Difference between revisions of "Part:BBa K4040023"
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<partinfo>BBa_K4040023 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4040023 SequenceAndFeatures</partinfo>
 
===Usage and Biology===
 
===Usage and Biology===
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The composite part consists of seven parts: the promoter UAS-PSV40 (<partinfo>BBa_K4040033</partinfo>), GFP Genera (<partinfo>E0840</partinfo>), which initiates the expression of coding sequence. pCMV(<partinfo>BBa_I712004</partinfo>) was used as the promoter. The indicator which detects the expression of the pathway and the expression component of CAR-FcRγ: CD8a signal peptide (<partinfo>BBa_K4040014</partinfo>), CR3022 scFv (<partinfo>BBa_K4040019</partinfo>), CD8 Hinge (<partinfo>BBa_K4040004</partinfo>), CD8TM (<partinfo>BBa_K4040007</partinfo>), FcRγ (<partinfo>BBa_K4040002</partinfo>).  When the GAL4-KRAb fragment from IL-6 complex receptor <partinfo>BBa_K4040021</partinfo> is released, it binds to the promoter UAS-PSV40 to inhibit the expression of CAR- FcRγ and reduce the transformation of macrophages into M1 state to alleviate the inflammatory response.  
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The composite part consists of two parts: the promoter UAS-PSV40 (<partinfo>BBa_K4040033</partinfo>), GFP Genera (<partinfo>E0840</partinfo>), which initiates the expression of coding sequence. When the GAL4-KRAB fragment from IL-6 complex receptor <partinfo>BBa_K4040021</partinfo> is released, it binds to the promoter UAS-PSV40 to inhibit the expression of CAR- FcRγ and reduce the transformation of macrophages into M1 state to alleviate the inflammatory response.  
 
[[File:T--NMU China--Expression of CARγ.jpg|600px|thumb|center|<b>Figure 1.</b>The coding of the CAR-gamma.]]
 
[[File:T--NMU China--Expression of CARγ.jpg|600px|thumb|center|<b>Figure 1.</b>The coding of the CAR-gamma.]]
 
[[File:T--NMU China--IL-6R.jpg|550px|thumb|center|<b>Figure 2.</b>The structure and downstream pathway of the composite IL6R.]]
 
[[File:T--NMU China--IL-6R.jpg|550px|thumb|center|<b>Figure 2.</b>The structure and downstream pathway of the composite IL6R.]]
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 +
As shown in Figure 2, synthetic receptors contained the gp130 followed by the TEV, IL-6R followed by the TCS, which is fused to Gal4-KRAB and IL-6R followed by the TCS, which is fused to tTA. The cDNA sequences containing the various fusion constructs were cloned into the pELNS vector with the promoter of CMV. Also the reporter GFP (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, and were added with different IL-6 concentration 24h post-transfection. Eighteen hours later, the mCherry and GFP fluorescence intensity was measured (figure. 3).
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[[File:T--NMU China--expressresult.jpg|600px|thumb|center|<b>Figure 3.</b>mCherry and GFP expressed by cells at different IL-6 concentrations.***P<0.001, *P<0.05 ]]
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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.
 
<!-- Add more about the biology of this part here
 
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Latest revision as of 01:06, 17 October 2021


pSV40-UAS-GFP Expression Cassette

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1115

Usage and Biology

The composite part consists of two parts: the promoter UAS-PSV40 (BBa_K4040033), GFP Genera (BBa_E0840), which initiates the expression of coding sequence. When the GAL4-KRAB fragment from IL-6 complex receptor BBa_K4040021 is released, it binds to the promoter UAS-PSV40 to inhibit the expression of CAR- FcRγ and reduce the transformation of macrophages into M1 state to alleviate the inflammatory response.

Figure 1.The coding of the CAR-gamma.
Figure 2.The structure and downstream pathway of the composite IL6R.

As shown in Figure 2, synthetic receptors contained the gp130 followed by the TEV, IL-6R followed by the TCS, which is fused to Gal4-KRAB and IL-6R followed by the TCS, which is fused to tTA. The cDNA sequences containing the various fusion constructs were cloned into the pELNS vector with the promoter of CMV. Also the reporter GFP (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, and were added with different IL-6 concentration 24h post-transfection. Eighteen hours later, the mCherry and GFP fluorescence intensity was measured (figure. 3).

Figure 3.mCherry and GFP expressed by cells at different IL-6 concentrations.***P<0.001, *P<0.05 

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.