Difference between revisions of "Part:BBa K1111013:Design"

 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1111013 short</partinfo>
 
<partinfo>BBa_K1111013 short</partinfo>
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===Design Notes===
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==Gibson Assembly Design ==
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Gibson Assembly
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''Insert:'' we amplified the streptavidin coding sequence received in a plasmid from Mark Howarth laboratory at Oxford university Dept. of Biochemistry.
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<br>''Backbone:'' Starting from the biobrick BBa_K523013 (INP fused with YFP), we amplified the whole sequence except the EYFP and added gibson overhang complementary to streptavidin ends.
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<!--<br>''Note:'' The streptavidin used is designed with three point mutations (N23A,S27D,S45A) to decrease the biotin binding affinity.-->
  
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==Primers==
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Streptavidin Dead PCR :
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<br>5' <font color = #0000FF>ATGGCTGAAGCTGGTATCACC</font> 3'
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<br>5' <font color = #0000FF>TTAGGAAGCAGCGGACGGTTTAAC</font> 3'
  
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BBa_K523013 PCR :
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<br>Fw: 5' <font color = #0000FF>ACCAAAGTTAAACCGTCCGCTGCTTCT</font><font color= red>AACATATCATAACGGAGTGATCGCAATG</font> 3'
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<br>Rev: 5' <font color = #0000FF>CCAGGTGCCGGTGATACCAGCTTCAGCCAT</font><font color= red>AGATCCCGCCACGCTGCT</font> 3'
  
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===Source===
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==Source==
  
 
Can be expressed in Escherichia Coli
 
Can be expressed in Escherichia Coli
  
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===References===
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==References and acknowledgements==
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Thanks to the '''Mark Howarth laboratory at Oxford university Dept. of Biochemistry''' [http://users.ox.ac.uk/~bioc0756/MyWebs/activesite/ReagentDistribution.htm] for providing us with the streptavidin cloning plasmid.
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<br>Thanks to the '''iGEM team of Edinburgh 2011''' that designed the Biobrick BBa_K523013 [https://parts.igem.org/Part:BBa_K523013].

Latest revision as of 21:41, 2 October 2013

Ice Nucleation Protein fused to Streptavidin Dead


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 1727
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 1727
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 1727
    Illegal NgoMIV site found at 1036
    Illegal AgeI site found at 1691
    Illegal AgeI site found at 1742
  • 1000
    COMPATIBLE WITH RFC[1000]


Gibson Assembly Design

Insert: we amplified the streptavidin coding sequence received in a plasmid from Mark Howarth laboratory at Oxford university Dept. of Biochemistry.
Backbone: Starting from the biobrick BBa_K523013 (INP fused with YFP), we amplified the whole sequence except the EYFP and added gibson overhang complementary to streptavidin ends.


Primers

Streptavidin Dead PCR :
5' ATGGCTGAAGCTGGTATCACC 3'
5' TTAGGAAGCAGCGGACGGTTTAAC 3'

BBa_K523013 PCR :
Fw: 5' ACCAAAGTTAAACCGTCCGCTGCTTCTAACATATCATAACGGAGTGATCGCAATG 3'
Rev: 5' CCAGGTGCCGGTGATACCAGCTTCAGCCATAGATCCCGCCACGCTGCT 3'

Source

Can be expressed in Escherichia Coli

References and acknowledgements

Thanks to the Mark Howarth laboratory at Oxford university Dept. of Biochemistry [http://users.ox.ac.uk/~bioc0756/MyWebs/activesite/ReagentDistribution.htm] for providing us with the streptavidin cloning plasmid.
Thanks to the iGEM team of Edinburgh 2011 that designed the Biobrick BBa_K523013 [1].