. After transfection, cells were cultured for 48 hours and examined under fluorescence
microscopy (Fig. 5A-I). Because of low G3BP1 expression level in GES-1 cells, there was no significant
difference between GES-1 cell lines transfected with all three plasmids (Fig. 5A-C). Because high expression
of G3BP1 in GC cells, the fluorescence of GFP was significantly decreased in AGS and MGC-803 cells
transfected with pCMV-EGFP-EIF3B-HSU compared with cells transfected with pCMV-EGFP and pCMV-EGFP-EIF3B-MUT
plasmids (Fig. 5D-I). In addition, we also measured the value of GFP fluorescence by plate reader
(SpectraMax i3) (Table 1 and Fig. 6). The result suggested that the pCMV-EGFP-EIF3B-HSU plasmid could show
the different expression of G3BP1 in gastric cancer cells.

![](https://static.igem.wiki/teams/5082/engineering-img-6.png)

Figure 5. The images of different gastric cells transfected with different plasmids.

Table 1. The value of EGFP fluorescence in cells.

![](https://static.igem.wiki/teams/5082/engineering-img-7.png)
![](https://static.igem.wiki/teams/5082/engineering-img-8.png)

Figure 6. Fluorescence value results of pCMV-EGFP-EIF3B-HSU (HSU), pCMV-EGFP-EIF3B-MUT (MUT), and
pCMV-EGFP (Ctrl) in three types of cells (GES-1, MGC-803, and AGS), \*\*\*\*P< 0.0001, two-tailed t-test.

4. The effect of pMIR-EIF3B-HSU sensor in gastric cancer cells

We also constructed the pMIR-EIF3B-HSU plasmid containing luciferase gene (another reporter) for our
experiment. The pMIR-EIF3B-MUT without HSU structure and the plasmid backbone pMIR have been served as
controls. pMIR-EIF3B-HSU (kkb website), pMIR-EIF3B-MUT (kkb website) (as negative control), or pMIR (kkb
website) (as negative control) (0.8 ug plasmids for each well) was transfected into cells in 24-well
plate, respectively. After transfection for 48 h, cells had been collected and lysed. The value of
luciferase activities were measured by plate reader (SpectraMax i3). Consistent with our previous data,
luciferase activity remained unchanged in GES-1 cells transfected with pMIR-EIF3B-HSU compared with
controls (Table 2 and Fig. 7). Luciferase activity was significantly decreased in AGS and MGC-803 cells
transfected with pMIR-EIF3B-HSU compared with cells transfected with pMIR and pMIR-EIF3B-MUT (Table 2
and Fig. 7). These data indicated pMIR-EIF3B-HSU can monitor the different expression of G3BP1 in
gastric cancer cells.

Table 2. The relative luciferase activity in cells.

![](https://static.igem.wiki/teams/5082/engineering-img-9.png)
![](https://static.igem.wiki/teams/5082/engineering-img-10.png)

Figure 7. Relative Luciferase activity results of pMIR-EIF3B-HSU (HSU), pMIR-EIF3B-MUT (MUT), and pMIR
(Ctrl) in three types of cells (GES-1, MGC-803, and AGS), \*\*P < 0.01, two-tailed t-test.

5. The effect of pCMV-EGFP-EIF3B-HSU sensor as a monitor to detect the expression of G3BP1

To test the effect of pCMV-EGFP-EIF3B-HSU as a “sensor” to detect G3BP1 expression, cells were transfected
with the same concentration of the pCMV-EGFP-EIF3B-HSU plasmid and different concentrations of pHAGE-G3BP1
plasmids which overexpress G3BP1 proteins. The plasmid map of pHAGE-G3BP1 is shown in Figure 8. We used the
plasmid of pHAGE-G3BP1 to quantify G3BP1 expression and calculate the copy numbers of G3BP1 by using the
formula listed below.

copies/μl= (6.02×1023)×(plasmids concentrations ng/μl×10-9)/(DNA length×660)

![](https://static.igem.wiki/teams/5082/engineering-img-11.png)

Figure 8. pHAGE-G3BP1 plasmid map.

The relationship between fluorescence value and pHAGE-G3BP1 plasmid concentration are shown in Table 3. The
standard curve of pCMV-EGFP-EIF3B-HSU sensor was made by Excel (Fig. 9). We found that the fluorescence
value of cells had a strong negative correlation with G3BP1 concentration. Based on the formula, the
correlation coefficient (R2 value) of pCMV-EGFP-EIF3B-HSU sensor was 0.9893. Therefore, fluorescence values
can reflect G3BP1 concentration. As luciferase and GFP are affected by G3BP1 proteins through similar
measures, luciferase activity, presumably, can also reflect G3BP1 concentration.

Table 3. The value of eGFP fluorescence in cells transfected with different concentration of pHAGE-G3BP1
plasmids.

![](https://static.igem.wiki/teams/5082/engineering-img-12.png)

![](https://static.igem.wiki/teams/5082/engineering-img-13.png)

Figure 9. The standard curve of pCMV-EGFP-EIF3B-HSU sensor.

References

[1] Ge, Y., Jin, J., Li, J., Ye, M., & Jin, X. (2022). The roles of G3BP1 in human diseases (review). Gene,
821, 146294. https://doi.org/10.1016/j.gene.2022.146294

[2] Xiong R, Gao J, Yin T. G3BP1 activates the TGF-β/Smad signaling pathway to promote gastric cancer. Onco
Targets Ther. 2019;12:7149-7156. https://doi.org/10.2147/OTT.S213728

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