Composite

Part:BBa_K3168005

Designed by: Eva Hanckmann, Harm van der Veer, Claire Michielsen   Group: iGEM19_TU_Eindhoven   (2019-09-12)
Revision as of 15:00, 26 September 2019 by CMichielsen (Talk | contribs)

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).

T--TU Eindhoven--dCas9-SmallBitNanoLuc.png

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.

T--TU Eindhoven--dCas9-SplitNL.png

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

Characterization

Expression
The fusion protein was successfully expressed in BL21(DE3) and purified with immobilized metal affinity chromatography (IMAC). The elution was collected in 5 fractions of 1.5 mL. The three elution samples with the highest concentrations were put on an SDS-PAGE gel to evaluate the purity and molecular weight of the samples. dCas9-SmallBitNanoLuc has a molecular weight of 162 kDa. As shown in figure 3, there are very obvious blobs of dCas9-SmallBitNanoLuc just above 150 kDa. There are other bands visible so the purity is not optimal. However, the contrast between the huge blobs and the other bands is very big.

T--TU Eindhoven--SDS-PAGE dCas9-SmallBitNanoLuc.png

Figure 3. SDS-PAGE gel of IMAC elution samples of dCas9-SmallBitNanoLuc (Elute 2: 20.79 μM, Elute 3: 7.298 μM, Elute 4: 12.08 μM).

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]


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Categories
Parameters
None