Part:BBa_K5302028

pBBR-INP-Z3C

This work is derived from pBBR1MCS-INP-mCherry, pUC19-miniZ-Z3C, and it has undergone codon optimization. This composite part combines INP(about 30kda) and Z3C(7kda), we succeeded in transferring this plasmid into Escherichia coli Nissle 1917 and let it express Z3C. The plasmid uses lac promotor and has kanamycin resistence.

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Z3C is derived from three helix 58-residue Z-domain of staphylococcal protein A, but it has three helix. Z3C also shows great affinity with VEGF(KD=41 nM). We used pBBR plasmid as a backbone and transfered Z3C into Escherichia coli Nissle 1917, and finally succeeded in expressing Z3C.

Figure 1. sequence of Z3C and its KD with VEGF

Figure 2. 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 4458
  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 4458
  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 4458
  Illegal BglII site found at 1803
  Illegal BamHI site found at 3195
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 4458
  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 4458
  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 5123
  Illegal AgeI site found at 4963
  
• 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