Difference between revisions of "Part:BBa K3168005"

Revision as of 15:00, 26 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.

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.

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