Difference between revisions of "Part:BBa K1997008"
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Let7f is one of the founding members of the let7 family. | Let7f is one of the founding members of the let7 family. | ||
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===Usage and Biology=== | ===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.<sup>1</sup> | ||
| + | |||
| + | 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.<sup>2</sup> | ||
| + | |||
| + | Zwiers identified a putative binding site for both LET7E and LET7F in the 3-prime UTR of IL23R that was abolished by a SNP associated with inflammatory bowel disease.<sup>3</sup> | ||
| + | ===Sequence and Features=== | ||
<!-- --> | <!-- --> | ||
<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K1997008 SequenceAndFeatures</partinfo> | <partinfo>BBa_K1997008 SequenceAndFeatures</partinfo> | ||
| + | ==Experimental Validation== | ||
| − | + | This part is validated through four ways: enzyme cutting, PCR, Sequence, and functional testing | |
| − | ===Functional | + | |
| − | + | ===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. | ||
| + | |||
| + | [[File:NUDT-008-1.jpg|300px|]] | ||
| + | |||
| + | ===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-7f was performed.The Electrophoresis was performed on a 2％ Agarose glu. | ||
| + | |||
| + | The result of the agarose electrophoresis was shown on the picture below. | ||
| + | |||
| + | |||
| + | |||
| + | [[File:T--NUDT_CHINA--let7f.jpg|300px|]] | ||
| + | |||
| + | |||
| + | ===References=== | ||
| + | 1.The tumor suppressor microRNA let-7 represses the HMGA2 oncogene.Lee YS, Dutta A. Genes Dev. 2007 May 1;21(9):1025-30. Epub 2007 Apr 16. | ||
| + | |||
| + | 2.RAS is regulated by the let-7 microRNA family. Johnson SM, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A, Labourier E, Reinert KL, Brown D, Slack FJ. Cell. 2005 Mar 11;120(5):635-47. | ||
| + | |||
| + | 3.Cutting edge: a variant of the IL-23R gene associated with inflammatory bowel disease induces loss of microRNA regulation and enhanced protein production. Zwiers A, Kraal L, van de Pouw Kraan TC, Wurdinger T, Bouma G, Kraal G. J Immunol. 2012 Feb 15;188(4):1573-7. doi: 10.4049/jimmunol.1101494. Epub 2012 Jan 18. | ||
Revision as of 04:54, 20 October 2016
let-7f
Let7f 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
Zwiers identified a putative binding site for both LET7E and LET7F in the 3-prime UTR of IL23R that was abolished by a SNP associated with inflammatory bowel disease.3
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-7f 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.The tumor suppressor microRNA let-7 represses the HMGA2 oncogene.Lee YS, Dutta A. Genes Dev. 2007 May 1;21(9):1025-30. Epub 2007 Apr 16.
2.RAS is regulated by the let-7 microRNA family. Johnson SM, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A, Labourier E, Reinert KL, Brown D, Slack FJ. Cell. 2005 Mar 11;120(5):635-47.
3.Cutting edge: a variant of the IL-23R gene associated with inflammatory bowel disease induces loss of microRNA regulation and enhanced protein production. Zwiers A, Kraal L, van de Pouw Kraan TC, Wurdinger T, Bouma G, Kraal G. J Immunol. 2012 Feb 15;188(4):1573-7. doi: 10.4049/jimmunol.1101494. Epub 2012 Jan 18.