Difference between revisions of "Part:BBa K1694015"

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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.
 
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.
 
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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.
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In order to easily change the various scFv DNA sequence, we added a ''NcoI'' restriction site between OmpA and scFv.
 
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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.
 
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.

Revision as of 05:32, 22 September 2015

OmpA-scFv(Anti-HER2)


Introduction:

Fig.1 Ompa-N-scfv(Anti-HER2)

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 easily change the various scFv DNA sequence, we added a NcoI restriction site between OmpA and scFv.
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.4 The 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.5 OmpA-N-scFv(Anti-HER2)


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 678
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 388
  • 1000
    COMPATIBLE WITH RFC[1000]