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Revision as of 15:14, 17 September 2015

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) and FadL-GBP, 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 test the GBP function by observing the green fluorescence on the gold chip. Terminator is (BBa_B0015),
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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