Difference between revisions of "Part:BBa K1997003"
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For such assay, E.coli carrying respective plasmid was cultured overnight under IPTG induction. Cells were then collected and lysed by high-pressure homogenizer. Once lysed and ultra-filtrated (to remove small molecules), with D-luciferin substrate solution was added into the cell lysate for the measurement of luciferase activity. | For such assay, E.coli carrying respective plasmid was cultured overnight under IPTG induction. Cells were then collected and lysed by high-pressure homogenizer. Once lysed and ultra-filtrated (to remove small molecules), with D-luciferin substrate solution was added into the cell lysate for the measurement of luciferase activity. | ||
| + | |||
| + | Chemo-luminescence assay showed significant variation between the PPI positive group and the PPI negative group. Thus then validated the function of this part. | ||
[[File:T--NUDT_CHINA--partsfig8.jpg|700px|]] | [[File:T--NUDT_CHINA--partsfig8.jpg|700px|]] | ||
| − | + | Figure 1. Evaluation of the Signal-Noise Ratio of split LUC system (A) Schematic representation of the evaluation protocol. The complete Zif-268 protein was introduced to simulate the condition where strong interaction among two proteins occur, whereas the split-zif protein was used to simulate the condition where no interaction exists. (B) Experimment showing the BLU under two different conditions. Relative BLU was calculated with normalization of BLU. This experiment was run in three parallel reactions, and the data represent results obtained from at least three independent experiments. **p<0.01. | |
===References=== | ===References=== | ||
Revision as of 07:28, 20 October 2016
sLuc-C
This is the C-terminal of split GFP reporter. It can be used for protein-protein interaction researches.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Experimental Validation
This part is validated through four ways: enzyme cutting, PCR, Sequence, and functional testing
Sequencing
This part is sequenced as correct after construction.
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.
Enzyme digestion test
Methods
After the assembly ,the plasmid was transferred into the Competent E. coli DH5α). After culturing overnight in LB,we minipreped the plasmid for cutting. The preparation of the plasmid was performed with TIANprep Mini Plasmid Kit from TIANGEN. The cutting procedure was performed with EcoRI and SpeI restriction endonuclease bought from TAKARA.
The plasmid was cutted in a 20μL system at 37 ℃ for 2 hours. The Electrophoresis was performed on a 1％ Agarose glu.
The result of the agarose electrophoresis was shown on the picture above.
Functional Test
This part is tested together with the part BBa_K1997002, in the composite part BBa_K19970021 and BBa_K19970022.
To proof the basic function of our split-luciferase reporting system, two devices, containing split-HRP fragments and a complete (or split as control) zinc finger protein, were built under control of a lac operon controlled T7 promoter. The complete zinc finger protein was to stimulate a PPI positive situation, while the split one was to stimulate a PPI negative situation.
For such assay, E.coli carrying respective plasmid was cultured overnight under IPTG induction. Cells were then collected and lysed by high-pressure homogenizer. Once lysed and ultra-filtrated (to remove small molecules), with D-luciferin substrate solution was added into the cell lysate for the measurement of luciferase activity.
Chemo-luminescence assay showed significant variation between the PPI positive group and the PPI negative group. Thus then validated the function of this part.
Figure 1. Evaluation of the Signal-Noise Ratio of split LUC system (A) Schematic representation of the evaluation protocol. The complete Zif-268 protein was introduced to simulate the condition where strong interaction among two proteins occur, whereas the split-zif protein was used to simulate the condition where no interaction exists. (B) Experimment showing the BLU under two different conditions. Relative BLU was calculated with normalization of BLU. This experiment was run in three parallel reactions, and the data represent results obtained from at least three independent experiments. **p<0.01.
References
[1]Gould SJ, Subramani S (Nov 1988). "Firefly luciferase as a tool in molecular and cell biology". Analytical Biochemistry. 175(1): 5–13. doi:10.1016/0003-2697(88)90353-3. PMID 3072883.
[2]Steghens JP, Min KL, Bernengo JC (Nov 1998). "Firefly luciferase has two nucleotide binding sites: effect of nucleoside monophosphate and CoA on the light-emission spectra". The Biochemical Journal. 336 ( Pt 1) (1): 109–13. PMC 1219848 . PMID 9806891.