Part:BBa_K1111012

Ice Nucleation Protein fused to Streptavidin Alive

Introduction

What it is : INP sequence fused with Streptavidin Alive by gibson assembly. The INP-streptavidin construct expresses streptavidin at the outer membrane of E.Coli. INP is an outer membrane protein foud in the genome of Pseudomonas syringae. It is under the control of Lac promoter and thus is constitutively expressed in E.Coli. The expression of this plasmid was tested by transforming cells and looking for microscopy at either of a fluorescent antibody again streptavidin or a fluorescent biotin (biotin and streptavidin have a strong affinity, their Kd is 10-15 M).

Usage and Biology

For us, the idea was to attach nanoparticles covered with biotin, using the strong affinity between both, but any experience implying biotin can be considered. Nanocapsules have a size comprised in a range from , which is quite big compared to a typical E:Coli cell size, we can therefore imagine attaching large proteins to these bacteria.

Acknowledgements

Thanks to the Mark Howarth Biochemistry department lab in Oxford for providing us the plasmids needed.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 1727
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1649
  
• 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]