Part:BBa_K1694014

OmpA-scFv(Anti-EGFR)

Introduction:

Fig.1 OmpA-N-scFv(Anti-EGFR)

To display scFv outside the E. coli, we used Lipoprotein-Outer membrane protein A (Lpp-OmpA). According to the paper reference, we chose the first 9 amino acid of Lpp and the 46~159 amino acid of OmpA. When designing XbaI-SpeI restriction site between Lpp-OmpA and scFv, it will cause a mixed site. Therefore, the NcoI restriction site rather than the EX-SP restriction site was designed.
In order to easily change various scFv DNA sequence, we added a NcoI restriction site between OmpA and scFv.
The Fig.2 showed how we design Lpp-OmpA-N and scFv into NcoI restriction site. First, we digest Lpp-OmpA part with EcoRI and NcoI, scFv part with NcoI and PstI. Then we ligated them together.

Fig.2 The combination of OmpA-N-scFv

Fig.3 OmpA-N-scFv

Introduction of basic parts:
Anti-EGFR

Experiment:

Fig.4 The PCR result of the Lpp-OmpA-N+scFv. The length of DNA sequence is 1181 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 OmpA-N-scFv. The length of OmpA-N-scFv DNA sequence is 1181 bp. In this PCR experiment, 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 successfully ligated the scFv sequence onto an ideal backbone.

Fig.5 OmpA-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]