Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Kim Luehmann
Date created: 2016-10-13 11:00:00
Date modified: 2016-10-18 01:51:50
TAL-effector Hax3-2xNG
| Types | DnaRegion |
| Roles | Coding CDS |
| Sequences | BBa_K2075023_sequence (Version 1) |
Description
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, 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 the Hax3-2xNG. The 12th and 13th amino acid of each of the 11.5 repeats from our TAL 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
Notes
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 the Hax3-2xNG. The 12th and 13th amino acid of each of the 11.5 repeats from our TAL 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
Source
Xanthomonas| Sequence Annotation | Location | Component / Role(s) |
| NLS SV40 N-Term Hax3 137 Hax3 C-Term Hax3 63 | 1,21 22,433 434,1611 1612,1803 | feature/cds CDS CDS feature/cds feature/cds CDS feature/cds CDS |