Reporter

Part:BBa_K4497028

Designed by: Till Gundlach   Group: iGEM22_Munich   (2022-10-09)
Revision as of 15:25, 12 October 2022 by Till Gundlach (Talk | contribs)


tTA Induceable EYFP Reporter

Components:

This part consists of the bidirectional Promoter Pbi-1 (BBa_K4497027), that is induced by the Transcription Factor tetracycline-inducable Transactivator (tTA). The promoter is made of Tetracycline repeat elements (TRE) with a minimal CMV promoter on both sides. The promoter induces expression of the downstream EYFP (BBa_K076004), allowing for an EYFP signal readout on tTA induction of the promoter.

We used the reporter as a means to read out the MESA receptor activation by our synthetic ligands for our planned quorum sensing loop system in mammalian cells/ CAR T cells. An induction of the receptor was measured using Flow cytometry.

Alternatives

As our MESA system incorporates a lot of fluorescent dyes to read out different aspects of our loop, we modified the reporter to miRFP680(Ex661/Em680) instead, which does not clash with our other measurements:


Reporter: EYFP (Ex513/Em527) Loop Ligand: 2xmCherry (Ex587/Em610), 2xmEGFP(Ex488/Em507) Transcription Factor: BFP (Ex381/Em445)


This allows for measurements of GFP in your samples

Origin

tTA Induceable EYFP Reporter can be ordered in the pBI-MCS plasmid from addgene: pBI-MCS-EYFP was a gift from Joshua Leonard (Addgene plasmid # 58855 ; http://n2t.net/addgene:58855 ; RRID:Addgene_58855)

The reporter is used in the corresponding paper: Modular Extracellular Sensor Architecture for Engineering Mammalian Cell-based Devices. Daringer NM, Dudek RM, Schwarz KA, Leonard JN. ACS Synth Biol. 2014 Mar 11. 10.1021/sb400128g PubMed 24611683 Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 465
    Illegal XbaI site found at 484
    Illegal SpeI site found at 490
    Illegal PstI site found at 471
    Illegal PstI site found at 700
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 465
    Illegal SpeI site found at 490
    Illegal PstI site found at 471
    Illegal PstI site found at 700
    Illegal NotI site found at 476
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 465
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 465
    Illegal XbaI site found at 484
    Illegal SpeI site found at 490
    Illegal PstI site found at 471
    Illegal PstI site found at 700
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 465
    Illegal XbaI site found at 484
    Illegal SpeI site found at 490
    Illegal PstI site found at 471
    Illegal PstI site found at 700
  • 1000
    COMPATIBLE WITH RFC[1000]



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