Difference between revisions of "Part:BBa K1789018:Design"

Revision as of 08:58, 18 September 2015

GFP_S1

Design Notes

This is the first experimental group of our project. Our theory is feasible if the functional parameter of this group is stronger than the group of negative control.

Split GFP is a technique that has been widely used in the research of protein-protein interaction. In our project, we demonstrated a prototype by fusing the Amino (or Carboxyl) Half of GFP with TALE1 (or TALE2/3).

By integrating the coding sequences of the TALE-fused proteins and the scaffold, three different plasmids can be constructed.

This prototype is designed to test if our system can achieve our goal of compartmentation by examining if the green florescent intensity raised observably.

Fluoroskan Ascent FL by Thermo can be used to detect the fluorescence intensity.

Source

We obtained it through the contribution of our basic subparts.

TALE2

This is a engineered TAL effector that can bind with the DNA sequence 5'-GATAAACACCTTTC-3'

Usage and Biology

The transcription activator-like (TAL) effectors is a family-III effector in Xanthomonas that helps when they infect various plant species (1). Different TALEs share a similar domain structure that enables them to bind the genome of the host cell and act as transcriptional effectors. 1.5 to 33.5 tandem repeats (TAL repeats), each of which can recognize one specific DNA base pair, were determined in the central DNA binding domain of the TALEs (2)(3). Each TAL repeat contains 33 to 35 highly conserved amino acids, among which, residues at positions 12 and 13 (also known as RVDs for repeat variable di-residues) confer DNA specificity. This structural characteristic allows the TAL effector being utilized in protein engineering applications. By physically fusing the TAL effector with the cleavage domain of FokI nucleases, TAL effector nucleases (TALENs) can be created. This nucleases were widely applied in Prokaryotic and Eukaryotic Cells. Other methods of engineering TAL effectors acting as transcriptional effectors were also reported.(右边放TALE结构图)

This part is designed to recognize the DNA binding motif 1 (BM1) in our project.

BM1 is sequenced as 5'-GGAGGCACCGGTGG-3'.

This BM exists in all of our scaffold systems (BBa_K178005, BBa_K178006, BBa_K178007). The sequences were chosen from Danio rerio CD154 gene in order to avoid homology with E.coli genome.

Sequence and Features

Sequence and Features

Experimental Validation