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

Revision as of 22:55, 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 IYS.jpg

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].

@@ Line 11: / Line 11: @@
 <br> Second, we perfomed a PCR on this linearized backbone to add gibson ovehangs complementary to the insert ends.
-<br>[[Image:Team-EPF-Lausanne_history_IYS.pdf]]
+<br>[[Image:Team-EPF-Lausanne_history_IYS.jpg]]
 ==Primers==