Difference between revisions of "Part:BBa K4040020"
 
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<partinfo>BBa_K4040020 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4040020 SequenceAndFeatures</partinfo>
 
===Usage and Biology===
 
===Usage and Biology===
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Based on the mechanism of Tango/MESA, we bind TEV-protase to GP130. And when the concentration of IL-6 rises to a certain level, IL-6 binds to mIL-6R (membrane-bound IL-6R), homodimerization of gp130 is induced. A high-affinity functional receptor complex of IL-6, IL-6R and gp130 is formed.  Then, TEV-protase connected behind is activated. TEV-protase binds to TCS and releases transcription factors on the other two receptors through enzyme digestion to initiate the subsequent pathway.  
+
Based on the mechanism of Tango/MESA, we bind TEV-protase to GP130. The MESA (modular extracellular sensor architecture) system is based on bringing intracellular membrane-anchored TEV (tobacco etch virus) protease into proximity to a membrane-anchored transcription factor in response to extracellular ligand binding. This is followed by cleavage and nuclear localization of the transcription factor and has been used to sense human VEGF (vascular endothelial growth factor)[1]. We used CMV as the promoter. When the concentration of IL-6 rises to a certain level, IL-6 binds to mIL-6R (membrane-bound IL-6R), homodimerization of gp130 is induced. A high-affinity functional receptor complex of IL-6, IL-6R and gp130 is formed.  Then, TEV-protase connected behind is activated. TEV-protase binds to TCS and releases transcription factors on the other two receptors through enzyme digestion to initiate the subsequent pathway.  
 +
 
 +
 
 +
[[File:T--NMU_China--gp130-tev.jpg|300px|thumb|left|<b>Figure. 1</b>The structure of gp130-TEV.]]
 +
[[File:T--NMU_China--IL-6R.jpg|550px|thumb|none|<b>Figure. 2</b>The structure and downstream activation of the composite IL-6R.]]
 +
 
 +
 
 +
 
 +
===Experimental results===
 +
1. Cell screening
 +
 
 +
As shown in Figure 2, synthetic receptors contained the gp130-TEV, IL-6R- TCS -Gal4-KRAB and IL-6R- TCS- tTA. The cDNA sequences containing the various fusion constructs were cloned into the pELNS vector with the promoter of CMV. Also the reporter GFP (<partinfo>E0840</partinfo>) was fused to the promoter UAS-pSV40 (<partinfo>BBa_K511003</partinfo>). We used mCherry (<partinfo>BBa_J06504</partinfo>) as the reporter and connected it to the pTET (<partinfo>BBa_K1061013</partinfo>), followed by PGK promoter and CD20. 1e7 cells were co-transfected with the plasmid. As shown in Figure 3, we screened 1e7 cells after transduction to obtain cells successfully expressing five cassettes for subsequent experiments. EGFR and HER2 positive cells were screened first (in the box), and GFP and Myc positive cells were screened after these cells were obtained (in the box).  CD20 positive cells were then screened (in the box).  The cells obtained were used in subsequent experiments to detect the effects of IL-6.  
 +
[[File:T--NMU_China--cell flow.png|600px|thumb|center|<b>Figure. 3</b>The structure of the Flow cytometry.]]
 +
2. Detection of IL-6 receptor effect
 +
 
 +
We added different concentrations of IL-6 to the above screened cells to detect mCherry and GFP fluorescence intensity, and the results were shown in Figure 4. MCherry fluorescence intensity represents pTET pathway expression, which is used to reflect CAR-MERTK expression in vivo.  GFP fluorescence intensity represents the expression of UAS-PSV40 pathway, corresponding to the expression of CAR-γ in vivo.  As can be seen from the figure, CAR-Ms change from M1 to M2 when IL-6 concentration is 2.5-12.5ng/mL. 
 +
[[File:T--NMU_China--expressresult.jpg|400px|thumb|center|<b>Figure. 4</b>The fluorescence intensity detected.]]
 +
===References===
 +
[1]Schwarz, K. A., Daringer, N. M., Dolberg, T. B. & Leonard, J. N. Rewiring human cellular input-output using modular extracellular sensors. Nat. Chem. Biol. 13, 202–209 (2017).
 +
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 12:04, 19 October 2021


Synthetic Receptor GP130-TEV

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1508
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

Based on the mechanism of Tango/MESA, we bind TEV-protase to GP130. The MESA (modular extracellular sensor architecture) system is based on bringing intracellular membrane-anchored TEV (tobacco etch virus) protease into proximity to a membrane-anchored transcription factor in response to extracellular ligand binding. This is followed by cleavage and nuclear localization of the transcription factor and has been used to sense human VEGF (vascular endothelial growth factor)[1]. We used CMV as the promoter. When the concentration of IL-6 rises to a certain level, IL-6 binds to mIL-6R (membrane-bound IL-6R), homodimerization of gp130 is induced. A high-affinity functional receptor complex of IL-6, IL-6R and gp130 is formed.  Then, TEV-protase connected behind is activated. TEV-protase binds to TCS and releases transcription factors on the other two receptors through enzyme digestion to initiate the subsequent pathway.  


Figure. 1The structure of gp130-TEV.
Figure. 2The structure and downstream activation of the composite IL-6R.


Experimental results

1. Cell screening

As shown in Figure 2, synthetic receptors contained the gp130-TEV, IL-6R- TCS -Gal4-KRAB and IL-6R- TCS- tTA. The cDNA sequences containing the various fusion constructs were cloned into the pELNS vector with the promoter of CMV. Also the reporter GFP (BBa_E0840) was fused to the promoter UAS-pSV40 (BBa_K511003). We used mCherry (BBa_J06504) as the reporter and connected it to the pTET (BBa_K1061013), followed by PGK promoter and CD20. 1e7 cells were co-transfected with the plasmid. As shown in Figure 3, we screened 1e7 cells after transduction to obtain cells successfully expressing five cassettes for subsequent experiments. EGFR and HER2 positive cells were screened first (in the box), and GFP and Myc positive cells were screened after these cells were obtained (in the box).  CD20 positive cells were then screened (in the box).  The cells obtained were used in subsequent experiments to detect the effects of IL-6.  

Figure. 3The structure of the Flow cytometry.

2. Detection of IL-6 receptor effect

We added different concentrations of IL-6 to the above screened cells to detect mCherry and GFP fluorescence intensity, and the results were shown in Figure 4. MCherry fluorescence intensity represents pTET pathway expression, which is used to reflect CAR-MERTK expression in vivo.  GFP fluorescence intensity represents the expression of UAS-PSV40 pathway, corresponding to the expression of CAR-γ in vivo.  As can be seen from the figure, CAR-Ms change from M1 to M2 when IL-6 concentration is 2.5-12.5ng/mL. 

Figure. 4The fluorescence intensity detected.

References

[1]Schwarz, K. A., Daringer, N. M., Dolberg, T. B. & Leonard, J. N. Rewiring human cellular input-output using modular extracellular sensors. Nat. Chem. Biol. 13, 202–209 (2017).