Difference between revisions of "Part:BBa K4497028"
| (2 intermediate revisions by the same user not shown) | |||
| Line 5: | Line 5: | ||
A tetracyclin-inducable Transactivator (tTA) inducable EYFP reporter. | A tetracyclin-inducable Transactivator (tTA) inducable EYFP reporter. | ||
| − | ==Design & Cloning | + | ==Design & Cloning== |
===Design=== | ===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. | 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. | ||
| Line 11: | Line 11: | ||
===Cloning=== | ===Cloning=== | ||
tTA Induceable EYFP Reporter was be received in the pBI-MCS plasmid from addgene: pBI-MCS-EYFP (Addgene plasmid # 58855, [1]) | tTA Induceable EYFP Reporter was be received in the pBI-MCS plasmid from addgene: pBI-MCS-EYFP (Addgene plasmid # 58855, [1]) | ||
| + | |||
==Usage== | ==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]]). | 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=== | ===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: | 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) | *Reporter: EYFP (Ex513/Em527) | ||
| Line 22: | Line 22: | ||
This makes it a good alternative to this reporter if your samples also contain GFP | This makes it a good alternative to this reporter if your samples also contain GFP | ||
| − | + | ||
| Line 34: | Line 34: | ||
<partinfo>BBa_K4497028 SequenceAndFeatures</partinfo> | <partinfo>BBa_K4497028 SequenceAndFeatures</partinfo> | ||
| + | ==References== | ||
| + | [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 | ||
<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
Latest revision as of 16:38, 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
Sequence and Features
- 10INCOMPATIBLE 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 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 465
- 23INCOMPATIBLE 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 - 25INCOMPATIBLE 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 - 1000COMPATIBLE WITH RFC[1000]
References
[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