Part:BBa_K5302021

pBBR-OmpA-miniZ

This work is derived from pBBR1MCS-OmpA-mCherry and pUC19-miniZ-Z3C, and it has undergone codon optimization. This composite part combines OmpA(21.4kda) and miniZ(3.8kda), we succeeded in transferring this plasmid into Escherichia coli Nissle 1917 and let it express miniZ. The plasmid uses lac promotor and has kanamycin resistence.

Figure 1. Colony PCR results of pBBR-OmpA-miniZ

Figure 2. SDS-PAGE analysis of pBBR1MCS-OmpA-miniZ expression in Escherichia coli Nissle 1917 （1）

Figure 3. SDS-PAGE analysis of pBBR1MCS-OmpA-miniZ expression in Escherichia coli Nissle 1917 （2）

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This part is derived from three helix 58-residue Z-domain of staphylococcal protein A. And through stabilizing mutations and the addition of a disulfide constraint the Z-domain is reengineered into a two-helix 34-residue “mini-Z” version that retains the parent's affinity. This is supposed to be more potent binders against VEGF. We used pBBR1MCS-2 plasmid as a backbone and transfered miniZ into Escherichia coli Nissle 1917, and finally succeeded in expressing miniZ.

Figure 4. sequence of miniZ and its KD with VEGF

Figure 5. Cartoon representation of the crystal structure of the mini-Z highlighting randomized residues shown as sticks and coloring the helices

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 4025
  Illegal XbaI site found at 3140
  Illegal PstI site found at 1967
  Illegal PstI site found at 3128
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 4025
  Illegal PstI site found at 1967
  Illegal PstI site found at 3128
  Illegal NotI site found at 1008
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 4025
  Illegal BglII site found at 1754
  Illegal BamHI site found at 3146
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 4025
  Illegal XbaI site found at 3140
  Illegal PstI site found at 1967
  Illegal PstI site found at 3128
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 4025
  Illegal XbaI site found at 3140
  Illegal PstI site found at 1967
  Illegal PstI site found at 3128
  Illegal NgoMIV site found at 2418
  Illegal NgoMIV site found at 2701
  Illegal NgoMIV site found at 4683
  Illegal AgeI site found at 4523
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1330
  Illegal SapI.rc site found at 2267
  Illegal SapI.rc site found at 2477