Part:BBa_K5302012

pBBR-INP-mCherry

This work is derived from pBBR1MCS-2, pUC19-INP and pET-21b(+), and it has undergone codon optimization. This composite part combines INP(30kda) and mCherry(26.7kda), and we succeeded in transferring this plasmid into Escherichia coli Nissle 1917 and let it express mCherry as a fluorescence labeling.The plasmid uses lac promotor and has kanamycin resistence. Ice nucleolin (Ice Nucleation Protein, INP) is a protein that catalyzes the formation of ice crystals by water molecules at relatively high temperatures (about-2 to-4 C). INP usually consists of three domains: an N-terminal domain, a C-terminal domain, and a repeat unit domain in the middle. Among them, the N-terminal domain is responsible for the interaction with the cell membrane, while the C-terminal domain is involved in protein secretion and stability. The INP interacts with water molecules through specific amino acid sequences of its domains to promote the formation of ice crystals. And the N-terminal domain of INP can serve as anchor motifs for the display of foreign proteins on the surface of bacteria.

Figure 1. INP construct

Figure 2. INP, InaK (N-domain+C-domain). In both panels (a and b), each protein domain is displayed with diferent colours. SP signal peptide, LP lipoprotein. OM outer membrane

Figure 3. Colony PCR results of pBBR1MCS-INP-mCherry

Figure 4. SDS-PAGE analysis of pBBR1MCS-INP-mCherry expression in Escherichia coli Nissle 1917

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 4229
  Illegal EcoRI site found at 4277
  Illegal EcoRI site found at 4550
  Illegal EcoRI site found at 4917
  Illegal XbaI site found at 3189
  Illegal PstI site found at 2016
  Illegal PstI site found at 3177
  Illegal PstI site found at 4538
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 4229
  Illegal EcoRI site found at 4277
  Illegal EcoRI site found at 4550
  Illegal EcoRI site found at 4917
  Illegal PstI site found at 2016
  Illegal PstI site found at 3177
  Illegal PstI site found at 4538
  Illegal NotI site found at 1057
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 4229
  Illegal EcoRI site found at 4277
  Illegal EcoRI site found at 4550
  Illegal EcoRI site found at 4917
  Illegal BglII site found at 1803
  Illegal BamHI site found at 3195
  Illegal XhoI site found at 4895
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 4229
  Illegal EcoRI site found at 4277
  Illegal EcoRI site found at 4550
  Illegal EcoRI site found at 4917
  Illegal XbaI site found at 3189
  Illegal PstI site found at 2016
  Illegal PstI site found at 3177
  Illegal PstI site found at 4538
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 4229
  Illegal EcoRI site found at 4277
  Illegal EcoRI site found at 4550
  Illegal EcoRI site found at 4917
  Illegal XbaI site found at 3189
  Illegal PstI site found at 2016
  Illegal PstI site found at 3177
  Illegal PstI site found at 4538
  Illegal NgoMIV site found at 2467
  Illegal NgoMIV site found at 2750
  Illegal NgoMIV site found at 3616
  Illegal NgoMIV site found at 5582
  Illegal AgeI site found at 4756
  Illegal AgeI site found at 5422
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1379
  Illegal SapI.rc site found at 2316
  Illegal SapI.rc site found at 2526