Difference between revisions of "Part:BBa K782021"

Revision as of 08:13, 26 September 2012

10x[TALB] operator_CMV promoter_fLuciferase

TALB label represents TAL effector 1297 from zebrafish experiments (Sander et al., 2011)

Introduction

Transcription activation like (TAL) effectors are DNA binding proteins with a high specifity, built from tandem repeats, with near identical seqences, differing only in two amino acids in each repeat called “repeat variable diresidue” (RVD), which determine the specifity for a single nucleotide.(Scholze and Boch, 2011)

We designed our construct with 10 binding sites for TALB, upstream of a CMV promoter (constitutive promoter for expression in mammalian cells). Downstream of the promoter we cloned fLuciferase (firefly luciferase) to function as a reporter.

Figure1:Schematic representation of our construct

Characterisation

HEK293T cells were transfected with the 10x[TALB] operator_CMV promoter_fLuciferase reporter and TALB:KRAB (Figure 2). Tests showed, successful repression of fLuciferase with TAL repressor. Tests showed that tested construct exhibited over 90% repression of the reporter plasmid (Figure 3).

Figure 2:Schematic representation of repression experiments. A: in the absence of a TALB:KRAB, fLuciferase is constituitively expressed. B: when a TALB:KRAB is present, it binds to its binding site upstream of the CMV promoter and represses transcription of fLuciferase with the KRAB domain.

Figure 3:Testing repression of reporter luciferase gene. White column is showing constantly expressed reporter (fLuc), blue column is showing repression with TALB:KRAB.

fLuciferase was contributed by host lab
Binding sites for TAL effectors were ordered from IDT.

References

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

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 914
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 341
Illegal NgoMIV site found at 701
Illegal NgoMIV site found at 1626
Illegal NgoMIV site found at 2970
Illegal NgoMIV site found at 2991
Illegal NgoMIV site found at 3306
Illegal AgeI site found at 206
Illegal AgeI site found at 541
Illegal AgeI site found at 566
Illegal AgeI site found at 901
Illegal AgeI site found at 2694
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 2876

@@ Line 14: / Line 14: @@
 ===Characterisation===
-HEK293T cells were transfected with the 10x[TALA] operator_CMV promoter_fLuciferase reporter and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782008 TALA:NLS:KRAB] (Figure 2). Tests showed, successful repression of fLuciferase with TAL repressor. Tests showed that tested construct exhibited over 90% repression of the reporter plasmid (Figure 3).
+HEK293T cells were transfected with the 10x[TALB] operator_CMV promoter_fLuciferase reporter and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782010 TALB:KRAB] (Figure 2). Tests showed, successful repression of fLuciferase with TAL repressor. Tests showed that tested construct exhibited over 90% repression of the reporter plasmid (Figure 3).
 [[Image:10×B_CMV_fLuc_shema2.png‎]]
-'''Figure 2:'''Schematic representation of repression experiments. A: in the absence of a TALA:NLS:KRAB, fLuciferase is constituitively expressed. B: when a TALA:NLS:KRAB is present, it binds to its binding site upstream of the CMV promoter and represses transcription of fLuciferase with the KRAB domain.
+'''Figure 2:'''Schematic representation of repression experiments. A: in the absence of a TALB:KRAB, fLuciferase is constituitively expressed. B: when a TALB:KRAB is present, it binds to its binding site upstream of the CMV promoter and represses transcription of fLuciferase with the KRAB domain.
-[[Image:Svn_12_talakgraf.png]]
+[[Image:Svn_12_talBKRAB_graf.png]]
-'''Figure 3:'''Testing repression of reporter luciferase gene. White column is showing constantly expressed reporter (fLuc), blue column is showing repression with TALA:KRAB.
+'''Figure 3:'''Testing repression of reporter luciferase gene. White column is showing constantly expressed reporter (fLuc), blue column is showing repression with TALB:KRAB.