Difference between revisions of "Part:BBa K782002"

Latest revision as of 21:30, 26 September 2012

4x[NicTAL]+4x[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 four consecutive specific binding sites for NicTAL12 and 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 NicTAL12:KRAB or TALD:KRAB on binding sites, a repression of reporter protein mCitrine occurs.

Single binding sequence for NicTAL12 is: TCTATCAATGATAGA

Single binding sequence for TALD is: TCGTCCAATAGCTTCTC

Figure 1. Shematic representation of four consecutive specific binding sites for NicTAL12 and TALD upstream of CMV promoter and reporter protein mCitrine.

Characterization

Results: Specific TAL 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
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 2: / Line 2: @@
 <partinfo>BBa_K782002 short</partinfo>
-==Introduction==
 * 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 [ ].
-[[Image:7xNicTAL+7xTALD.png]]
+==Introduction==
-Figure 1. Shematic representation of four consecutive specific binding sites for NicTAL and TALD
+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.
-upstream of CMV promoter and reporter protein mCitrine.
+Our construct contains [https://parts.igem.org/Part:BBa_K782068 four consecutive specific binding sites] for [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782007 NicTAL12] and [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_K782011 NicTAL12:KRAB] or [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 NicTAL12 is: TCTATCAATGATAGA
-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, that ensure inducible expression of reporter protein mCitrine. mCitrine is yellow fluorescent protein.
+Single binding sequence for TALD is: TCGTCCAATAGCTTCTC
-Single binding sequence for NicTAL is:  ACTCTATCAATGATAGAGT
-Single binding sequence for TALD is: TCGTCCAATAGCTTCTC
+[[Image:7xNicTAL+7xTALD.png]]
+'''Figure 1.''' Shematic representation of four consecutive specific binding sites for NicTAL12 and TALD
+upstream of CMV promoter and reporter protein mCitrine.
+==Characterization==
+Results: Specific TAL [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782025 binding sites] were further characterized with other 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.
@@ Line 29: / Line 39: @@
 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.