Difference between revisions of "Part:BBa K1111015:Design"

 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1111015 short</partinfo>
 
<partinfo>BBa_K1111015 short</partinfo>
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===Design Notes===
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==Gibson Assembly Design ==
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Obtained by gibson assembly using PCR products from BBa_K283010 and BBa_K523013 Biobricks.
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''Insert:'' we amplified the entire streptavidin sequence from the Biobrick BBa_K283010 designed by iGEM 2009 group HKU-HKBU.
 +
<br>
 +
<br>''Backbone:'' Starting from the biobrick BBa_K523013 (INP fused with YFP), we first amplified the sequence adding a linker after EYFP for the EYFP-strep futur junction.
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<br> Second, we perfomed a PCR on this linearized backbone to add gibson ovehangs complementary to the insert ends.
 +
 
 +
[[File:Team-EPF-Lausanne_history_IYS.pdf]]
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==Primers==
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<br><font color = #00FF00>Binds to Insert </font>
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<br><font color= #FF00FF>Binds to Backbone</font>
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<br><font color = #0000FF>Linker</font>
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<br>
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Streptavidin BBa_K283010 PCR :
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<br>5' <font color = #00FF00>ATGGCTGAAGCTGGTATCACC </font> 3'
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<br>5' <font color = #00FF00>TTAGGAAGCAGCGGACGGTTTAAC</font> 3'
  
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BBa_K523013 first PCR to add linker :
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<br>Fw: 5' <font color = #0000FF>GCTACCGCTGCCGCTACC</font><font color= #FF00FF>CTTGTACAGCTCGTCCATGCC</font> 3'
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<br>Rev: 5' <font color= #FF00FF>TAATACTAGCAACATATCATAACGGAGTGATCG</font> 3'
  
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BBa_K523013 second PCR to add overhangs :
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<br>Fw: 5' <font color = #00FF00>CCAGGTGCCGGTGATACCAGCTTCAGCCAT</font><font color = #0000FF>GCTACCGCTGCCGCTACC</font><font color= #FF00FF>CTT</font> 3'
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<br>Rev: 5' <font color = #00FF00>ACCAAAGTTAAACCGTCCGCTGCTTCCTAA</font><font color= #FF00FF>TAATACTAGCAACATATCATAACGGAGTGATCGC</font> 3'
  
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===Source===
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==Source==
  
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To be used in E. Coli
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Can be expressed in Escherichia Coli.
  
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===References===
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==References and acknowledgements==
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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].
 +
<br>Thanks to the '''iGEM 2009 group HKU-HKBU''' that designed the Biobrick BBa_K283010 [https://parts.igem.org/Part:BBa_K283010].

Latest revision as of 23:02, 28 October 2013

INP_EYFP_Streptavidin Fusion Protein


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 2384
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1036
    Illegal AgeI site found at 2426
    Illegal AgeI site found at 2477
  • 1000
    COMPATIBLE WITH RFC[1000]


Gibson Assembly Design

Insert: we amplified the entire streptavidin sequence from the Biobrick BBa_K283010 designed by iGEM 2009 group HKU-HKBU.

Backbone: Starting from the biobrick BBa_K523013 (INP fused with YFP), we first amplified the sequence adding a linker after EYFP for the EYFP-strep futur junction.
Second, we perfomed a PCR on this linearized backbone to add gibson ovehangs complementary to the insert ends.

File:Team-EPF-Lausanne history IYS.pdf

Primers


Binds to Insert
Binds to Backbone
Linker

Streptavidin BBa_K283010 PCR :
5' ATGGCTGAAGCTGGTATCACC 3'
5' TTAGGAAGCAGCGGACGGTTTAAC 3'

BBa_K523013 first PCR to add linker :
Fw: 5' GCTACCGCTGCCGCTACCCTTGTACAGCTCGTCCATGCC 3'
Rev: 5' TAATACTAGCAACATATCATAACGGAGTGATCG 3'

BBa_K523013 second PCR to add overhangs :
Fw: 5' CCAGGTGCCGGTGATACCAGCTTCAGCCATGCTACCGCTGCCGCTACCCTT 3'
Rev: 5' ACCAAAGTTAAACCGTCCGCTGCTTCCTAATAATACTAGCAACATATCATAACGGAGTGATCGC 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].