Difference between revisions of "Part:BBa K1896018"
 
Line 12: Line 12:
 
since it is less likely to cause aggregation.[2]
 
since it is less likely to cause aggregation.[2]
  
−
+++ insert movie +++
+
<html>
 +
<center>
 +
<iframe width="560" height="315" src="https://www.youtube.com/embed/ckfOn9_UYuM" frameborder="0" allowfullscreen></iframe><br><p>When crude cell lysate of a culture of <i>E. coli</i> expressing mGFPuv2 is added to supercooled water, no reaction takes place. When crude cell lysate containing INP_RC-mSA2 is added, ice is formed instantly. This demonstrates that the cytoplasmatic INP_RC fusion protein is still active as an ice nucleating protein.</p>
 +
</center>
 +
</html>
  
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 20:30, 19 October 2016


INP_RC-mSA2 generator

This part contains the INPRC-mSA2 fusion protein controlled by a weak to medium constitutive promoter and RBS.

Usage and Biology

INPRC is a truncated Ice Nucleating Protein from which the N-terminal domain has been removed. The truncated INP is still capable of catalysing the formation of ice crystals, but is no longer localised to the outer cell membrane.[1] The mSA2 protein has been fused to INPRC for use in protein adhesion experiments performed by the UGent Belgium 2016 iGEM team. MSA2 is a monomeric variant of streptavidin that retains the biotin-binding capabilities and is better suited for protein fusions since it is less likely to cause aggregation.[2]


When crude cell lysate of a culture of E. coli expressing mGFPuv2 is added to supercooled water, no reaction takes place. When crude cell lysate containing INP_RC-mSA2 is added, ice is formed instantly. This demonstrates that the cytoplasmatic INP_RC fusion protein is still active as an ice nucleating protein.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 203
    Illegal NgoMIV site found at 1715
    Illegal NgoMIV site found at 1859
    Illegal NgoMIV site found at 2123
    Illegal NgoMIV site found at 2267
    Illegal NgoMIV site found at 2315
    Illegal NgoMIV site found at 2435
    Illegal AgeI site found at 1379
    Illegal AgeI site found at 3254
    Illegal AgeI site found at 3314
  • 1000
    COMPATIBLE WITH RFC[1000]


References

  1. Green, R. L., Corotto, L. V., & Warren, G. J. (1988). Deletion mutagenesis of the ice nucleation gene from Pseudomonas syringae S203. Molecular and General Genetics MGG, 215(1), 165-172.
  2. Mann, J. K., Demonte, D., Dundas, C. M., & Park, S. (2016). Cell labeling and proximity dependent biotinylation with engineered monomeric streptavidin. Technology, 1-7.