Part:BBa_K811005

INPNC-MCS

See BBa_K811003 for details. INPNC with 5' BamHI and 3' PstI cloning sites on the C terminus with intervening GS linker domain. This construct can be used for the surface display of large proteins by ligating your gene of interest between the BamHI and PstI cut sites. To design a forward PCR primer compatible with this biobrick, please use the following attachment as a 5' overhang: AGGCGGATCCGGT-GENE. This will include the BamHI cut site and will also ensure that your insert is in-frame with the INPNC-GSlinker so that it will properly be displayed on the surface.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 952
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 72
  Illegal NgoMIV site found at 405
  Illegal AgeI site found at 823
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

Ice nucleation protein (INP) is a protein found in Xanthomonas campestris pc. campestris BCRC 12846. It functions as, as its namesake suggests, causing ice nucleation and formation. However, recent studies have utilized INP for its surface display properties. In nature, the protein is anchored in the membrane through a glycosylphosphatidylinositol (GPI) anchor, a relatively rare occurance in prokaryotes.

The INP protein is composed of a N-terminal region that appears to interact with the phospholipid membrane, a C-terminus hydrophillic region that is exposed to the outside membrane, as well as a central 8, 16, or 48 amino acid motif that is responsible for INP's ice nucleation properties. However, this central amino acid motif is not necessary for INP's surface display properties. Therefore, scientists truncated the proteamin, retaining only the N (179 aa) and C termini (49 aa) to produce INPNC.

This truncated protein retains INP's membrane display abilities, and also contains a GS amino acid linker followed by a site containing multiple restriction sites for the easy ligation of additional DNA for INPNC fusion experiments and surface display of desired proteins.

Characterization

Please see BBa_K811003 and BBa_K811004 for full characterization.