Part:BBa_K1997006
let-7a
Let7a is one of the founding members of the let7 family.
Usage and Biology
Let7 is one of the founding members of the miRNA family. It was first found in C.elegans. There are several human let7 have the same base sequence with C.elegans let7, let7a1 is one in this group. The expression of let7 is barely detectable in embryonic stages, but it increases after differentiation and in mature tissues.1
HRAS, KRAS, and NRAS, these three human RAS genes have several let-7 complementary sites in their Untranslated Regions. It allows let-7 miRNA to control their function. The level of let7 in lung tumor’s cell is much lower than in normal cell. The expression of the RAS proteins was significantly higher in lung tumors, it suggests a possible mechanism for let-7 in cancer.2
Compared with cirrhotic livers, the level of LET7A1 was reduced about 70%in cancered livers. It was also happened in all HCC-derived cell lines examined. It may shows that the low level of the expression of LET7A1 promotes the development of cancer.
Sequence and Features
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
In vitro transcription experiment of let-7a was performed.The Electrophoresis was performed on a 2％ Agarose glu.
The result of the agarose electrophoresis was shown on the picture below.
References
[1] Day, R. N. & Davidson, M. W.The fluorescent protein palette: tools for cellular imaging. Chem Soc Rev 38, 2887-2921, doi:10.1039/b901966a (2009).
[2] Pfleger, K. D.& Eidne, K. A. Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET). Nature methods 3,165-174, doi:10.1038/nmeth841 (2006).
[3] Kodama, Y. &Hu, C. D. An improved bimolecular fluorescence complementation assay with a high signal-to-noise ratio.Biotechniques 49, 793-805, doi:10.2144/000113519(2010).
[4] Cabantous, S. et al. A new protein-protein interaction sensor based on tripartite split-GFP association. Scientific reports 3, 2854, doi:10.1038/srep02854 (2013).
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