Team:NCTU Formosa/Composite Part

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Composite Parts

Composite Part

BBa_K1694013 OmpA-anti-VEGF BBa_K1694014 OmpA-anti-EGFR BBa_K1694015 OmpA-anti-HER2		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.
BBa_K1694023 Pcons+RBS+OmpA-anti-VEGF BBa_K1694024 Pcons+RBS+OmpA-anti-EGFR BBa_K1694025 Pcons+RBS+OmpA-anti-HER2		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. Having this part, we can co-transform with other parts in order to produce color as the detection signal. In addition, by co-transforming these different types of E. coli with different fluorescence or color as signals, we are able to create a platform which can detect multimarker and consequently achieve combination therapy.
BBa_K1694033 Pcons+RBS+OmpA-anti-VEGF+RBS+GFP+Ter BBa_K1694034 Pcons+RBS+OmpA-anti-EGFR+RBS+GFP+Ter BBa_K1694035 Pcons+RBS+OmpA-anti-HER2+RBS+GFP+Ter BBa_K1694044 Pcons+RBS+OmpA-anti-EGFR+RBS+RFP+Ter BBa_K1694045 Pcons+RBS+OmpA-anti-HER2+RBS+BFP+Ter		The most commonly constructing way of composite parts is to ligate the required parts together. We also provide this kind of parts. At the back of the Lpp-OmpA-scFv part, we ligated the weaker ribosome biding site (BBa_B0030), different fluorescent protein and terminator (BBa_J61048) to make it continuously and simultaneously express the fluorescence and the scFv. We used a weak ribosome binding site to ensure that scFv's production will not be affected.
BBa_K1694053 Pcons+RBS+OmpA-anti-VEGF+RBS+amilCP+Ter BBa_K1694054 Pcons+RBS+OmpA-anti-EGFR+RBS+amilCP+Ter BBa_K1694055 Pcons+RBS+OmpA-anti-HER2+RBS+amilCP+Ter		Chromoprotein is another example of what can be added when making your own probe. We constructed this part as the Pcons+RBS+OmpA-scFv+RBS+fluorescent protein+Terminator part.
BBa_K1694027 Pinduce+RBS+FadL-GBP		By ligating the induced promoter (BBa_R0010), ribosome binding site (BBa_B0034), FadL-GBP and terminator (BBa_B0005), we can continuously display the GBP on the E. coli outer membrane. Then we co-transform the OmpA-scFv parts and this FadL-GBP parts into the same E. coli, hence allowing our E. coli to bind on gold chips and detect antigens simultaneously.
BBa_K1694037 Pinduce+RBS+FadL-GBP+RBS+GFP+Ter		With this part, we can testt the GBP function by observing the green fluorescence on the gold chip.

Reference
[1] C Hartmann et al. (2010) Peptide mimotopes recognized by antibodies cetuximab and matuzumab induce a functionally equivalent anti-EGFR immune responsehttp://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
[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 surfacehttp://www.ncbi.nlm.nih.gov/pubmed/19228193

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NCTU_Formosa APOllO
National Chaio Tung University
Engineering Building 6 EF455, 1001 University Road, Hsinchu 300, Taiwan, ROC.