Difference between revisions of "Part:BBa K1997025"
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This part codes a fusion protein of sHRP-C and dCas9 | This part codes a fusion protein of sHRP-C and dCas9 | ||
| − | |||
===Usage and Biology=== | ===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. <sup>1 </sup> | ||
| + | |||
| + | HRP was used as a reporter,the enzymatic activity of split fragments would be reconstituted when they were reassembled. <sup>2 </sup> | ||
| + | |||
| + | In our project, we used engineered E.coli to produce the fusion protein of dCas9 and split-HRP fragments. | ||
| + | |||
| + | |||
| + | ===Sequence and Features=== | ||
<!-- --> | <!-- --> | ||
<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K1997025 SequenceAndFeatures</partinfo> | <partinfo>BBa_K1997025 SequenceAndFeatures</partinfo> | ||
| + | ==Experimental Validation== | ||
| − | + | This part is validated through two ways: PCR and functional testing | |
| − | ===Functional | + | |
| − | + | ===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. | ||
| + | |||
| + | [[File:NUDT-025-1.jpg|300px|]] | ||
| + | |||
| + | |||
| + | ===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%. | ||
| + | |||
| + | [[File:NUDT-024-2.jpg|700px|]] | ||
| + | |||
| + | Figure 1. 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. | ||
Latest revision as of 01:14, 21 October 2016
P+R->sHRP-C->dCas9->Ter
This part codes a fusion protein of sHRP-C 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1681
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 3960
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 334
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.
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%.
Figure 1. 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.