Part:BBa_K4040024

pTET-mCherry-PGK-CD20 Expression Cassette

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 1649
  Illegal PstI site found at 2560
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 1649
  Illegal PstI site found at 2560
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 1649
  Illegal PstI site found at 2560
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 1649
  Illegal PstI site found at 2560
  Illegal NgoMIV site found at 2407
  Illegal AgeI site found at 1115
  Illegal AgeI site found at 1227
  Illegal AgeI site found at 1514
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2654
  Illegal BsaI.rc site found at 2723
  Illegal BsaI.rc site found at 2729
  

Usage and Biology

The composite part consists of seven parts: the promoter pTET(BBa_K1061013) which initiates the expression of coding sequence. Fluorescent marker mCherry (BBa_J04450) which detects the expression of the pathway and the expression component of CAR-MERTK: CD8a signal peptide (BBa_K4040014), CR3022 scFv (BBa_K4040019), CD8 Hinge (BBa_K4040004), CD8TM (BBa_K4040007), MERTK(BBa_K4040002).  When the tTA fragment from IL-6 complex receptor BBa_K4040022 is released, it binds to the promoter TET to promote the expression of CAR-MERTK and facilitate the transformation of macrophages into M2 state to alleviate the inflammatory response.

Figure 1.The coding of the CAR-MERTK.

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.