Difference between revisions of "Part:BBa K1694014"

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[[File:EG14.png|200px|thumb|right|'''Fig.1''' Ompa-N-scfv(Anti-EGFR)]]
 
[[File:EG14.png|200px|thumb|right|'''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.
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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.
 
<br>
 
<br>
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.<br>
+
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.<br>
 
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 22:39, 19 September 2015

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]