Composite

Part:BBa_K1997024

Designed by: Chushu Zhu   Group: iGEM16_NUDT_CHINA   (2016-10-13)


P+R->sHRP-N->dCas9->Ter

This part codes a fusion protein of sHRP-N and dCas9

Usage and Biology

A catalytically dead Cas9 (dCas9) formed a DNA recognition complex which can bind any sequence when co-expressed with a guide RNA. 1

HRP was used as a reporter,the enzymatic activity of split fragments would be reconstituted when they were reassembled. 2

In our project, we used engineered E.coli to produce the fusion protein of dCas9 and split-HRP fragments.


Sequence and Features

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 415
    Illegal NheI site found at 2038
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 657
    Illegal BamHI site found at 4317
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Experimental Validation

This part is validated through two ways: PCR and functional testing

PCR

Methods

The PCR is performed with Premix EX Taq by Takara.

F-Prime: 5’- GAATTCGCGGCCGCTTCTAGAATGC-3’

R-Prime: 5’- GGACTAGTATTATTGTTTGTCTGCC-3’

The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1% Agarose glu. The result of the agarose electrophoresis was shown on the picture below.

NUDT-024-1.jpg


Functional Test

The plasmid was transformed into the E.coli BL21 (DE3) competent cells. After a overnight culture with (or without, as control group) 0.5mM IPTG induction, cells were collected and lysed by high pressure homogenizer. Subsequent purification was performed by nickel-nitrilotriacetic acid agarose affinity chromatography according to the standard protocol. As examined by SDS-PAGE and Western blots (probed with an anti-His-tag antibody), both of these proteins were successfully expressed and purified as a high degree of purity up to 90%.

NUDT-024-2.jpg

Figure 2. Prokaryotic expression and protein purification of split-HRP-dCas9 fusion proteins.

(A) Expression and purification of sHRP-C-dCas9 protein. (B) Expression and purification of sHRP-N-dCas9 protein. Western blots were probed with an anti-His-tag antibody. “-” represents the un-induced control group, “+” represents the induced group.

References

1. Lei S. Qi, Matthew H. Larson, Luke A. Gilbert et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 2013, 152: 1173-1183. 2. Kathryn E. Luker, Matthew C. P. Smith, et al. Kinetics of regulated protein–protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. PNAS, 2004, 101: 12288-12293.

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