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https://2015.igem.org/Team:NCTU_Formosa/more_details
 
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===Background===
 
  
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Cancers,the last thing that human beings would like to get involved. Everyone wishes to have clean bills of health no matter in what age. According to National Cancer Institute (NCI), in 2015,the top cancers are lung cancer, colorectal cancer, breast cancer, melanoma and thyroid cancer, the estimated number of new cases of these cancers are 724,410. The estimated death cases of these cancers are 260,360. So, if it was inevitable for some situations to suffer cancers, and we would all be desperate to have the most effective and appropriate therapies to beat up cancers. Therefore, <B>targeted drug</b>, a precise therapy directly identifying the cancer cells has been invented.
 
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[[File:NCTU_Formosa_Background 1.jpg |right|400px|thumb|Fig.1 the usage rate of targeted drug therapy]]
 
  
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==== The usage rate of targeted drug therapies ====
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Targeted drug therapy becomes widely used over time. As we known that targeted drug therapies are <B>more precisely attack cancer cells</B>, which can <B>increase the treatment efficiency</B> by a large margin. In Figure 1, in 2003, targeted drug therapy is not commonly used compared with other therapies, accounting for only 11% usage. Over one decade, it is estimated that the usage of targeted drug therapy dramatically <b>increases to 46%</b>. It becomes the top therapy in cancer treatments among others. According to the statistics, the usage of targeted drugs therapy does have effective treatment in cancer, decreasing the morality of cancer and increasing the five-year survival rate after treatment.
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還缺一張圖 (資料來源?)標於圖下
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Link: Learn more about the advantages of targeted drug
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==== The crucial points of targeted drugs therapy ====
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Researchs have indicated that <B>utilizing different targeted drugs to treat cancers simultaneously attain greater effects</B> than only using one targeted drug, called combination therapy. However, targeted drugs therapy must treat patients in the proper condition to reach the best effect of the therapy. What’s worse, the improper usage of targeted drugs therapy would not only waste money and medical resources but also cause the invalid treatment result to patients.Above all, <B>defining whether to use the targeted drugs might be the crucial point of targeted drugs therapy</B>. That is, if doctor can prescribe the proper targeted drugs that specific to every patient’s condition, it will reach to a direct <b>personalized medicine</b> method for using targeted drugs therapy <BR>
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Therefore, NCTU_Formosa focuses on creating a <B>multimarker diagnosis platform</B> for helping doctors to judge whether to use monoclonal antibody targeted drugs by innovative method directly.
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===Overview===
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In 2015, NCTU_Formosa created an APOllO organization, which stands for Almighty probe of scFv organization, to develop a new product. We want to help doctor to select the candidates for targeted drug therapies and determine what kinds of targeted drugs can be used in a cheaper way. If the diagnosis cost become inexpensive, there will be more people can afford, therefore more people can be prescribe and treated, decreasing the mortality rate.
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<B>What’s special in 2015:</B>
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<b>APOllO E.Cotector</b>, our hit product, is to display part of monoclonal antibody targeted drugs, called scFv, outside the <i>E.coli</i> outer membrane and to express the fluorescent protein at the same time. It can stain the tissue biopsies by antibody-antigen interaction and perform fluorescence. Then it can help doctor determine the targeted drug therapy. Furthermore, by using various kinds of APOllO E.Cotectors with different scFv and fluorescent proteins at the same time, we can identify multimarker to achieve combination therapy.
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<B>APOllO E.Cotector Plus</b>, is another powerful helper, which has not only displayed scFv but also Gold binding polypeptide, GBP, on the outer membrane of <i>E.Coli</i>. Our E.Cotector Plus can immobilize on gold, applied on any sensor used gold as transducer, to test the concentration of specific antigens in serum and to give more information about the patients’ condition.
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===APOllO E.Cotector===
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To help doctors with innovated methods to judge whether to use <b>monoclonal antibody targeted drugs</b> more directly ,we redesigned the FDA approved monoclonal antibody targeted drugs, such as Bevacizumab (Avastin<sup>®</sup> anti-VEGF)<sup>[1]</sup>, Cetuximab (Erbitux<sup>®</sup> anti-EGFR)<sup>[2]</sup> and Trastuzumab (Herceptin<sup>®</sup> anti-HER2)<sup>[3]</sup> into recombinant antibodies : scFv (single chain variable fragment).
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For detecting the specific molecules (“targeted molecules”), which are specific to these scFv of targeted drugs, scFv are displayed on the surfaces of E.coli by using a transmembrane fusion protein, Lpp-OmpA<sup>[5]</sup> respectively; that is one APOllO E.Cotector displays one kind of targeted drugs of scFv each time. Take an example, while the anti-EGFR scFv (originate from Cetuximab) is displayed on the surface of E.coli, we can detect EGFR by this APOllO E.Cotector. Furthermore, as APOllO E.Cotector expressed fluorescence protein at the same time, the specific molecules (“targeted molecules”) that correspond to those scFv of targeted drugs can be identified individually in direct by those different colors APOllO E.Cotector, In a simple word, because of the APOllO E.Cotector that display scFv of targeted drugs can we identify the corresponding specific molecules (“targeted molecules”) ,then achieve to offer a prescription of monoclonal antibody targeted drug in direct .
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[[File:NCTU Formosa APOllO E.Cotecter 1.jpg|center|400px|thumb|Fig.1 Concept of APOllO E.Cotecter: Displaying single chain variable fragment(scFv) of antibody drugs on the surface of <i>E.coli</i> ]]
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==== The introduction of scFv ====
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scFv is a fusion protein of the variable regions of the heavy chains(VH) and light chains (VL) of antibody connected by a flexible linker peptide. scFv still reserve completely functional antigen-binding fragment and specificity of the original immunoglobulin; that is ,the property of specificity of antibody to antigen has being maintained. Moreover, scFv is only 20 percent the size as antibody <sup>[4]</sup>, therefore it will not cause stress to E.coli for displaying it
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====Cell Staining: Direct Multimarker diagnosis =====
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In clinical situation, doctors may stain cancer tissue slides by specific antibody to judge whether to use targeted drugs therapy. As we utilizing those fluorescence APOllO E.Cotectors, which respectively display scFv of targeted drugs, such as anti-EGFR scFv , anti-VEGF scFv and anti-HER2 scFv to stain the cancer cells, they may specify each of the targeted molecules –EGFR,VEGFR or HER2 on the cell directly in same time. Therefore, doctors may obtain results to define whether these monoclonal antibody targeted drugs is proper to use for the patient or not. That is, by this application, we will offer a multimarker diagnosis for doctors to judge whether to use combination targeted drugs
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====The reasons why APOllO E.Cotector may be a directly multimarker diagnosis method ====
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1.The design of scFv of targeted drugs <BR>
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2.The usage of Cell Staining <BR> 
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[[File:NCTU Formosa APOllO E.Cotecter 2.jpg|center|400px|thumb|Fig.2 THE CONCEPT OF OUR APPLICATION ]]
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[1] DrugBank: Bevacizumab (DB00112) http://www.drugbank.ca/drugs/DB00112 <BR>
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[2] DrugBank: Cetuximab (DB00002) http://www.drugbank.ca/drugs/DB00002 <BR>
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[3] DrugBank: Trastuzumab (DB00072) http://www.drugbank.ca/drugs/DB00072 <BR>
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[4] Single Chain Epidermal Growth Factor Receptor Antibody Conjugated Nanoparticles for in vivo Tumor Targeting and Imaging <BR>http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3626261/ <BR>
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[5] C Hartmann et al. (2010) Peptide mimotopes recognized by antibodies cetuximab and matuzumab induce a functionally equivalent anti-EGFR immune response<BR>
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http://www.nature.com/onc/journal/v29/n32/pdf/onc2010195a.pdf<BR>
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===APOllO E.Cotector Plus===
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While imaging tests and tissue biopsies are the most common methods for diagnosing cancer, serum tests can also help doctors identify the cancer by the usage of certain indicators, the antigens. Via measuring the levels of antigens, our APOllO E.Cotector Plus can help doctors to suggest the diagnosis as the reference for prescription of target therapy<sup>[1]</sup>.
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<td>[[File:APOllO_E.Cotector_Plus.jpg|center|thumb|400px|Fig-1 APOllO E.Cotector Plus contains dual display system. One system displayed the transmembrane protein Lpp-OmpA fused with the sequence of scFv, which can bind with targeted antigens. The other system expressed transmembrane protein FadL fused with gold binding polypeptide (GBP) for the purpose of specifically binding on the gold chip.]]</td>
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<td>[[File:E.Cotector_Plus.jpg|center|thumb|400px|Fig-2  (a)When targeted antigens in patients’ serum attach to scFv on APOllO E.Cotector Plus, the signal change of mass on the gold surface will be caught by the machines, such as Quartz crystal microbalance (QCM), Surface plasmon resonance Spectroscopy (SPR). Therefore, we can quantize and evaluate the level of binding antigens, and give the more accurate diagnosis for doctors. (b)A biosensor is an analytical device, which can be divided into three elements including the sensitive biological element, the transducer and the detector with data evaluation device.]]</td> 
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====Our novel design ====
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In our APOllO E.Cotector Plus design, we engineered the E.coli to display dual display system. One of the system displayed scFv by the transmembrane protein Lpp-OmpA. Meanwhile, the other system expressed transmembrane protein FadL fused with gold binding polypeptide (GBP) for the purpose of specifically binding on the gold chip, the material commonly used in biosensors<sup>[2]</sup>. By processing our E.Cotector Plus, with gold binding polypeptide connecting to the gold chip, we simplified the procedures of the self-assemble modification (SAM), the technique to immobilize antibodies on the gold surface<sup>[3]</sup>. We skipped the procedure of the gold surface decoration with sulfur bond and omitted the carbon chains attaching to antibody, which may occasionally block the binding sites of scFv, further increasing the efficiency of antibody-antigen interactions.
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Combined with the idea of the biosensor, we deemed our E.cotectorplus played the role of biological recognition part and the gold chip acted as the transducer part. Therefore, our E.cotectorplus, which is able to attach on gold chip, can be regarded as the platform for precise physicochemical nanoscale detectors, such as Quartz crystal microbalance, Surface plasmon resonance Spectroscopy, Dual-polarization interferometry, ellipsometry, etcetera.
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With our powerful APOllO E.Cotector Plus , we solved the time-consuming and sophisticated process of gold surface modification. What’s more, by coupling our E.Cotector Plus to precise measurement instruments, we can achieve a huge boost to provide more sensitive and specific scFv detecting techniques options, and give more reliable diagnosis for doctors to apply monoclonal antibody target drugs.
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====The introduction of  gold binding polypeptide====
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The gold binding polypeptide, abbreviated as GBP, is the three-repeated of following 14 aminoacid sequences: [MHGKTQATSGTIQS], which was developed in an <i>E. coli</i> cell-surface display system<sup>[4]</sup>. According to the paper, the binding sequence of GBP does not contain cysteine which can form a covalent thiol linkage with gold, the linkage to the gold surface in Self-Assembled Monolayers (SAMs)<sup>[5]</sup>. The mechanism of the connection between GBP and gold metal plane remains unknown. By using Molecular Dynamics (MD), it indicates that GBP, with an antiparallel β-sheet structure, can recognize gold surface via OH-binding. It is likely that the hydroxyl, together with amine, ligands on GBP recognize the atomic lattice of gold, aligning the molecule along the variants of a six-fold axis on the Au (111) surface<sup>[6]</sup>.
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====The utilize of gold chip in biosensor====
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The gold is the best choice for our biosensor substrate because of its advantages of stability to external environment, the excellent capability of transducing electronic signals, the sensitive physicochemical properties and, most important of all, the specific interaction with gold binding polypeptide.
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Reference <BR>
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[1]Blood Tests and Biomarkers http://www.asbestos.com/mesothelioma/blood-test.php<BR>
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[2]Development of a whole-cell biosensor by cell surface display of a gold-binding polypeptide on the gold surface Tae Jung Park1,2, Shun Zheng1,2, Yeon Jae Kang2 & Sang Yup Lee1,2,3, Oxford University press, FEMS Microbiology Letters (2009)<BR>
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[3]Biosensor surface chemistry for oriented protein immobilization and biochip patterning Linköping Studies in Science and Technology Licentiate Thesis No. 1573 (2013)<BR>
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[4] Molecular characterization of a prokaryotic polypeptide sequence that catalyzes Au crystal formation, John L. Kulp III,a Mehmet Sarikayab and John Spencer Evans, Journal of Materials Chemistry(2004)<BR>
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[5] Adsorption of genetically engineered proteins studied by time-of-flight secondary ion mass spectrometry (TOF-SIMS). Part A: data acquisition and principal component analysis (PCA), Noriaki Suzuki,1 Lara Gamble,2 Candan Tamerler,3 Mehmet Sarikaya,1 David G. Castner2,4 (2007)<BR>
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[6] Assembly of Gold-Binding Proteins for Biomolecular Recognition, Zareie HM1,2* and Sarikaya M3, Austin Journal of Biosensors & Bioelectronics (2015)<BR>
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===Summary===
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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 the 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.
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<UL>E.Cotectors marked the tumor cells by displaying scFv on its outer membranes and fluorescence proteins:
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<LI> Simultaneously marked multiple kinds of overexpressed unique antigens on the cells.<br></LI>
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<LI> Amplified the signal by E. coli expressing fluorescence proteins.</LI>
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E.Cotectors Plus detected the antigens in the serum by dual-displaying scFv and gold binding peptides on their outer membranes:
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<LI> An innovative indicator to combine synthetic biology and numerous precision measurement technology.</LI>
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<LI> Achieve the extraordinary degree of precision in detecting concentration of antigens in the serum.</LI> 
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<LI> Enhance the process yield in immobilization of antibodies on the medium gold surface.</LI>
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Want to see more, please see Achievements page.
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===Biobrick Design===
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====Lpp-OmpA-scFv====
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[[File:NCTU Formosa Lpp-ompA-scfv 1.png|center|thumb|400px|Fig-1 Our Basic Biobrick Lpp-OmpA-scFv.]]
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To display the antibody on the <i>E.coli</i> <B>outer membrane</B>, we used Lipoprotein-Outer membrane protein A (Lpp-OmpA). According to the paper reference <sup>[1]</sup>, 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 <B>change any scFv DNA sequence</B> just by NcoI restriction enzyme.
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[[File:NCTU Formosa Lpp-ompA-scfv 2.png|center|thumb|400px|Fig-2 Our Composite Biobrick]]
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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 <i>E.coli</i> outer membrane continuously. At the back of this part, we have added fluorescent proteins as the reporters.
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[[File:NCTU Formosa Lpp-ompA-scfv 3.png|center|thumb|400px|Fig-3 Our Composite Biobrick for cell staining.]] 
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In our current work, we chose three targeted drugs, <B>Avastin (Bevacizumab, anti-VEGF)<sup>[2]</sup>, Erbitux (Cetuximab, anti-EGFR)<sup>[3]</sup> and Herceptin (Trastuzumab, anti-HER2)<sup>[4]</sup></B> from Drugbank, selecting their single chain variable fragments (scFv) to use, which is short and it will not give too much stress to <i>E.coli</i>.
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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 <B>apply our <i>E.coli</i> to cell staining</B>. 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 <i>E.coli</i> with different fluorescence, we are able to create a platform which can detect <B>multimarker</B>.
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Reference<BR>
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[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<BR>
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[2] DrugBank: Bevacizumab (DB00112) http://www.drugbank.ca/drugs/DB00112 <BR>
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[3] DrugBank: Cetuximab (DB00002) http://www.drugbank.ca/drugs/DB00002 <BR>
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[4] DrugBank: Trastuzumab (DB00072) http://www.drugbank.ca/drugs/DB00072<BR>
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====FadL-GBP====
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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.
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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 <sup>[1]</sup>, signal peptide included.
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[[File:NCTU Formosa Lpp-ompA-scfv 4.png|center|thumb|400px|Fig-3 Our Composite Biobrick for GBP]] 
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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 <i>E.coli</i> outer membrane so that our <i>E.coli</i> can bind on gold chip to apply on many measuring instruments.
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Reference<BR>
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[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
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===Result===
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====Cell Staining====
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====GBP test====
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===Modeling===
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===Safety===
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Latest revision as of 01:34, 19 September 2015



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