Difference between revisions of "Team:NCTU Formosa/Project"
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===Summary=== | ===Summary=== | ||
| + | This year, our hit project, E.Cotector is to assist the medical practitioners to choose the appropriate targeted drug therapies for various conditions of patients. Before doctors prescribing the targeted drugs for cancer patients, E.Cotector can mark the tumor cells or test the antigens in serum by part of monoclonal antibodies (scFv) which is a kind of targeted drug directly binding with antigens. APOllO organization provided an advanced method in selecting personalized therapy for every particular patient. | ||
| + | |||
| + | E.Cotectors marked the tumor cells by displaying scFv on its outer membranes and fluorescence proteins: | ||
| + | <UL> | ||
| + | <LI> Simultaneously marked multiple kinds of overexpressed unique antigens on the cells.<br></LI> | ||
| + | Amplified the signal by E. coli expressing fluorescence proteins.<RB> | ||
| + | E.Cotectors detected the antigens in the serum by dual-displaying scFv and gold binding peptides on their outer membranes: | ||
| + | An innovative indicator to combine synthetic biology and numerous precision measurement technology. | ||
| + | Achieve the extraordinary degree of precision in detecting concentration of antigens in the serum. | ||
| + | Enhance the process yield in immobilization of antibodies on the medium gold surface. | ||
| + | Want to see more, please see Achievements page. | ||
| + | |||
===Biobrick Design=== | ===Biobrick Design=== | ||
====Lpp-OmpA-scFv==== | ====Lpp-OmpA-scFv==== | ||
Revision as of 15:55, 12 August 2015
Contents
Project
TESTTESTTEST
DESIGN
WERWERWER
scFv
GBP
Summary
This year, our hit project, E.Cotector is to assist the medical practitioners to choose the appropriate targeted drug therapies for various conditions of patients. Before doctors prescribing the targeted drugs for cancer patients, E.Cotector can mark the tumor cells or test the antigens in serum by part of monoclonal antibodies (scFv) which is a kind of targeted drug directly binding with antigens. APOllO organization provided an advanced method in selecting personalized therapy for every particular patient.
E.Cotectors marked the tumor cells by displaying scFv on its outer membranes and fluorescence proteins:
- Simultaneously marked multiple kinds of overexpressed unique antigens on the cells.
Amplified the signal by E. coli expressing fluorescence proteins.
E.Cotectors detected the antigens in the serum by dual-displaying scFv and gold binding peptides on their outer membranes:
An innovative indicator to combine synthetic biology and numerous precision measurement technology. Achieve the extraordinary degree of precision in detecting concentration of antigens in the serum. Enhance the process yield in immobilization of antibodies on the medium gold surface. Want to see more, please see Achievements page.
Biobrick Design
Lpp-OmpA-scFv
To display the antibody outside 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, Lpp-OmpA then fused the single chain variable fragment (scFv) C-terminally. 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 outside the E.coli outer membrane continuously. At the back of this part, we have added fluorescent proteins as the reporters.
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.
Reference
[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
FadL-GBP
Gold binding peptide (GBP) is a kind of peptide 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 and threonine, 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.
By ligating the constitutive promoter (BBa_J23110) ribosome binding site (BBa_B0034), FadL-GBP and terminator (BBa_J61048), we can continuously display the GBP outside the E.coli outer membrane so that our E.coli can bind on gold chip to apply on many measuring instruments.
Reference
[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
Result
Cell Staining
GBP test
SAFETY
===HUMAN PRACTICE===