Part:BBa_K4367015

PYL-nTEV
PYL-nTEV 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 PYL-nTEV 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 a stepping stone for troubleshooting what 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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 601
  Illegal BamHI site found at 961
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 14
  Illegal BsaI.rc site found at 968
  

References

[1] Tina Fink, Jan Lonzarić, Arne Praznik, Tjaša Plaper, Estera Merljak, Katja Leben, Nina Jerala, Tina Lebar, Žiga Strmšek, Fabio Lapenta, Mojca Benčina & 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