Part:BBa_K5082014

pMIR -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 pMIR-EI3B-HSU plasmid to utilize G3BP1’s HSU mRNA-degrading activity to detect G3BP1 concentration and thereby diagnose GC.

Design

The pMIR-EIF3B-HSU plasmid is constructed by fusing the EIF3B-HSU sequence (BBa_K5082005) into the pMIR vector backbone (BBa_K5082010). The EIF3B-HSU sequence is inserted downstream the luciferase reporter gene contained in the plasmid backbone. Upon transcription, the EIF3B-HSU sequence will form a highly structured 3’UTR (HSU) structure downstream the luciferase gene. G3BP1 proteins can bind with HSU structures and recruit proteins to degrade HSU mRNA. Therefore, by detecting luciferase concentrations, we can indirectly measure G3BP1 concentration and thereby diagnose GC. The plasmid map for pMIR-EIF3B-HSU is shown in Figure 1.

                       
                              Figure 1. pMIR-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 pMIR-EIF3B-HSU plasmid into the three cell lines, we cultured the cells under identical conditions for 48 hours. Afterward, we lysed the cells and measured their luciferase activity using SpectraMax i3. The results are shown in Figure 2 and Table 1.

                       
   Figure 2. Luciferase activity of cell lines transfected with pMIR-EIF3B-HSU plasmid measured using SpectraMax i3.

    Table 1. Luciferase activity of cell lines transfected with pMIR-EIF3B-HSU plasmid measured using SpectraMax i3.
                        

As shown in Figure 2, the luciferase activity in the GES-1 cell line is much higher than that of the two GC cell lines. This trend suggests that the GES-1 cell line contains a higher luciferase protein concentration than the two GC cell lines. Given the same concentration of pMIR-EIF3B-HSU plasmids, the higher luciferase 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.

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 XbaI site found at 7431
  Illegal SpeI site found at 5443
  Illegal PstI site found at 2146
  Illegal PstI site found at 5700
  Illegal PstI site found at 7443
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7419
  Illegal SpeI site found at 5443
  Illegal PstI site found at 2146
  Illegal PstI site found at 5700
  Illegal PstI site found at 7443
  Illegal NotI site found at 191
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 5189
  Illegal BamHI site found at 2891
  Illegal XhoI site found at 7425
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 7431
  Illegal SpeI site found at 5443
  Illegal PstI site found at 2146
  Illegal PstI site found at 5700
  Illegal PstI site found at 7443
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 7431
  Illegal SpeI site found at 5443
  Illegal PstI site found at 2146
  Illegal PstI site found at 5700
  Illegal PstI site found at 7443
  Illegal NgoMIV site found at 2597
  Illegal NgoMIV site found at 5807
  Illegal NgoMIV site found at 7151
  Illegal NgoMIV site found at 7172
  Illegal AgeI site found at 1921
  Illegal AgeI site found at 4025
  Illegal AgeI site found at 5308
  Illegal AgeI site found at 6875
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 3851
  Illegal BsaI.rc site found at 1534
  Illegal SapI.rc site found at 2446
  Illegal SapI.rc site found at 2656
  Illegal SapI.rc site found at 7057