Part:BBa_K4367014
ABI-cTEV
ABI-cTEV is a part of the ABI/PYL-splitTEV system, which is used as a control for other experiments. Essentially, the complementation of split TEV can be induced with the addition of abscisic acid to the system.
Description
The design of ABI-cTEV is essentially identical to what was used in this reference [1], but with the addition of a 6xHis-tag and Part-3-Overhangs for Modular Cloning. PYL and ABI can dimerize if abscisic acid is present [1]. When PYL-nTEV dimerises with ABI-cTEV, the split TEVp halves can complement each other and regain enzymatic function. More information can be found at BBa_K4367010 (TEVp).
Usage
It was planned to use the ABI/PYL-splitTEV system as a control for the dCas9-splitTEV system, as stepping stones for troubleshooting which parts could be dysfunctional. For example, if the ABI/PYL system works but not the dCas9-splitTEV system, it is shown that the testing conditions can allow for complementation of split TEV, and the problem is perhaps because of the dCas9 or gRNA.
Future design considerations
If for some reason the ABI/PYL system would not be desirable, there is an alternative: The proteins FKBP and FRB, which uses the chemical rapamycin to induce their dimerisation [1].
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 952
Illegal BamHI site found at 1034
Illegal BamHI site found at 1363 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 14
Illegal BsaI.rc site found at 1370
Illegal SapI.rc site found at 1270
References
[1] Tina Fink, Jan Lonzarić, Arne Praznik, TjaÅ¡a Plaper, Estera Merljak, Katja Leben, Nina Jerala, Tina Lebar, Žiga StrmÅ¡ek, Fabio Lapenta, Mojca BenÄina & Roman Jerala (2019). Design of fast proteolysis-based signaling and logic circuits in mammalian cells. Available at: https://www.nature.com/articles/s41589-018-0181-6
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