Difference between revisions of "Part:BBa K1111014:Design"
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<partinfo>BBa_K1111014 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1111014 SequenceAndFeatures</partinfo>
  
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==Design Notes==
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==Gibson Assembly Design ==
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To assemble this part, we asked for the Biobrick BBa_K283010 from iGEM 2009 group HKU-HKBU ([https://parts.igem.org/Part:BBa_K283010 Biobrick page]).  
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''Insert:'' we amplified the streptavidin coding from the Biobrick BBa_K283010 designed by iGEM 2009 group HKU-HKBU.
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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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<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.
  
 
==Primers==
 
==Primers==
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PCR of streptavidin alive :
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Streptavidin iGEM PCR :
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5' ATGGCTGAAGCTGGTATCACC 3'
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<br>5' <font color = #0000FF>ATGGCTGAAGCTGGTATCACC</font> 3'
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5' TTAGGAAGCAGCGGACGGTTTAAC 3'
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<br>5' <font color = #0000FF>TTAGGAAGCAGCGGACGGTTTAAC</font> 3'
  
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Overlapping PCR of BBa_K523013 :  
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BBa_K523013 PCR :  
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5' accaaagttaaaccgtccgctgcttcctaacatatcataacggagtgatcgcaatg 3'
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<br>Fw: 5' <font color = #0000FF>ACCAAAGTTAAACCGTCCGCTGCTTCT</font><font color= red>AACATATCATAACGGAGTGATCGCAATG</font> 3'
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5' ccaggtgccggtgataccagcttcagcCATagatcccgccacgctgct 3'
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<br>Rev: 5' <font color = #0000FF>CCAGGTGCCGGTGATACCAGCTTCAGCCAT</font><font color= red>AGATCCCGCCACGCTGCT</font> 3'
  
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==References and Acknowledgements==
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==Source==
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Thanks to the iGEM group that designed this Biobrick.
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Can be expressed in Escherichia Coli.
  
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==References and acknowledgements==
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===Source===
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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].
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<br>Thanks to the '''iGEM 2009 group HKU-HKBU''' that designed the Biobrick BBa_K283010 [https://parts.igem.org/Part:BBa_K283010].
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Can be expressed in Escherichia Coli
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===References===
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Latest revision as of 21:42, 2 October 2013

Ice Nucleation Protein fused to Streptavidin BBa_K283010


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1649
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    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 from the Biobrick BBa_K283010 designed by iGEM 2009 group HKU-HKBU.
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 iGEM 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 iGEM team of Edinburgh 2011 that designed the Biobrick BBa_K523013 [1].
Thanks to the iGEM 2009 group HKU-HKBU that designed the Biobrick BBa_K283010 [2].