Composite

Part:BBa_K5082013

Designed by: Fangyuan Duan   Group: iGEM24_YiYe-China   (2024-09-15)

pHAGE G3BP1 expression plasmid

Usage and Biology

Ras-GTPase-activating protein SH3 domain binding protein 1 is a cytoplastic multifunctional binding protein [1]. It is involved in many biological reactions including RNA metabolism, cell proliferation, apoptosis, metastasis, differentiation. Regarding the role of G3BP1 in RNA metabolism, it can bind with highly structured 3’ UTR (HSU) mRNA and degrade the HSU mRNA [1]. Therefore, G3BP1 is an effective regulator in expression of genes with HSU mRNA. The mechanism of G3BP1 on HSU mRNA is shown in Figure 1. 

                               phag3-1.png
                   Figure 1. G3BP1 binds to HSU mRNA and facilitates degradation.

G3BP1 plays important roles in multiple diseases and is considered a potential biomarker. In cancer cells, G3BP1’s roles include promoting stress granule assembly and affecting ribosomal stability [2]. To our interest, upregulated G3BP1, and subsequently higher stress granule levels, activates the TGF-β/Smad signaling pathway can promote gastric cancer (GC)[3]. Therefore, G3BP1 can be a potential biomarker for GC. In our experiment, we utilized the HSU mRNA-degrading activity of G3BP1. By connecting HSU mRNA sequences downstream reporter genes, reporter protein expression levels reflected G3BP1 concentrations and thus enables GC diagnosis. To set up a model to determent the amount of G3BP1 in cells, we clone the full-length G3BP1 cDNA and made a construct to overexpress G3BP1. We designed the specific primers of human G3BP1 based on the sequences of G3BP1 cDNA and amplified 1401 bp fragment by PCR.

Design

We designed two sensors to detect G3BP1 concentrations and thus diagnose GC. To test how well the systems reflect G3BP1 concentrations, we fused the G3BP1 (BBa_K5082007) into the pHAGE vector backbone (BBa_K5082009), producing the pHAGE-G3BP1 plasmid. The plasmid map for pHAGE-G3BP1 is shown in Figure 2. 

                                 phage-2.png

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 3 and Table 1. 

          Table 1. The value of eGFP fluorescence in cells transfected with different concentration of pHAGE-G3BP1 plasmids.

                           phage-3.png
                        
                            phage-4.png
                       Figure 3. Fluorescence value against pHAGE-G3BP1 concentration.

As shown in Figure 3 and Table 1, 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] Sidibé, Hadjara et al. “The multi-functional RNA-binding protein G3BP1 and its potential implication in neurodegenerative disease.” Journal of neurochemistry vol. 157,4 (2021): 944-962. doi:10.1111/jnc.15280 [3] 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 2028
    Illegal EcoRI site found at 2972
    Illegal SpeI site found at 2212
    Illegal SpeI site found at 3227
    Illegal PstI site found at 3484
    Illegal PstI site found at 8435
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2028
    Illegal EcoRI site found at 2972
    Illegal SpeI site found at 2212
    Illegal SpeI site found at 3227
    Illegal PstI site found at 3484
    Illegal PstI site found at 8435
    Illegal NotI site found at 2879
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2028
    Illegal EcoRI site found at 2972
    Illegal BglII site found at 473
    Illegal BglII site found at 4743
    Illegal BglII site found at 4809
    Illegal BglII site found at 4850
    Illegal BamHI site found at 2859
    Illegal XhoI site found at 2871
    Illegal XhoI site found at 8975
    Illegal XhoI site found at 9032
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2028
    Illegal EcoRI site found at 2972
    Illegal SpeI site found at 2212
    Illegal SpeI site found at 3227
    Illegal PstI site found at 3484
    Illegal PstI site found at 8435
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2028
    Illegal EcoRI site found at 2972
    Illegal SpeI site found at 2212
    Illegal SpeI site found at 3227
    Illegal PstI site found at 3484
    Illegal PstI site found at 8435
    Illegal NgoMIV site found at 1146
    Illegal NgoMIV site found at 4627
    Illegal AgeI site found at 3092
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 455
    Illegal BsaI site found at 4832
    Illegal BsaI site found at 6036
    Illegal BsaI.rc site found at 3982
    Illegal SapI.rc site found at 1107
    Illegal SapI.rc site found at 7118


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