Part:BBa_K1789010

TALE3

This is a engineered TAL effector that can bind with the DNA sequence 5'-CTTTCCACAAATAG-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. This part is designed to recognize the REVERSED sequence of DNA binding motif 2 (BM2) in our project. REVERSED BM2 is sequenced as 5'-CTTTCCACAAATAG-3'. This BM exists in scaffold BBa_K1789005. The sequences were chosen from Danio rerio CD154 gene in order to avoid homology with E.coli genome.

Sequence and Features

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 2083
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 565
  Illegal BsaI.rc site found at 973
  Illegal BsaI.rc site found at 1279
  

Experimental Validation

Sequencing

This part is sequenced as correct after construction.

ChIP-PCR Analysis

To evaluate whether TALE1 protein can effectively target the binding motifs on plasmid DNA scaffold, the ChIP-PCR analysis was conducted. For this experiment, the plasmid of pSB1C3-Plac-RBS-TALE3-GFP2-Ter-Scaffold1 was constructed, interpret into E.coli BL21 (DE3), and subsequently induced expression by IPTG. Bacterial lysis samples were cross-linked in 1% formaldehyde without ultrasonic treatment due to the small size of binding plasmid, and immunoprecipitated with anti-GFP polyclonal antibody. Because the binding motifs of TALEs are containing highly repeated sequences, and their flanking sequences are also homologous to the other parts of the harboring plasmid, the primers used for ChIP-PCR were forward P3 and reverse P4 for GFP2 amplification (Fig. 1).

Fig. 1 A schematic showing the primers and the plasmid regions tested in ChIP assays. P3/P4 was designed for TALE3-GFP2 ChIP assay.

As shown in Fig 2, a 251 bp of DNA fragments was amplified from the precipitates of TALE2-GFP3-Scaffold1 using anti-GFP antibody. However, the negative control immunoprecipitations using no antibody (beads only) or normal rabbit IgG showed no amplification signal. The amplified fragment was confirmed by sequencing. These results indicate that TALE3-GFP2 can specifically binds to the corresponding plasmid DNA binding motifs in vivo.

Fig. 2 Determination of the binding abilities of TALE3-GFP2 to corresponding DNA scaffolds. Input indicates an aliquot of total DNA. Antibodies used for immunoprecipitation are indicated above the lanes.

References

1. J. Boch, U. Bonas, Xanthomonas AvrBs3 family-type III effectors: discovery and function. Annual review of phytopathology 48, 419 (2010).

2. J. Boch et al., Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326, 1509 (Dec 11, 2009).

3. M. J. Moscou, A. J. Bogdanove, A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501 (Dec 11, 2009).