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Part:BBa_K782007

Designed by: Fedja Pavlovec   Group: iGEM12_Slovenia   (2012-09-19)

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 that NicTAL10 part deposited in the Registry by the Slovenian iGEM2010 team is not functional. Part was missing subdomain in DNA-binding domain, so our team added the missing subdomain on the N terminus, since at that time the requirement for the minimal DNA binding domain has not been known. Apart from adding the missing N terminal aminoacids, we added a HIS tag also on the N terminus and NLS on the C terminus of NicTAL12 (Figure 2). This construct was later for designing TAL-based activator and repressor that were experimentaly verified 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:KRAB. HEK293T cells were cotransfected with NicTAL12:KRAB repressor under the control of a CMV promoter, and with a firefly luciferase reporter plasmid (BBa_K782023) containing 12 DNA-binding sites for NicTAL repressor upstream the CMV promoter. Check detailed results on iGEM 2012 team Slovenia wiki.

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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