Difference between revisions of "Part:BBa K3168005"

Revision as of 12:21, 23 September 2019

dCas9-SmallBitNanoLuc

This composite part is made up of two basic parts (figure 1). The first basic part (BBa_K3168000) codes for a catalytically dead CRISPR associated protein 9 (dCas9). dCas9 binds to a specific double-stranded DNA sequence which is determined by the guide RNA. The second basic part (BBa_K3168003), which is fused to dCas9, consists of a (GGS)₅ linker and the small bit of NanoLuc. When the small bit of NanoLuc forms a complex with the large bit, blue light is emitted. Thus this composite part is part of a Split-NanoLuc detection system, which targets a specific sequence on dsDNA and sends out a bioluminescent signal upon binding of this specific sequence (figure 2).

Figure 1. Schematic representation of dCas9-SmallBitNanoLuc.

Usage and Biology

dCas9 in combination with guide RNA forms a dsDNA recognition complex. A stronger bioluminescent signal is created when dCas9-LargeBitNanoLuc and dCas9-SmallBitNanoLuc bind in close proximity.

Figure 2. Schematic representation of dCas9-Split-NanoLuc system.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1099
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 3378
Illegal BamHI site found at 4189
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 2: / Line 2: @@
 ===dCas9-SmallBitNanoLuc===
-This composite part is made up of two basic parts. The first basic part [https://parts.igem.org/wiki/index.php?title=Part:BBa_K3168000 (BBa_K3168000)] codes for a catalytically dead CRISPR associated protein 9 (dCas9). This protein binds to a specific target on double stranded DNA. This specific target is determined by the guide RNA, which makes the target sequence of this protein very modular. The second basic part [https://parts.igem.org/wiki/index.php?title=Part:BBa_K3168003 (BBa_K3168003)], which is fused to dCas9, consist of a (GGS)<sub>5</sub> linker and the small bit of NanoLuc. When the small bit of NanoLuc forms a complex with the large bit, blue light is emitted. Thus this composite part is part of a Split-NanoLuc detection system, which targets a specific sequence on dsDNA and sends out a bioluminescent signal.
+This composite part is made up of two basic parts (figure 1). The first basic part [https://parts.igem.org/wiki/index.php?title=Part:BBa_K3168000 (BBa_K3168000)] codes for a catalytically dead CRISPR associated protein 9 (dCas9). dCas9 binds to a specific double-stranded DNA sequence which is determined by the guide RNA. The second basic part [https://parts.igem.org/wiki/index.php?title=Part:BBa_K3168003 (BBa_K3168003)], which is fused to dCas9, consists of a (GGS)<sub>5</sub> linker and the small bit of NanoLuc. When the small bit of NanoLuc forms a complex with the large bit, blue light is emitted. Thus this composite part is part of a Split-NanoLuc detection system, which targets a specific sequence on dsDNA and sends out a bioluminescent signal upon binding of this specific sequence (figure 2).
 [[File:T--TU_Eindhoven--dCas9-SmallBitNanoLuc.png|300px|]]
@@ Line 9: / Line 9: @@
 ===Usage and Biology===
-dCas9 in combination with guide RNA forms a dsDNA recognition complex. A stronger bioluminescent signal is created when dCas9-LargBitNanoLuc and dCas9-SmallBitNanoLuc bind in close proximity.
+dCas9 in combination with guide RNA forms a dsDNA recognition complex. A stronger bioluminescent signal is created when dCas9-LargeBitNanoLuc and dCas9-SmallBitNanoLuc bind in close proximity.
 [[File:T--TU_Eindhoven--dCas9-SplitNL.png|800px|]]