Difference between revisions of "Part:BBa K1997009"
Zhuchushu13 (Talk | contribs) |
|||
| Line 5: | Line 5: | ||
Let7g is one of the founding members of the let7 family. | Let7g is one of the founding members of the let7 family. | ||
| − | |||
===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> | ||
| + | |||
| + | The ectopic expression of Let7g induced cell cycle arrest and cell death in murine lung cancer cells with an oncogenic Kras mutation.<sup>2</sup> In mouse tumors, let7g reduce the level of Ras family and Hmga2 protein. Some lung tumors are caused by the mutations of oncogenic KRas. There are some evidences show that let7g can suppress lung tumor. Ectopic expression of an oncogenic Kras mutant in mouse cells largely rescued Let7g-mediated tumor suppression, whereas ectopic expression of Hmga2 was less effective.<sup>3</sup> | ||
| + | |||
| + | ===Sequence and Features=== | ||
<!-- --> | <!-- --> | ||
<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K1997009 SequenceAndFeatures</partinfo> | <partinfo>BBa_K1997009 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-009-1.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-7g was performed.The Electrophoresis was performed on a 2％ Agarose glu. | ||
| + | |||
| + | The result of the agarose electrophoresis was shown on the picture below. | ||
| + | |||
| + | [[File:NUDT-009-2.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.Suppression of non-small cell lung tumor development by the let-7 microRNA family. Kumar MS, Erkeland SJ, Pester RE, Chen CY, Ebert MS, Sharp PA, Jacks T. Proc Natl Acad Sci U S A. 2008 Mar 11;105(10):3903-8. doi: 10.1073/pnas.0712321105. Epub 2008 Feb 28. | ||
| + | |||
| + | 3.http://www.omim.org/entry/612102 | ||
Revision as of 05:00, 20 October 2016
let-7g
Let7g 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
The ectopic expression of Let7g induced cell cycle arrest and cell death in murine lung cancer cells with an oncogenic Kras mutation.2 In mouse tumors, let7g reduce the level of Ras family and Hmga2 protein. Some lung tumors are caused by the mutations of oncogenic KRas. There are some evidences show that let7g can suppress lung tumor. Ectopic expression of an oncogenic Kras mutant in mouse cells largely rescued Let7g-mediated tumor suppression, whereas ectopic expression of Hmga2 was less effective.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-7g 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.Suppression of non-small cell lung tumor development by the let-7 microRNA family. Kumar MS, Erkeland SJ, Pester RE, Chen CY, Ebert MS, Sharp PA, Jacks T. Proc Natl Acad Sci U S A. 2008 Mar 11;105(10):3903-8. doi: 10.1073/pnas.0712321105. Epub 2008 Feb 28.
3.http://www.omim.org/entry/612102