Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Yao Xiao
Date created: 2014-10-09 11:00:00
Date modified: 2015-05-08 01:09:47
left arm->TALE03->right arm
| Types | DnaRegion |
| Roles | Coding CDS |
| Sequences | BBa_K1311002_sequence (Version 1) |
Description
This part is one of the product of the TALE assembly strategy. The TALE assembly strategy uses the Golden Gate cloning method, which is based on the ability of type IIS enzymes to cleave outside of their recognition site. When type IIS recognition sites are placed to the far 5??? and 3??? end of any DNA fragment in inverse orientation, they are removed in the cleavage process, allowing two DNA fragments flanked by compatible sequence overhangs, termed fusion sites, to be ligated seamlessly. Since type IIS fusion sites can be designed to have different sequences, directional assembly of multiple DNA fragments is feasible. Using this strategy, DNA fragments can be assembled from undigested input plasmids in a one-pot reaction with high efficiency.We chose the native TALE AvrBs3 as a scaffold for customized assembly of TALE constructs. The central DNA binding domain of AvrBs3 is formed by 17.5 tandemly arranged 34 amino acid repeats, with the last half repeat showing similarity to only the first 20 amino acids of a full repeat.
In a single Golden Gate cloning reaction, cloning efficiency is significantly reduced for assembly of 17 repeat modules. Therefore, we split the assembly in two successive steps to obtain TALE01, TALE 02, etc. At the former cloning steps, first 10 repeats and 11~17 are separately assembled in two vectors. The preassembly vectors confer SpecR and encode a lacZ-α fragment for blue/white selection. On both sides of the lacZ-α fragment a type IIS recognition sequence - BsaI - is positioned. After preassembly of the 10 and 7 and last repeats using BsaI, the intermediate blocks are released via Esp3I and cloned into the final assembly vector (modified pTAL1). Modified pTAL1 confers AmpR, and allows plasmid replication in E.coli. In this way we got TALE03. The vector pTAL1 also contains all elements of the final TALE expression construct, except the repeat modules. TALE03 can be directly used on targeting a certain DNA sequence.