Team:NCTU Formosa/Parts
To display the antibody on the E.coli outer membrane, we used Lipoprotein-Outer membrane protein A (Lpp-OmpA). According to the paper reference [1], We chose the first 9 amino acids of Lpp to be the signal peptide, and the 46-159 amino acids of OmpA to be the anchor, the C-terminally of Lpp-OmpA then fused the single chain variable fragment (scFv). We added a NcoI restriction side between OmpA and scFv so that we can change any scFv DNA sequence just by NcoI restriction enzyme.
By ligating the constitutive promoter (BBa_J23101), strong ribosome binding site (BBa_B0034) and Lpp-OmpA-scFv, we were able to display scFv on the E.coli outer membrane continuously. At the back of this part, we have added fluorescent proteins as the reporters.
Figure 3.Our Composite Biobrick for cell staining.
In our current work, we chose three targeted drugs, Avastin (Bevacizumab, anti-VEGF)[2], Erbitux (Cetuximab, anti-EGFR)[3] and Herceptin (Trastuzumab, anti-HER2)[4] from Drugbank, selecting their single chain variable fragments (scFv) to use, which is short and it will not give too much stress to E.coli.
At the back of Lpp-OmpA-scFv part, we ligated the weaker ribosome biding site (BBa_B0030), different fluorescent protein and terminator (BBa_J61048) to make it continuously express the fluorescence and the scFv at the same time so that we can apply our E.coli to cell staining. The reason why we used the weak ribosome biding site so that the expression of scFv will not be affected. In addition, by combining these different types of E.coli with different fluorescence, we are able to create a platform which can detect multimarker.
[1] C Hartmann et al. (2010) Peptide mimotopes recognized by antibodies cetuximab and matuzumab induce a functionally equivalent anti-EGFR immune response http://www.nature.com/onc/journal/v29/n32/pdf/onc2010195a.pdf
[2] DrugBank: Bevacizumab (DB00112) http://www.drugbank.ca/drugs/DB00112
[3] DrugBank: Cetuximab (DB00002) http://www.drugbank.ca/drugs/DB00002
[4] DrugBank: Trastuzumab (DB00072) http://www.drugbank.ca/drugs/DB00072
Gold binding polypeptide (GBP) is a kind of polypeptide which can bind on gold, usually used to immobilize protein on gold surface. The mechanism of how GBP bind the gold is not so understood, but its polar side-chains, such as serine, threonine and OH-binding, seem to interact with gold. We used a 42 amino acids long GBP, which contain three 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 the first 384 amino acids to link with GBP [1], signal peptide included.
Figure 4. Our Composite Biobrick for GBP
By ligating the constitutive promoter (BBa_J23110) ribosome binding site (BBa_B0034), FadL-GBP and terminator (BBa_J61048), we can continuously display the GBP on the E.coli outer membrane so that our E.coli can bind on gold chip to apply on many measuring instruments.
[1] 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
NCTU_Formosa APOllO
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