Difference between revisions of "Part:BBa K782007"

Revision as of 13:41, 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 DNA binding sequence for NicTAL:TCTATCAATGATAGA

Characterization

We found out that NicTAL part deposited in the Registry by the Slovenian iGEM2010 team is 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 terminus. Apart from adding missing N terminal aminoacids, we added HIS tag also on N terminus and NLS on C terminus 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.

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 [http://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators 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