Revision as of 06:40, 22 September 2015

OmpA-scFv(Anti-HER2)

Introduction:

Fig.1 OmpA-N-scFv (anti-HER2)

To display the <a href="https://parts.igem.org/Part:BBa_K1694005"> scFv</a> 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.
In order to easily change various scFv DNA sequence, we added a NcoI restriction site between OmpA and scFv. 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.

The Fig.2 showed how we design NcoI restriction site. First, we digest Lpp-OmpA part with EcoRI and NcoI, scFv part with NcoI PstI. Then we ligated them together.

Fig.2 The combination of OmpA-N-scFv

Fig.3 OmpA-N-scFv

Experiment:

Fig.4 The PCR result of the Lpp-OmpA-N+scFv. The DNA sequence length is 1026 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 length of OmpA-N-scFv DNA sequence is 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 successfully ligated the scFv sequence onto an ideal backbone.

Fig.5 OmpA-N-scFv (anti-HER2)

Sequence and Features