Part:BBa_K2075022

Vector iGEM_02_Hax3_2xNN

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).

Our aim is to circulate the TAL-effector so the proteins are solid during the purification. To do that we use the Vector construct of the igem Team Heidelberg 2014 with their intein mechanism ( see here http://2014.igem.org/Team:Heidelberg). But we changed the intein linker sequence because we want to optimize and stabilize the circulation. Another new point is the repeat side of the TAL-effector we cloned into the vector of team Heidelberg. If you want to read more about our project concept have a look by Project. We designed four Vectors with different circularize TAL-effectors. Thereby we used the mechanism of Team Heidelberg but a sepcial intein linker sequence. All of them have a TAL-effector sequence, a Strep Tag, the TEV side and the intein Sequence. Two of them have also a gfp sequence and everyone have a special TAL-effector sequence in order to binds another dna sequence.

This composite part includes an Intein and linker, an eGFP, a TAL-effector, 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 an eGFP and the TAL-effector in frame. There is also a strep XT tag and the TEV Site. 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 XT tag the protein can be purified even if it is circularised. The Strep XT 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 eGFP gives us the opportunity to detect the connected TAL-effector. The TAL-effector is Hax3-2xNN. The 12th and 13th amino acid of each of the 11.5 repeats from our TAL Hax3 – 2xNN are: NI HD NI HD HD HD NN NN NS HD NI They bind the DNA sequence: A C A C C C G/A G/A N C A.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2636
  
• 1000
  COMPATIBLE WITH RFC[1000]