Difference between revisions of "Part:BBa K1694014"
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In order to easily change various scFv DNA sequence, we added a ''NcoI'' restriction site between OmpA and.<br> | In order to easily change various scFv DNA sequence, we added a ''NcoI'' restriction site between OmpA and.<br> | ||
− | Fig.2 showed how we | + | The Fig.2 showed how we integrate Lpp-OmpA-N and scFv into NcoI restriction site. We use restriction enzyme ''NcoI'' to digest the upstream and downstream parts. After ligating two digest product, there are no mixed site in Lpp-OmpA-N-scFv. |
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Revision as of 05:54, 22 September 2015
OmpA-scFv(Anti-EGFR)
Introduction:
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.
In order to easily change various scFv DNA sequence, we added a NcoI restriction site between OmpA and.
The Fig.2 showed how we integrate Lpp-OmpA-N and scFv into NcoI restriction site. We use restriction enzyme NcoI to digest the upstream and downstream parts. After ligating two digest product, there are no mixed site in Lpp-OmpA-N-scFv.
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 OmpA-N-scFv. The DNA sequence length of OmpA-N-scFv 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 successful ligated the scFv sequence onto an ideal backbone.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 388
- 1000COMPATIBLE WITH RFC[1000]