Difference between revisions of "Part:BBa K1789004"
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<partinfo>BBa_K1789004 short</partinfo> | <partinfo>BBa_K1789004 short</partinfo> | ||
| − | + | In order to check the functions of our system that whether two frames standing near enough so that enzymes could react easily, we divided the sequence of GFP into two parts called N-fragment and C-fragment. This is the latter. This works like this way: only when supplied with the proper distance for tow enzymes will it produce light output. | |
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==Usage and Biology== | ==Usage and Biology== | ||
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After standardization, we used PCR to proved it. | After standardization, we used PCR to proved it. | ||
| − | The forward primer sequence is 5’-CGGAATTCGCGGCCGCTTCTAGAAAGAATGGAATCC-3’. | + | The forward primer sequence is 5’-CGGAATTCGCGGCCGCTTCTAGAAAGAATGGAATCC-3’. The reverse primer sequence is 5’-GGACTAGTTTATTATTTGTATAGTTC -3’. |
[[File:GFP2_PCR|500px|]] | [[File:GFP2_PCR|500px|]] | ||
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we also sent it to sequencing, from the reporter we can proved that it's right. | we also sent it to sequencing, from the reporter we can proved that it's right. | ||
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Revision as of 09:19, 18 September 2015
GFP2
In order to check the functions of our system that whether two frames standing near enough so that enzymes could react easily, we divided the sequence of GFP into two parts called N-fragment and C-fragment. This is the latter. This works like this way: only when supplied with the proper distance for tow enzymes will it produce light output.
Usage and Biology
Bimolecular fluorescence complementation (BiFC) means two non-fluorescent complementary fragments of the fluorescent protein can reassemble to form a fluorescent complex and restore fluorescence when they are fused to two proteins that interact with each other.
BiFC analysis has been used to study interactions among a wide range of proteins in many cell types. The study of interactions and post-translational modification of the protein makes people master the biological regulatory mechanism more. Interactions in protein are also highly valued, so there have been a number of related technologies having different characteristics and applications[1-2].
Such as GFP,after the sequence of certain sites in interchanges with the amino-terminal or carboxy-terminal sequence loop, it could still be able to fold correctly to form the structure of the chromophore and maintain fluorescence properties[3-5]
Sequence and Features
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2083
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1177
Illegal BsaI.rc site found at 1381
Experimental Validation
After standardization, we used PCR to proved it.
The forward primer sequence is 5’-CGGAATTCGCGGCCGCTTCTAGAAAGAATGGAATCC-3’. The reverse primer sequence is 5’-GGACTAGTTTATTATTTGTATAGTTC -3’.
As can be seen from the picture, it's length is right.
we also sent it to sequencing, from the reporter we can proved that it's right.
References
[ 1] Drewes G, Bouwmeester T.Global approaches to protein – protein interaction[ J ]. Curr Opin Cell Biol, 2003, 15(2): 199-205.
[ 2] Collura V, Boissy G. From protein -protein complexes to interactomics [ J ].Subcell Biochem, 2007, 43: 135-83.
[ 3] M isteli T, Spector DL. Application of the green fluorescent protein in cell biology and biotechnology[ J ].Nat Biotechnol, 1997, 15( 10): 961 - 964.
[ 4] Baird G. S, Zacharias DA, Tsien RY. Circular permutation and receptor insertion within green fluorescent proteins[ J ]. Proc Natl Acad Sci USA , 1999, 96( 20): 11241-11246.
[ 5] Ghosh I, Hamilton AD, Regan L. Antiparallel leucinezipper –directed protein reassembly: application to the green fluorescent protein[ J ]. J Am Chem Soc, 2000, 122: 5658-5659.