Composite

Part:BBa_K3168004

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-LargeBitNanoLuc

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_K3168002), which is fused to dCas9, consists of a (GGS)5 linker and the large bit of NanoLuc. When the large bit of Split-NanoLuc forms a complex with the small bit and the substrate Furimazine is present, blue light is emitted. dCas9-LargeBit itself is slightly bioluminescent when the substrate is added. However, the intensity is more bright when a complex is formed with the small bit. 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-LargeBit.png

Figure 1. Schematic representation of dCas9-LargeBitNanoLuc.

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.

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-LargeBitNanoLuc has a molecular weight of 178 kDa. As shown in figure 3, there are very obvious blobs of dCas9-LargeBitNanoLuc 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-LargeBitNanoLuc.png

Figure 3. SDS-PAGE gel of IMAC elution samples of dCas9-LargeBitNanoLuc (Elute 2: 17.90 μM, Elute 3: 6.37 μM, Elute 4: 2.88 μM).

References

Dixon, A. S., Schwinn, M. K., Hall, M. P., Zimmerman, K., Otto, P., Lubben, T. H., ... & Wood, M. G. (2015). NanoLuc complementation reporter optimized for accurate measurement of protein interactions in cells. ACS chemical biology, 11(2), 400-408.

Zhang, Y., Qian, L., Wei, W., Wang, Y., Wang, B., Lin, P., ... & Cheng, S. (2016). Paired design of dCas9 as a systematic platform for the detection of featured nucleic acid sequences in pathogenic strains. ACS synthetic biology, 6(2), 211-216.

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 4630
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//awards/composite_part/nominee
Parameters
None