Team:Hannover/Composite Part
Composite Part
A TALE is always binding to DNA by winding itself around it. Cyclic TALEs cannot bind to the DNA because their cyclic structure is inhibiting the bonding. This is why cyclization is a form of TALE regulation, it makes itself more stable.
This could also be commercially used for drug delivery. If the cyclic bond is irreversible and a protease can cut the protein, then the TALE regains full transcriptional activity (Lonzarić, 2016). To prove this statement, we inserted a TEV cleavage site from the tobacco etch virus into the vector which enables induced linearization of the protein after expression with ProTEV Plus protease (Promega).
This composite part includes an Intein, a linker, a TAL-effector Ax7L-DS, a TEV Site and a Strep XT Tag. They are all translated into a protein together.
The Intein and linker are important in the circularization of the amino acid sequence between the N- and C-terminal parts. Between the Intein sites are the TAL-effector, TEV site and the Strep XT tag.
When it is translated, the Intein site reacts and forms a circularized protein. All the parts in between N- and C-Terminal Intein are a part of the protein circle. In this circle, the whole protein gains more stability.
With the help of the Strep XT tag the protein can be purified even if it is circularised. The Tag is also useful for the detection of the protein with an immunostain.
The TEV Site creates an opportunity to linearise the protein again with the help of the TEV protease. The linearised TAL can now bind to the specific DNA sequence.
The 12th and 13th amino acid of each of the repeats from our TALE Ax7L-DS are: NI NN HD NI HD NG NI NG NI NG NI NI NI HD HD HD HD HD
They bind to the DNA sequence: A G/A C A C T A T A T A A A C C C C C
This composite part includes an Intein, a linker, a TAL-effector Ax7L-DS, a TEV Site and a Strep II Tag. They are all translated into a protein together.
The Intein and linker are important in the circularization of the amino acid sequence between the N- and C-terminal parts. Between the Intein sites are the TAL-effector, TEV site and the Strep II tag.
When it is translated, the Intein site reacts and forms a circularised protein. All the parts in between N- and C-Terminal Intein are a part of the protein circle. In this circle, the whole protein gains more stability.
With the help of the Strep II tag the protein can be purified even if it is circularized. The Tag is also useful for the detection of the protein with an immunostain.
The TEV Site creates an opportunity to linearise the protein again with the help of the TEV protease. The linearised TAL can now bind to the specific DNA sequence.
The 12th and 13th amino acid of each of the repeats from our TALE Ax7R-RR 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
This composite part includes an Intein, a linker, an eGFP, a TAL-effector, a TEV Site and a Strep XT Tag. They are all translated into a protein together.
The Intein and linker are important in the circularization of the amino acid sequence between the N- and C-terminal parts. Between the Intein sites are the TAL-effector, TEV site and the Strep XT tag.
When it is translated, the Intein site reacts and forms a circularised protein. All the parts in between N- and C-Terminal Intein are a part of the protein circle. In this circle, the whole protein gains more stability.
With the help of the Strep XT tag the protein can be purified even if it is circularized. The Tag is also useful for the detection of the protein with an immunostain.
The TEV Site creates an opportunity to linearise the protein again with the help of the TEV protease. The linearised TAL can now bind to the specific DNA sequence.
The 12th and 13th amino acid of each of the 11.5 repeats from our TALE Hax3 – 2xNG (B) are: NI HD NI HD HD HD NG NG NS HD NI NG.
They bind the DNA sequence: A C A C C C T T N C A T
This composite part includes an Intein, a linker, an eGFP, a TAL-effector, a TEV Site and a Strep XT Tag. They are all translated into a protein together.
The Intein and linker are important in the circularization of the amino acid sequence between the N- and C-terminal parts. Between the Intein sites are the TAL-effector, TEV site and the Strep XT tag.
When it is translated, the Intein site reacts and forms a circularised protein. All the parts in between N- and C-Terminal Intein are a part of the protein circle. In this circle, the whole protein gains more stability.
With the help of the Strep XT tag the protein can be purified even if it is circularized. The Tag is also useful for the detection of the protein with an immunostain.
The TEV Site creates an opportunity to linearise the protein again with the help of the TEV protease. The linearised TAL can now bind to the specific DNA sequence.
The 12th and 13th amino acid of each of the 11.5 repeats from our TALE 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.
