Part:BBa_K5082012

pCMV-EGFP-EIF3B-MUT

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. To validate our theory, we deleted and altered a few nucleotides in the EIF3B-HSU sequence (BBa_K5082005) to form the EIF3B-MUT sequence (BBa_K5082006). Afterward, we constructed the pCMV-EGFP-EIF3B-MUT plasmid.

Design

The pCMV-EGFP-EIF3B-MUT plasmid is constructed by fusing the EIF3B-MUT sequence into the pCMV-EGFP vector backbone (BBa_K5082008). The EIF3B-MUT sequence is inserted downstream the GFP reporter gene contained in the plasmid backbone. Upon transcription, the EIF3B-MUT sequence will not form a highly structured 3’UTR (HSU) structure downstream the GFP gene due to the deletion and alteration of specific nucleotides. Therefore, G3BP1 proteins will not affect mRNA degradation and translation. The plasmid map for pCMV-EGFP-EIF3B-MUT is shown in Figure 1.

                     
                       Figure 1. pCMV-EGFP-EIF3B-MUT 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-MUT 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-MUT 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-MUT plasmid measured using SpectraMax i3.

    Table 1. Fluorescence values of cell lines transfected with pCMV-EGFP-EIF3B-MUT plasmid measured using SpectraMax i3.
                   

As shown in Figure 2, Figure 3 and Table 1, there is no significant difference in fluorescence between all three cell lines. This suggests that the different G3BP1 concentrations among the cell lines do not have an effect on mRNA without HSU regions, proving that our theory was correct.

Reference

References

[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

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1684
  Illegal XbaI site found at 1721
  Illegal SpeI site found at 270
  Illegal PstI site found at 1689
  Illegal PstI site found at 3043
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1684
  Illegal NheI site found at 916
  Illegal SpeI site found at 270
  Illegal PstI site found at 1689
  Illegal PstI site found at 3043
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1684
  Illegal BglII site found at 1664
  Illegal BglII site found at 6170
  Illegal BamHI site found at 1715
  Illegal XhoI site found at 1668
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1684
  Illegal XbaI site found at 1721
  Illegal SpeI site found at 270
  Illegal PstI site found at 1689
  Illegal PstI site found at 3043
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1684
  Illegal XbaI site found at 1721
  Illegal SpeI site found at 270
  Illegal PstI site found at 1689
  Illegal PstI site found at 3043
  Illegal NgoMIV site found at 2153
  Illegal NgoMIV site found at 3494
  Illegal NgoMIV site found at 3777
  Illegal AgeI site found at 925
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 903
  Illegal BsaI.rc site found at 5302
  Illegal SapI site found at 4222
  Illegal SapI.rc site found at 3343
  Illegal SapI.rc site found at 3553