Difference between revisions of "Part:BBa K782007"

(Characterization)
(Characterization)
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==Characterization==
 
==Characterization==
  
We discovered that [https://parts.igem.org/Part:BBa_K323214 NicTAL ] part deposited in the Registry by the Slovenian iGEM2010 team  was missing subdomain in DNA-binding domain, so[http://2012.igem.org/Team:Slovenia our team] added this 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  [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782066 VP16] and  [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782011 KRAB] domain.  
+
We discovered that [https://parts.igem.org/Part:BBa_K323214 NicTAL ] part deposited in the Registry by the Slovenian iGEM2010 team  was missing subdomain in DNA-binding domain, so [http://2012.igem.org/Team:Slovenia our team] added this 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  [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782066 VP16] and  [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782011 KRAB] domain.  
  
 
[[Image:Svn_12_NicTAL10vs12.PNG | 600 px]]
 
[[Image:Svn_12_NicTAL10vs12.PNG | 600 px]]

Revision as of 08:08, 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).

Svn 12 NicTAL12 .png

Figure 1: Schematic representation of the construct.

Single binding sequence for NicTAL:TCTATCAATGATAGA

Characterization

We discovered that NicTAL part deposited in the Registry by the Slovenian iGEM2010 team was missing subdomain in DNA-binding domain, so [http://2012.igem.org/Team:Slovenia our team] added this 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]