Difference between revisions of "Part:BBa K1111013:Design"

(→‎References and acknowledgements)
(→‎Primers)
Line 10: Line 10:
  
 
==Primers==
 
==Primers==
−
PCR of streptavidin dead :
+
Streptavidin Alive PCR :
−
5' ATGGCTGAAGCTGGTATCACC 3'
+
<br>5' <font color = #0000FF>ATGGCTGAAGCTGGTATCACC</font> 3'
−
5' TTAGGAAGCAGCGGACGGTTTAAC 3'
+
<br>5' <font color = #0000FF>TTAGGAAGCAGCGGACGGTTTAAC</font> 3'
  
−
Overlapping PCR of BBa_K523013 :  
+
BBa_K523013 PCR :  
−
5' accaaagttaaaccgtccgctgcttcctaacatatcataacggagtgatcgcaatg 3'
+
<br>Fw: 5' <font color = #0000FF>ACCAAAGTTAAACCGTCCGCTGCTTCT</font><font color= red>AACATATCATAACGGAGTGATCGCAATG</font> 3'
−
5' ccaggtgccggtgataccagcttcagcCATagatcccgccacgctgct 3'
+
<br>Rev: 5' <font color = #0000FF>CCAGGTGCCGGTGATACCAGCTTCAGCCAT</font><font color= red>AGATCCCGCCACGCTGCT</font> 3'
  
  

Revision as of 21:02, 2 October 2013

Ice Nucleation Protein fused to Streptavidin Dead


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 1727
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 1727
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 1727
    Illegal NgoMIV site found at 1036
    Illegal AgeI site found at 1691
    Illegal AgeI site found at 1742
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

To assemble this part, we orderd a plasmid containing the streptavidin dead. Three point mutations have been made, N23A,S27D,S45A, in order to decrease the biotin-streptavidin Kd. This streptavidin is monovalent. We amplified only the coding sequence of streptavidin of the plasmid by PCR. Also, we did a PCR with overlapping ends corresponding to streptavidin on the Biobrick BBa_K523013 (INP fused with YFP). This PCR was keeping all the plasmid including RBS and Lac promoter but excluding YFP. The linearized plasmid and the insert were then assembled by Gibson assembly.

Primers

Streptavidin Alive PCR :
5' ATGGCTGAAGCTGGTATCACC 3'
5' TTAGGAAGCAGCGGACGGTTTAAC 3'

BBa_K523013 PCR :
Fw: 5' ACCAAAGTTAAACCGTCCGCTGCTTCTAACATATCATAACGGAGTGATCGCAATG 3'
Rev: 5' CCAGGTGCCGGTGATACCAGCTTCAGCCATAGATCCCGCCACGCTGCT 3'


Source

Can be expressed in Escherichia Coli

References and acknowledgements

Thanks to the Mark Howarth laboratory at Oxford university Dept. of Biochemistry (link) for providing us with the streptavidin cloning plasmid. Thanks also to the iGEM team of Edinburgh 2011 that designed the Biobrick BBa_K523013.