Difference between revisions of "Part:BBa K1111016:Design"

 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1111016 short</partinfo>
 
<partinfo>BBa_K1111016 short</partinfo>
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===Design Notes===
+
==Gibson Assembly Design ==
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Obtained by gibson assembly using PCR products from BBa_K283010 and BBa_K523013 Biobricks.
+
''Insert:'' we amplified the streptavidin sequence from the Biobrick BBa_K283010 designed by iGEM 2009 group HKU-HKBU, thus removing the stop codon.
 +
<br>''Backbone:'' Starting from the biobrick BBa_K523013 (INP fused with YFP), we first amplified the sequence adding a linker after INP for the INP-strep futur junction.
 +
<br> Second, we perfomed a PCR on this linearized backbone to add gibson ovehangs complementary to the insert ends.
 +
 
 +
==Primers==
 +
<font color = #00FF00>Binds to Insert </font>
 +
<font color= #FF00FF>Binds to Backbone</font>
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<font color = #00FFFF>Linker</font>
 +
<br>
 +
 
 +
Streptavidin iGEM PCR :
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<br>5' <font color = #00FF00>ATGGCTGAAGCTGGTATCACCGG </font> 3'
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<br>5' <font color = #00FF00>GGAAGCAGCGGACGGTTTAACTTTG</font> 3'
 +
 
 +
BBa_K523013 first PCR to add linker :
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<br>Fw: 5' <font color = #00FFFF>GCTACCGCTGCCGCTACC</font><font color= #FF00FF>TTTCACTTCGATCCAATCATCATCTTC</font> 3'
 +
<br>Rev: 5' <font color= #FF00FF>GGTACTAGCAGCAGCATTGCGAGC</font> 3'
  
 +
BBa_K523013 second PCR to add overhangs :
 +
<br>Fw: 5' <font color = #00FF00>CCAGGTGCCGGTGATACCAGCTTCAGCCAT</font><font color = #00FFFF>GCTACCGCTGCCGCTAC</font><font color= #FF00FF>CTTT</font> 3'
 +
<br>Rev: 5' <font color = #00FF00>TTCACCAAAGTTAAACCGTCCGCTGCTTCC</font><font color= #FF00FF>GGTACTAGCAGCAGCATTGCGAGC</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===
+
==References and acknowledgements==
 +
<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].

Revision as of 22:24, 28 October 2013

INP_Streptavidin_EYFP Fusion Protein


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1616
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1036
    Illegal AgeI site found at 1658
    Illegal AgeI site found at 1709
  • 1000
    COMPATIBLE WITH RFC[1000]


Gibson Assembly Design

Insert: we amplified the streptavidin sequence from the Biobrick BBa_K283010 designed by iGEM 2009 group HKU-HKBU, thus removing the stop codon.
Backbone: Starting from the biobrick BBa_K523013 (INP fused with YFP), we first amplified the sequence adding a linker after INP for the INP-strep futur junction.
Second, we perfomed a PCR on this linearized backbone to add gibson ovehangs complementary to the insert ends.

Primers

Binds to Insert Binds to Backbone Linker

Streptavidin iGEM PCR :
5' ATGGCTGAAGCTGGTATCACCGG 3'
5' GGAAGCAGCGGACGGTTTAACTTTG 3'

BBa_K523013 first PCR to add linker :
Fw: 5' GCTACCGCTGCCGCTACCTTTCACTTCGATCCAATCATCATCTTC 3'
Rev: 5' GGTACTAGCAGCAGCATTGCGAGC 3'

BBa_K523013 second PCR to add overhangs :
Fw: 5' CCAGGTGCCGGTGATACCAGCTTCAGCCATGCTACCGCTGCCGCTACCTTT 3'
Rev: 5' TTCACCAAAGTTAAACCGTCCGCTGCTTCCGGTACTAGCAGCAGCATTGCGAGC 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].