Difference between revisions of "Part:BBa K1694013"

 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1694013 short</partinfo>
 
<partinfo>BBa_K1694013 short</partinfo>
  
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We selected the single chain variable fragments (scFv) of monoclonal antibodies Cetuximab
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<h1>'''Introduction:'''</h1>
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[[File:ompascfv.png|200px|thumb|left|'''fig.1''' ompascfv]]
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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.
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<br>
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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 vario us scFv DNA sequence using the NcoI restriction enzyme.
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<h1>'''Experiment:'''</h1>
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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. showed the correct size of the scFv, and proved that we successful ligated the scFv sequence onto an ideal backbone.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 02:52, 19 September 2015

OmpA-scFv(Anti-VEGF)

Introduction:

fig.1 ompascfv

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 vario us scFv DNA sequence using the NcoI restriction enzyme.

Experiment:

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. showed the correct size of the scFv, and proved that we successful ligated the scFv sequence onto an ideal backbone.

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]