Part:BBa_K5302014

pBBR-intimin-mCherry

This work is derived from pBBR1MCS-2, pUC19-intimin-spytag and pET-21b(+)-mCherry, and it has undergone codon optimization. This composite part combines intimin-spytag(approximately 49.5kda) 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. intimin is an important outer membrane protein that includes several different domains: a lysozyme domain located in the periplasm, a β barrel domain embedded in the membrane, and an extracellular region at the C terminal or a "passenger" domain. This "passenger" domain is linked to the β barrel domain by a short linker sequence. intimin is classified as a reverse autotransporter, a subclass of the type 5 secretion system that transports the extracellular region or "passenger" of the C terminal through a compartment of the N terminal β barrel located in the outer membrane. The "passenger" domain of intimin forms a rod-like extension, consisting of four tandem bacterial immunoglobulin-like domains (D00-D0-D1-D2) with a C-terminal C-like domain (D3). The extension and rigidity of the D00-D0 domain of Intimin increases the possible radius of interaction with the protein, which improves intimin presentation after secretion to the bacterial surface.

Figure 1. structure of intimin, from genbank

Figure 2. Colony PCR results of pBBR1MCS-intimin-mCherry

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

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  INCOMPATIBLE WITH RFC[12]

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• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 4483
  Illegal EcoRI site found at 4531
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  Illegal PstI site found at 2016
  Illegal PstI site found at 3494
  Illegal PstI site found at 3742
  Illegal PstI site found at 4189
  Illegal PstI site found at 4792
  Illegal NgoMIV site found at 2467
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• 1000
  INCOMPATIBLE WITH RFC[1000]

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