Difference between revisions of "Part:BBa K782002"

Revision as of 16:19, 25 September 2012

4x[NicTAL]+4x[TALD] operator_CMV promoter_mCitrine

TALD label represents TAL effector 1295 from zebrafish experiments (Sander et al., 2011).

Introduction

Transcription activation like (TAL) effectors are bacterial plant pathogen transcription factors that bind to DNA by recognizing a specific DNA sequence in which each base pair binds a single tandem repeat in in the TAL DNA-binding domain. A tandem TAL repeat contains 33 to 35 amino acids, where the 12th and the 13th amino acid, called a “repeat variable diresidue” (RVD) are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011). All TAL repeats have almost identical sequences, differing only in the RVDs. This modularity of TAL effector binding domains therefore makes them a perfect tool to target specific DNA sequences by designing specific binding domains for a selected TAL effector. We designed four consecutive specific binding sites for NicTAL and TALD upstream of CMV promoter (Figure 1), that cause a repression of reporter protein mCitrine, after binding of NicTAL12:KRAB or TALD:KRAB. mCitrine is yellow fluorescent protein.

Single binding sequence for NicTAL is: TCTATCAATGATAGA

Single binding sequence for TALD is: TCGTCCAATAGCTTCTC

Figure 1. Shematic representation of four consecutive specific binding sites for NicTAL and TALD upstream of CMV promoter and reporter protein mCitrine.

mCitrine was provided from host lab.
Binding sites for TAL effectors were ordered from IDT.

References

Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. Curr. Opin. Microbiol. 14, 47-53.

Sander, J. D., Cade, L., Khayter, C., Reyon, D., Peterson, R. T., Joung, J. K., and Yeh, J.-R. J. (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nature Biotechnology 29, 697–698

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 270
Illegal XhoI site found at 900
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 35
Illegal AgeI site found at 235
1000
COMPATIBLE WITH RFC[1000]

@@ Line 1: / Line 1: @@
 __NOTOC__
 <partinfo>BBa_K782002 short</partinfo>
-==Introduction==
 * TALD label represents TAL effector 1295 from zebrafish experiments (Sander et al., 2011).
+==Introduction==
 Transcription activation like (TAL) effectors are bacterial plant pathogen transcription factors that bind to DNA by recognizing a specific DNA sequence in which each base pair binds a single tandem repeat in in the TAL DNA-binding domain. A tandem TAL repeat contains 33 to 35 amino acids, where the 12th and the 13th amino acid, called a “repeat variable diresidue” (RVD)  are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011).  All TAL repeats have almost identical sequences, differing only in the RVDs. This modularity of TAL effector binding domains therefore makes them a perfect tool to target specific DNA sequences by designing specific binding domains for a selected TAL effector. We designed four consecutive specific binding sites for NicTAL and TALD upstream of CMV promoter (Figure 1), that cause a repression of reporter protein mCitrine, after binding of [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782011 NicTAL12:KRAB] or [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782009 TALD:KRAB]. mCitrine is yellow fluorescent protein.