Part:BBa_K5302011

pBBR-OmpA-mCherry

This work is derived from pBBR1MCS-2, pUC19-OmpA and pET-21b(+)-mCherry, and it has undergone codon optimization. This composite part combines OmpA(21.4kda) 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. OmpA (outer membrane protein A) is a major protein in the outer membrane of Gram-negative bacteria. OmpA is a transmembrane protein with multiple functions, including the maintenance of outer membrane integrity and the involvement in bacterial-host interactions. The OmpA protein consists of three domains: an eight-stranded β -barrel N-terminal domain, a flexible linker connecting the two domains, and a spherical C-terminal domain located in the periplasmic space. The N-terminal domain of OmpA is embedded in the outer membrane, while the C-terminal domain interacts with the peptidoglycan layer of the cell wall to help fix the outer membrane in place.

Figure 1. Colony PCR results of pBBR1MCS-OmpA-mCherry

Figure 2. structure of OmpA, from genbank

Figure 3. SDS-PAGE analysis of pBBR1MCS-OmpA-mCherry expression in Escherichia coli Nissle 1917

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 3884
  Illegal EcoRI site found at 3932
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  Illegal PstI site found at 4193
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 3884
  Illegal EcoRI site found at 3932
  Illegal EcoRI site found at 4205
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  Illegal PstI site found at 2016
  Illegal PstI site found at 3177
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• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 3884
  Illegal EcoRI site found at 3932
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• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 3884
  Illegal EcoRI site found at 3932
  Illegal EcoRI site found at 4205
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  Illegal XbaI site found at 3189
  Illegal PstI site found at 2016
  Illegal PstI site found at 3177
  Illegal PstI site found at 4193
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 3884
  Illegal EcoRI site found at 3932
  Illegal EcoRI site found at 4205
  Illegal EcoRI site found at 4608
  Illegal XbaI site found at 3189
  Illegal PstI site found at 2016
  Illegal PstI site found at 3177
  Illegal PstI site found at 4193
  Illegal NgoMIV site found at 2467
  Illegal NgoMIV site found at 2750
  Illegal NgoMIV site found at 5266
  Illegal AgeI site found at 4411
  Illegal AgeI site found at 5106
  
• 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