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Revision as of 08:08, 26 September 2012

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 binding sequence for NicTAL:TCTATCAATGATAGA

Characterization

We discovered that NicTAL part deposited in the Registry by the Slovenian iGEM2010 team was missing subdomain in DNA-binding domain, so[http://2012.igem.org/Team:Slovenia our team] added this 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.

Results obtained by testing NicTAL12:KRAB repressor and activator, proved that NicTAL12 binds to its binding sites better than NicTAL10.

Figure 3: Results of testing NicTAL:KRAB repressor demonstrated better binding ability of NicTAL12.

References

Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. Curr. Opin. Microbiol. 14, 47-53.

Sequence and Features