Part:BBa_K782007
NicTAL12:NLS DNA binding domain
Introduction
TAL effectors (TALEs) are bacterial plant pathogen transcription factors, that bind to DNA by specifically recognizing one base pair with a single tandem repeat in their DNA-binding domain. A tandem TALE repeat contains 33 to 35 amino acids, where the 12th and 13th amino acid, called a “repeat variable diresidue” (RVD), are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011).
Figure 1: Schematic representation of the construct.
Single DNA binding sequence for NicTAL:TCTATCAATGATAGA
Characterization
We found out NicTAL part deposited in the Registry by the Slovenian iGEM2010 team was not functional. Part was missing subdomain in DNA-binding domain, so [http://2012.igem.org/Team:Slovenia our team] added the missing part on N terminal sequence. Apart from adding N terminal sequence, we added HIS tag on N terminal end and NLS on terminal C end of NicTAL12 (Figure 2). This construct was later used for designing TAL-based activator and repressor by adding VP16 and KRAB domain.
Figure 2: Sequence alignment of NicTAL10 and NicTAL12 showing differences between constructs.
Figure 3: Results obtained by testing NicTAL12:KRAB repressor proved better binding ability since NicTAL12 gave much better results than NicTAL10.
References
Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. Curr. Opin. Microbiol. 14, 47-53.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2133
Illegal XhoI site found at 1224 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |