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Part:BBa_K782007

Designed by: Fedja Pavlovec   Group: iGEM12_Slovenia   (2012-09-19)
Revision as of 09:53, 26 September 2012 by UrosZupancic (Talk | contribs)

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).

Svn 12 NicTAL12--.png

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 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.

Svn 12 NicTAL10vs12.PNG

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.

Svn 12 NicTAL graf.png

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 2133
    Illegal XhoI site found at 1224
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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