Difference between revisions of "Part:BBa K1789003"

 
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<partinfo>BBa_K1789003 short</partinfo>
 
<partinfo>BBa_K1789003 short</partinfo>
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This part is the Amino Half of GFP with termination codon
 
This part is the Amino Half of GFP with termination codon
  
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==Usage and Biology==
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===Usage and Biology===
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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.
 +
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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]
 +
 +
In our project, we plan to fuse the GFP1 to the C-terminal of TALE1 protein. However, the sample of BBa_ I715019 provided by the 2015 DNA distribution does not have a termination codon on its 3’ terminal to stop the translation. To fix this, we designed a pair of primers to add a termination codon on the 3’ terminal of BBa_ I715019 to further extend its usage.
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==Sequence and Features==
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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===Functional Parameters===
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==Functional Parameters==
 
<partinfo>BBa_K1789003 parameters</partinfo>
 
<partinfo>BBa_K1789003 parameters</partinfo>
 
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==Experimental Validation==
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After standardization, we used PCR to proved it.
 +
 +
The forward primer sequence is 5’- GAATTCGCGGCCGCTTCTAGAATGC-3’. The reverse primer sequence is 5’- GGACTAGTATTATTGTTTGTCTGCC-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.
 +
 +
 +
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===References===
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[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.

Revision as of 09:51, 18 September 2015

GFP1(with termination codon)

This part is the Amino Half of GFP with termination codon

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]

In our project, we plan to fuse the GFP1 to the C-terminal of TALE1 protein. However, the sample of BBa_ I715019 provided by the 2015 DNA distribution does not have a termination codon on its 3’ terminal to stop the translation. To fix this, we designed a pair of primers to add a termination codon on the 3’ terminal of BBa_ I715019 to further extend its usage.

Sequence and Features

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



Experimental Validation

After standardization, we used PCR to proved it.

The forward primer sequence is 5’- GAATTCGCGGCCGCTTCTAGAATGC-3’. The reverse primer sequence is 5’- GGACTAGTATTATTGTTTGTCTGCC-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.