Part:BBa_K5082011
pCMV-EGFP-EIF3B-HSU
Usage and Biology
Previously, it has been reported that gastric cancer (GC) tissue cells overexpress the oncogene G3BP1 [1]. Meanwhile, G3BP1 proteins have been proved to have HSU mRNA degrading activity [2]. Based on these findings, we designed and constructed the pCMV-EGFP-EI3B-HSU plasmid to utilize G3BP1’s HSU mRNA-degrading activity to detect G3BP1 concentration and thereby diagnose GC.
Design
The pCMV-EGFP-EIF3B-HSU plasmid is constructed by fusing the EIF3B-HSU sequence (Part Name) into the pCMV-EGFP vector backbone (Part Name). The EIF3B-HSU sequence is inserted downstream the GFP reporter gene contained in the plasmid backbone. Upon transcription, the EIF3B-HSU sequence will form a highly structured 3’UTR (HSU) structure downstream the GFP gene. G3BP1 proteins can bind with HSU structures and degrade HSU mRNA. Therefore, by detecting GFP protein concentrations, we can indirectly measure G3BP1 concentration and thereby diagnose GC. The plasmid map for pCMV-EGFP-EIF3B-HSU is shown in Figure 1.
Figure 1. pCMV-EGFP-EIF3B-HSU plasmid map.
Results
In our experiments, we used three cell lines. We used the GES-1 cell line to simulate healthy stomach cells and used the MGC-803 and AGS cell lines to simulate GC tissue cells. After transfecting the pCMV-EGFP-EIF3B-HSU plasmid into the three cell lines, we cultured the cells under identical conditions for 48 hours. Afterward, we qualitatively observed their fluorescence under fluorescence microscopes and quantitatively measured their fluorescence values using SpectraMax i3. The quantitative observation results are shown in Figure 1. The qualitative measurement results are shown in Figure 2 and Table 1.
Figure 2. Fluorescence of cell lines transfected with pCMV-EGFP-EIF3B-HSU plasmid observed under fluorescence microscopes. (A) GES-1 cell line. (B) MGC-803 cell line. (C) AGS cell line.
Figure 3. Fluorescence values of cell lines transfected with pCMV-EGFP-EIF3B-HSU plasmid measured using SpectraMax i3.
Table 1. Fluorescence values of cell lines transfected with pCMV-EGFP-EIF3B-HSU plasmid measured using SpectraMax i3.
As shown in Figure 2, the fluorescence in the GES-1 cell line was much higher than that of the two GC cell lines. This trend is more obvious in the quantitative measurement shown in Figure 3 and Table 1. Based on the trend shown in both Figures, the GES-1 cell line contains a higher GFP protein concentration than the two GC cell lines. Given the same concentration of pCMV-EGFP-EIF3B-HSU plasmids, the higher GFP protein concentrations in the GES-1 cell line indicates more mRNA translation, longer-lasting mRNA and thus lower G3BP1 concentrations, as compared to the two GC cell lines. This is consistent with our hypothesis, validating our theory and proving that our sensor is effective. We then transfected different concentrations of the pHAGE-G3BP1 plasmid into the GES-1 healthy stomach mucosal cell line. Meanwhile, we transfected identical amounts of pCMV-EGFP-EIF3B-HSU plasmids into all the groups and cultured the cells for another 48 hours under identical conditions. The results are shown in Figure 4 and Table 2.
Table 2. The value of eGFP fluorescence in cells transfected with different concentration of pHAGE-G3BP1 plasmids.
Figure 4. Fluorescence value against pHAGE-G3BP1 concentration.
As shown in Figure 4, fluorescence value of the cells had a strong negative linear correlation with the plasmid concentration of pHAGE-G3BP1. This suggests that sensors created by fusing HSU structures downstream reporter genes can successfully reflect G3BP1 concentrations, proving the reliability of our designed systems.
Reference
[1] Ge, Yidong et al. “The roles of G3BP1 in human diseases (review).” Gene vol. 821 (2022): 146294. doi:10.1016/j.gene.2022.146294
[2] Xiong, Rui et al. “G3BP1 activates the TGF-β/Smad signaling pathway to promote gastric cancer.” OncoTargets and therapy vol. 12 7149-7156. 2 Sep. 2019, doi:10.2147/OTT.S213728
Sequence and Features
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Illegal PstI site found at 3088 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1729
Illegal BglII site found at 1709
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Illegal XhoI site found at 1713 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1729
Illegal XbaI site found at 1766
Illegal SpeI site found at 315
Illegal PstI site found at 1734
Illegal PstI site found at 3088 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1729
Illegal XbaI site found at 1766
Illegal SpeI site found at 315
Illegal PstI site found at 1734
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