Coding

Part:BBa_K1111015:Design

Designed by: Sandra Elisabeth Chaudron, Caroline Desmuget and Mareike Apelt   Group: iGEM13_EPF_Lausanne   (2013-10-28)

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].