Part:BBa_K2075021

TAL-effector Ax7R-RR

Designing cyclic TALEs allows a regulation of those proteins, because of topological problems. A TALE is always winding itself around the DNA to bind. If the protein is cyclic, this is no longer possible and the TALE-bond is inhibited. This could also be used for appliances concerning drug delivery. If the cyclic bond is irreversible and a protease can cut the protein, the TALE regains full transcriptional activity (Lonzarić, 2016). To prove this statement, we inserted a TEV cleavage site from the tobacco each virus into the vector which enables induced linearization of the protein after expression with ProTEV Plus protease (Promega).

This composite part includes an Intein and linker, the TAL-effector Ax7L-DS, a TEV Site and a Strep Tag. They translated all together. The intein and linker are important to circularice the aminoacid sequence between the N- and C-terminal parts. Between the Intein sites is the TAL-effector and the TEV Site. There is also a strep II tag. When it is translated the intein sites react and form a circularised protein. All the parts between N- and C-Terminal Intein is part of the protein circle. In this circle the hole protein gains more stability.

With the help of the strep TX tag the protein can be purified even if it is circularised. The Strep II Tag is also usefull to detect the protein with an immunostain. The TEV Site give the opportunity to linearise the protein again wit the help oft he TEV protease. The linearised TAL can bind the specific DNA Sequence. The 12. And 13. Aminoacid of each of the 11.5 repeats from our TAL Hax3 – 2xNN are: NG NN NI NI NN NN HD NG NG NN NI NG NN NI NN HD NG They bind the DNA sequence: T G/A A A G/A G/A C T T G/A A T G/A A G/A C T

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 2939
  Illegal BamHI site found at 2345
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2284
  Illegal NgoMIV site found at 2879
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 2359