Difference between revisions of "Part:BBa K1111012:Design"

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<partinfo>BBa_K1111012 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1111012 SequenceAndFeatures</partinfo>
  
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==Design Notes==
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==Gibson Assembly Design ==
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To assemble this part, we orderd a plasmid containing the streptavidin alive.
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Insert: we amplified the streptavidin coding sequence received in a plasmid from .... lab.
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We amplified only the coding sequence of streptavidin of the plasmid by PCR. Also, we did a PCR with overlapping ends corresponding to streptavidin on the '''Biobrick BBa_K523013''' (INP fused with YFP). This PCR was keeping all the plasmid including RBS and Lac promoter but excluding YFP. The linearized plasmid and the insert were then assembled by Gibson assembly.
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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==
 
==Primers==

Revision as of 18:52, 2 October 2013

Ice Nucleation Protein fused to Streptavidin Alive


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 1727
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1649
  • 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 .... lab. 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

PCR of streptavidin alive : 5' ATGGCTGAAGCTGGTATCACC 3' 5' TTAGGAAGCAGCGGACGGTTTAAC 3'

Overlapping PCR of BBa_K523013 : 5' accaaagttaaaccgtccgctgcttcctaacatatcataacggagtgatcgcaatg 3' 5' ccaggtgccggtgataccagcttcagcCATagatcccgccacgctgct 3'

References and acknowledgements

Thanks to the Mark Howarth laboratory at Oxford university Dept. of Biochemistry (link) for providing us with the streptavidin cloning plasmid. Thanks also to the iGEM team of Edinburgh 2011 that designed the Biobrick BBa_K523013.