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Revision as of 10:39, 18 September 2015

Basic Parts

Basic Parts

BBa_K1694002
Lpp-OmpA-NcoI		 To display the scFv to the E. coli outer membrane, we chose Lipoprotein-Outer membrane protein A (Lpp-OmpA). According to the paper reference [1], we used the first 9 amino acids of Lpp as the signal peptide, and the 46-159 amino acids of OmpA as the anchor. All the sequences is from DH5-alpha strain itself[4].
The C-terminal of Lpp-OmpA, located on the outer membrane, is then be fused with the scFv part.
In order to change scFv part easily, we provide a basic part which contain NcoI restriction site.
BBa_K1694010
Lpp-OmpA		 We also provide Lpp-OmpA without NcoI restriction site.
BBa_K1694003
scFv of anti-VEGF
BBa_K1694004
scFv of anti-EGFR
BBa_K1694005
scFv of anti-HER2
In our library of customized E.Cotectors, we chose two targeted drugs, Avastin (Bevacizumab, anti-VEGF)[1], Erbitux (Cetuximab, anti-EGFR)[2] from Drugbank, and a monoclonal antibody, anti-HER2[3]. We select the part of single chain variable fragments (scFv), which is short and will not give too much stress for E. coli expressing it.
BBa_K1694006
GBP
BBa_K1694007
FadL-GBP 		Gold binding polypeptide (GBP) is a kind of polypeptide which can bind on gold, and is usually used to immobilize proteins on gold surfaces. The mechanism of how GBP binds the gold is not clearly understood, but its polar side-chains, such as those of serine and threonine, seem to interact with gold.
We used a 42 amino acids long GBP, which contain 3 repeated amino acid sequences [MHGKTQATSGTIQS]. To display GBP on cell surface, we used Long-chain fatty acid transport protein (FadL) as a transmembrane protein, selecting its first 384 amino acids to link with GBP [5], signal peptide included.
Reference
 [1] C Hartmann et al. (2010) Peptide mimotopes recognized by antibodies cetuximab and matuzumab induce a functionally equivalent anti-EGFR immune response
[2] DrugBank: Bevacizumab (DB00112)
[3] DrugBank: Cetuximab (DB00002)
[4] DrugBank: Trastuzumab (DB00072)
[5] Tae Jung Park et al. (2009) Development of a whole-cell biosensor by cell surface display of a gold-binding polypeptide on the gold surface


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