Part:BBa_K1694014

OmpA-scFv(Anti-EGFR)

Introduction:

Fig.1 Ompa-N-scfv(Anti-EGFR)

To display the antibody outside the E. coli, we used Lipoprotein-Outer membrane protein A (Lpp-OmpA). According to the paper reference [1], We chose the first 9 amino acid of Lpp, and the 46~159 amino acid of OmpA.
In order to change the scFv parts easily, we added a NcoI restriction site between OmpA and scFv so that we can change various scFv DNA sequence using the NcoI restriction enzyme.
The Fig.2 showed how we combine Lpp-OmpA-N and scFv together, first we use restriction enzyme NcoI to digest the upstream and downstream parts. After ligate two digest product, there are no M site in Lpp-OmpA-N-scFv.

Fig.2 The combination of Ompa-N-scfv

Fig.3 Ompa-N-scfv

Experiment:

Fig.4The PCR result of the Lpp-OmpA-N+scFv. The DNA sequence length is around 1000~1200 bp, so the PCR products should appear at 1200~1400 bp.

After receiving the DNA sequences from the gene synthesis company, we recombined each OmpA-N-scFv gene to PSB1C3 backbones and conducted a PCR experiment to check the size of each of the OmpA-N-scFvs. The DNA sequence length of the OmpA-N-scFvs are around 1000~1200 bp. In this PCR experiment, the OmpA-N-scFv products size should be near at 1200~1400 bp. The Fig.4 showed the correct size of the scFv, and proved that we successful ligated the scFv sequence onto an ideal backbone.

Fig.5OmpA-N-scFv(Anti-EGFR)

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 388
  
• 1000
  COMPATIBLE WITH RFC[1000]