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Part:BBa_K782007

Designed by: Fedja Pavlovec   Group: iGEM12_Slovenia   (2012-09-19)
Revision as of 10:06, 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 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.

Svn 12 NicTAL10vs12.PNG

Figure 2: Sequence alignment of NicTAL10 and NicTAL12 showing differences between constructs.



Svn 12 NicTAL graf.png

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


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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