Part:BBa_K5302029

pBBR-INP-miniZ

This work is derived from pBBR1MCS-INP-mCherry and pUC19-miniZ-Z3C, and it has undergone codon optimization. This composite part combines INP(about 30kda) 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-INP-miniZ

Figure 2. SDS-PAGE analysis of pBBR1MCS-INP-miniZ expression in Escherichia coli Nissle 1917

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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 3. sequence of miniZ and its KD with VEGF

Figure 4. 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 4383
  Illegal XbaI site found at 3189
  Illegal PstI site found at 2016
  Illegal PstI site found at 3177
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 4383
  Illegal PstI site found at 2016
  Illegal PstI site found at 3177
  Illegal NotI site found at 1057
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 4383
  Illegal BglII site found at 1803
  Illegal BamHI site found at 3195
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 4383
  Illegal XbaI site found at 3189
  Illegal PstI site found at 2016
  Illegal PstI site found at 3177
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 4383
  Illegal XbaI site found at 3189
  Illegal PstI site found at 2016
  Illegal PstI site found at 3177
  Illegal NgoMIV site found at 2467
  Illegal NgoMIV site found at 2750
  Illegal NgoMIV site found at 3616
  Illegal NgoMIV site found at 5048
  Illegal AgeI site found at 4888
  
• 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