Difference between revisions of "Part:BBa K1111016:Design"
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==Gibson Assembly Design ==
 
==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.
 
''Insert:'' we amplified the streptavidin sequence from the Biobrick BBa_K283010 designed by iGEM 2009 group HKU-HKBU, thus removing the stop codon.
 +
<br>
 
<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>''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.
 
<br> Second, we perfomed a PCR on this linearized backbone to add gibson ovehangs complementary to the insert ends.
 +
 +
[[File:Team-EPF-Lausanne_history_ISY.pdf]]
  
 
==Primers==
 
==Primers==
−
<font color = #00FF00>Binds to Insert </font>
+
<br><font color = #00FF00>Binds to Insert </font>
−
<font color= #FF00FF>Binds to Backbone</font>
+
<br><font color= #FF00FF>Binds to Backbone</font>
−
<font color = #00FFFF>Linker</font>
+
<br><font color = #0000FF>Linker</font>
 
<br>
 
<br>
  
−
Streptavidin iGEM PCR :
+
Streptavidin BBa_K283010 PCR :
 
<br>5' <font color = #00FF00>ATGGCTGAAGCTGGTATCACCGG </font> 3'
 
<br>5' <font color = #00FF00>ATGGCTGAAGCTGGTATCACCGG </font> 3'
 
<br>5' <font color = #00FF00>GGAAGCAGCGGACGGTTTAACTTTG</font> 3'
 
<br>5' <font color = #00FF00>GGAAGCAGCGGACGGTTTAACTTTG</font> 3'
  
 
BBa_K523013 first PCR to add linker :  
 
BBa_K523013 first PCR to add linker :  
−
<br>Fw: 5' <font color = #00FFFF>GCTACCGCTGCCGCTACC</font><font color= #FF00FF>TTTCACTTCGATCCAATCATCATCTTC</font> 3'
+
<br>Fw: 5' <font color = #0000FF>GCTACCGCTGCCGCTACC</font><font color= #FF00FF>TTTCACTTCGATCCAATCATCATCTTC</font> 3'
 
<br>Rev: 5' <font color= #FF00FF>GGTACTAGCAGCAGCATTGCGAGC</font> 3'
 
<br>Rev: 5' <font color= #FF00FF>GGTACTAGCAGCAGCATTGCGAGC</font> 3'
  
 
BBa_K523013 second PCR to add overhangs :  
 
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>Fw: 5' <font color = #00FF00>CCAGGTGCCGGTGATACCAGCTTCAGCCAT</font><font color = #0000FF>GCTACCGCTGCCGCTAC</font><font color= #FF00FF>CTTT</font> 3'
 
<br>Rev: 5' <font color = #00FF00>TTCACCAAAGTTAAACCGTCCGCTGCTTCC</font><font color= #FF00FF>GGTACTAGCAGCAGCATTGCGAGC</font> 3'
 
<br>Rev: 5' <font color = #00FF00>TTCACCAAAGTTAAACCGTCCGCTGCTTCC</font><font color= #FF00FF>GGTACTAGCAGCAGCATTGCGAGC</font> 3'
−
 
  
 
==Source==
 
==Source==

Latest revision as of 22:59, 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.

File:Team-EPF-Lausanne history ISY.pdf

Primers


Binds to Insert
Binds to Backbone
Linker

Streptavidin BBa_K283010 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].