Part:BBa_K5302025

pBBR-INP-8IIU

This work is derived from pUC19-8IIU and pBBR1MCS-INP-mCherry, and it has undergone codon optimization. This composite part combines INP(30kda) and 8IIU(13.789kda), we succeeded in transferring this plasmid into Escherichia coli Nissle 1917 and let it express 8IIU. The plasmid uses lac promotor and has kanamycin resistence.

Figure 1. Colony PCR results of pBBR-INP-8IIU （1）

Figure 2. Colony PCR results of pBBR-INP-8IIU （2）

Figure 3. Colony PCR results of pBBR-INP-8IIU （3）

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8IIU is an anti-VEGF nanobody, and it has 2 chains, chainA sequence: mdvqlvesgg glvqpggslr lscaasgrtf ssysmgwfrq apgkerefvv aiskggykyd avslegrfti srdnakntvy lqinslrped tavyycassr aygssrlrla dtyeywgqgt lvtvss. chainB sequence:mdvqlvesgg glvqpggslr lscaasgrtf ssysmgwfrq apgkerefvv aiskggyky avslegrfti srdnakntvy lqinslrped tavyycassr aygssrlrla dtyeywgqgt lvtvss. These two chains all show great affinity with VEGF, we used pBBR plasmid as a backbone and transfered 8IIU into Escherichia coli Nissle 1917, and finally succeeded in expressing 8IIU.

Figure 4. 8IIU chainA features, from genbank

Figure 5. 8IIU chainB features, from genbank

Figure 6. 8IIU structure, from genbank

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 4325
  Illegal EcoRI site found at 4659
  Illegal XbaI site found at 3189
  Illegal PstI site found at 2016
  Illegal PstI site found at 3177
  Illegal PstI site found at 4427
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 4325
  Illegal EcoRI site found at 4659
  Illegal PstI site found at 2016
  Illegal PstI site found at 3177
  Illegal PstI site found at 4427
  Illegal NotI site found at 1057
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 4325
  Illegal EcoRI site found at 4659
  Illegal BglII site found at 1803
  Illegal BamHI site found at 3195
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 4325
  Illegal EcoRI site found at 4659
  Illegal XbaI site found at 3189
  Illegal PstI site found at 2016
  Illegal PstI site found at 3177
  Illegal PstI site found at 4427
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 4325
  Illegal EcoRI site found at 4659
  Illegal XbaI site found at 3189
  Illegal PstI site found at 2016
  Illegal PstI site found at 3177
  Illegal PstI site found at 4427
  Illegal NgoMIV site found at 2467
  Illegal NgoMIV site found at 2750
  Illegal NgoMIV site found at 3616
  Illegal NgoMIV site found at 5324
  Illegal AgeI site found at 5164
  
• 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