Difference between revisions of "Part:BBa K782014"

Latest revision as of 21:33, 26 September 2012

12x[TALD] operator_CMV promoter_mCitrine

TALD label represents TAL effector 1295 from zebrafish experiments (Sander et al., 2011).
DNA binding sites for individual TAL effectors are indicated with square brackets [ ].

Introduction

Transcription activation like (TAL) effectors are proteins able to specifically bind desired DNA sequence. The central domain of the protein is constructed from variable number of tandem repeats differing only in two amino acids. The 12th and the 13th amino acid are called a “repeat variable diresidue” (RVD) and are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011). This modularity of TAL effector binding domains therefore makes them a perfect tool to target specific DNA sequences.

Our construct contains 12 specific binding sites for TALD upstream of CMV promoter. Downstream of CMV promoter we cloned yellow fluorescent protein mCitrine an easy detectable monomer with excitation maximum at 516 nm and emission maximum at 529 nm (Figure 1). After binding of TALD:KRAB on binding sites, a repression of reporter protein mCitrine occurs.

Single binding sequence for TALD is: TCGTCCAATAGCTTCTC

Figure 1. Shematic representation of twelve specific binding site for TALD upstream of CMV promoter and reporter protein mCitrine.

Characterization

Results: 12x[TALD] binding sites were further characterized with other reporter constructs.

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

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
INCOMPATIBLE WITH RFC[10]
Unknown
12
INCOMPATIBLE WITH RFC[12]
Unknown
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 465
Illegal XhoI site found at 1095
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 214
Illegal AgeI site found at 446
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 * TALD label represents TAL effector 1295 from zebrafish experiments (Sander et al., 2011).
+* DNA binding sites for individual TAL effectors are indicated with square brackets [ ].
 ==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 twelve specific binding sites for TALD:KRAB upstream of CMV promoter (Figure 1). After binding of TALD:KRAB on binding sites, a repression of reporter protein mCitrine occurs. mCitrine is yellow fluorescent protein.
+Transcription activation like (TAL) effectors are proteins able to specifically bind desired DNA sequence. The central domain of the protein is constructed from variable number of tandem repeats differing only in two amino acids. The 12th and the 13th amino acid are called a “repeat variable diresidue” (RVD) and are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011). This modularity of TAL effector binding domains therefore makes them a perfect tool to target specific DNA sequences.
-Single binding sequence for [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782009 TALD:KRAB] is: TCGTCCAATAGCTTCTC
+Our construct contains [https://parts.igem.org/Part:BBa_K782072 12 specific binding sites] for [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782005 TALD] upstream of CMV promoter. Downstream of CMV promoter we cloned yellow fluorescent protein mCitrine an easy detectable monomer with excitation maximum at 516 nm and emission maximum at 529 nm (Figure 1).
+After binding of [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782009 TALD:KRAB] on binding sites, a repression of reporter protein mCitrine occurs.
+Single binding sequence for TALD is: TCGTCCAATAGCTTCTC
-[[Image:12xD-pCMV-mCit.png]]
-'''Figure 1.''' Shematic representation of twelve specific binding site for TALD:KRAB
+[[Image:12dmcit.png]]
+'''Figure 1.''' Shematic representation of twelve specific binding site for TALD
 upstream of CMV promoter and reporter protein mCitrine.
+==Characterization==
+Results: 12x[TALD] binding sites were further characterized with other [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782031 reporter constructs].
 * mCitrine was provided from host lab.
-* Binding sites for TAL effectors were ordered from IDT.
+* Binding sites for TAL effectors were ordered from GeneArt.
 ==References==
@@ Line 25: / Line 35: @@
 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
+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.