Difference between revisions of "Part:BBa K4497028"

Revision as of 16:15, 13 October 2022

tTA Induceable EYFP Reporter

A tetracyclin-inducable Transactivator (tTA) inducable EYFP reporter.

Design & Cloning=

Design

This part consists of the bidirectional Promoter Pbi-1 (Part: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.

Cloning

tTA Induceable EYFP Reporter was be received in the pBI-MCS plasmid from addgene: pBI-MCS-EYFP (Addgene plasmid # 58855, [1])

Usage

The reporter was used previously by Daringer et al. for readout of tTA release by their MESA receptor system. Since our MESA system is based on theirs, we also decided to use this reporter. The reporter was transfected into HEK293T or COS7 cels together with the MESA system to test its functionality (for Results see Part:Bba_K4497028).

Alternatives

As our MESA system incorporated a lot of fluorescent dyes to read out different aspects of our loop, we modified the reporter to express miRFP680(Ex661/Em680)(Part:BBa_K4497030) instead of EYFP, which did not clash with our other measurements:

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

This makes it a good alternative to this reporter if your samples also contain GFP

[1] 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]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K4497028 short</partinfo>
-'''Components'''
+A tetracyclin-inducable Transactivator (tTA) inducable EYFP reporter.
-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.
+==Design & Cloning===
+===Design===
+This part consists of the bidirectional Promoter Pbi-1 ([[Part: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.
+===Cloning===
-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.
+tTA Induceable EYFP Reporter was be received in the pBI-MCS plasmid from addgene: pBI-MCS-EYFP (Addgene plasmid # 58855, [1])
+==Usage==
-'''Alternatives'''
+The reporter was used previously by Daringer et al. for readout of tTA release by their MESA receptor system. Since our MESA system is based on theirs, we also decided to use this reporter. The reporter was transfected into HEK293T or COS7 cels together with the MESA system to test its functionality (for Results see [[Part:Bba_K4497028]]).
+===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 (BBa_K4497030), which does not clash with our other measurements:
+As our MESA system incorporated a lot of fluorescent dyes to read out different aspects of our loop, we modified the reporter to express miRFP680(Ex661/Em680)([[Part:BBa_K4497030]]) instead of EYFP, which did not clash with our other measurements:
@@ Line 18: / Line 20: @@
 *Loop Ligand: 2xmCherry (Ex587/Em610), 2xmEGFP(Ex488/Em507)
 *Transcription Factor: BFP (Ex381/Em445)
+This makes it a good alternative to this reporter if your samples also contain GFP
+[1] 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
-This makes it a good alternative to this reporter if your samples also contain GFP
-'''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
 <!-- Add more about the biology of this part here
 ===Usage and Biology===