
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 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.
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: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
- 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 |