Coding

Part:BBa_K1111016:Design

Designed by: Sandra Elisabeth Chaudron, Caroline Desmurget and Mareike Apelt   Group: iGEM13_EPF_Lausanne   (2013-10-28)
Revision as of 22:35, 28 October 2013 by Sandelisa90 (Talk | contribs) (→‎Primers)

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 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].