Difference between revisions of "Team:NCTU Formosa/Project"
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====Skype meetup ==== | ====Skype meetup ==== | ||
| − | We had 4 engaging internet discussions with teams that were KAIT_ Japan, Amoy_ China, TU Delft, and Oxford. We always learned so much from each team’s amazing idea. After exchanging ideas, we were amazed by them and what’s more, as we talked to different people from different culture, culture shock and multicultural interaction experience were something we were excited about. With all these cross-cultural meet ups, our lives had been spiced up. | + | We had 4 engaging internet discussions with teams that were KAIT_ Japan, Amoy_ China, TU Delft, and Oxford. We always learned so much from each team’s amazing idea. After exchanging ideas, we were amazed by them and what’s more, as we talked to different people from different culture, culture shock and multicultural interaction experience were something we were excited about. With all these cross-cultural meet ups, our lives had been spiced up.<br> |
| − | Skype meeting with Amoy on May 13 | + | Skype meeting with Amoy on May 13<br> |
| − | Skype meeting with KAIT_Japan on May 20 | + | Skype meeting with KAIT_Japan on May 20<br> |
| − | Skype meeting with TU_Delft on August 4 | + | Skype meeting with TU_Delft on August 4<br> |
====Mini Conference with Mingdao & HSNU==== | ====Mini Conference with Mingdao & HSNU==== | ||
Revision as of 15:41, 15 August 2015
Contents
Background
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. Nowadays, the top cancers include lung cancer, colorectal cancer, breast cancer, melanoma and thyroid cancer. According to National Cancer Institute(NCI), in 2015, the estimated number of new cases of these cancers are 724,410. The estimated death cases of these cancers are 260,360.So, it is 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. Eventually, targeted drug, a precise therapy directly identifying the cancer cells has been invented.
Targeted drug therapies becomes widely used over time. As we known that targeted drug therapies are more precisely attack cancer cells, which can increase the treatment efficiency by a large margin. In this chart (Fig.1), in 2003, targeted drug therapy is not commonly used compared with other therapies, accounting for only 11% usage. Over Ten years, it is estimated that the usage of targeted drug therapy dramatically increases to 46%. 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.
One of a kind of targeted drugs therapy is monoclonal antibody. It blocks the growth and spread of cancer by interfering with specific molecules("molecular targets") that are involved in the growth, progression, and spread of cancers. Furthermore, the research also indicated that utilizing different targeted drugs to treat cancers simultaneously attain greater effects than only using one targeted drug. 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, defining whether to use the targeted drugs might be the crucial point of targeted drugs therapy.Therefore,NCTU_Formosa focuses on creating a multimarker diagnosis platform for helping doctors to judge whether to use monoclonal antibody (mAb) targeted drugs by innovative methods directly.
Overview
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 drug can be used.
APOllO E.Cotector, our hit product, is to present a part of monoclonal antibody targeted drug, called scFv, outside the E.coli outer membrane and the fluorescent protein at the same time. It can be applied on tissue biopsies staining. Furthermore, by using various kinds of APOllO E.Cotector with different scFv and fluorescent protein at the same time, we can identify multimarker to achieve combination therapy.
APOllO E.Cotector Plus, is another powerful helper, which has not only scFv but also Gold binding polypeptide, GBP, on outer membrane. It can immobilize on gold, applied on any sensor used gold as transducer, to test the serum and to give more information about the patients’ condition.
scFv of targeted drugs
We redesigned the FDA approved monoclonal antibody targeted drugs, such as Bevacizumab (Avastin®)[1], Cetuximab (Erbitux®)[2] and Trastuzumab (Herceptin®)[3] into recombinant antibodies : scFv (single chain variable fragment). scFv is a fusion protein of the variable regions of the heavy chains (VH)and light chains (VL) of immunoglobulin connected by a flexible linker peptide. Surprisely, scFv could keep completely functional antigen-binding fragment and specificity of the original immunoglobulin. What’s more, scFv is only 20 percent the size as immunoglobulin [4] ; therefore it will not cause stress to E.coli for displaying it.
APOllO E.Cotector
Concept: Displaying single chain variable fragment(scFv) of antibody drugs on the surface of E.coli
APOllO E.Cotector displays these scFv sequences on the surfaces of E.coli by using the Lpp-OmpA, the transmembrane protein[5]. The scFv antibody fragments, we displayed on the surface are anti-VEGF (Bevacizumab)[2], anti-EGFR (Cetuximab)[3] and anti-HER2 (Trastuzumab)[4].Under APOllO E.Cotector, the specific molecules ("molecular targets") that correspondence to the scFv of targeted drugs can be identified. As the result ,we canhelp doctors to judge whether to use monoclonal antibody (mAb) targeted drugs directly .
Cell Staining
In clinical situation, doctors may stain cancer tissue slides to judge whether to use targeted drugs therapy; however, existing the limitation of indirect diagnosis. We applied APOllO E.Cotectors with diverse fluorescence proteins respectively to stain cancer cells. By this application, we will offer a multimarker diagnosis for doctors to judge whether to use combination targeted drugs therapy in direct.
[1] DrugBank: Bevacizumab (DB00112) http://www.drugbank.ca/drugs/DB00112
[2] DrugBank: Cetuximab (DB00002) http://www.drugbank.ca/drugs/DB00002
[3] DrugBank: Trastuzumab (DB00072) http://www.drugbank.ca/drugs/DB00072
[4] Single Chain Epidermal Growth Factor Receptor Antibody Conjugated Nanoparticles for in vivo Tumor Targeting and Imaging
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3626261/
[5] 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
APOllO E.Cotector Plus
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 tumor antigens. Via measuring the levels of tumor antigens, our APOllO E.Cotector Plus can help doctors to suggest the diagnosis as the reference for prescription of target therapy[1].
Our novel design ── the APOllO E.Cotector Plus
In our APOllO E.Cotector Plus design, we engineered the E.coli to express dual display system. One of the system displayed scFv by the transmembrane protein Lpp-OmpA, which can transport scFv onto the surface of the cell, as mentioned before. Meanwhile, the other system expressed transmembrane protein FadL fused with three-repeated gold binding peptide (GBP) for the purpose of specifically binding on the gold chip, the material commonly used in biosensors. By processing our E.cotector Plus, with gold binding peptide connecting to the gold chip, we simplified the procedures of the self-assemble modification (SAM), the technique to immobilize antibodies on the gold surface. 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 affinity of antibody-antigen interactions[2][3] .
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, etc.
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 options of the sensitive and specific detecting techniques for scFv, and give more reliable diagnosis for doctors to apply monoclonal antibody target drugs.
The introduction of gold binding polypeptide
The gold binding polypeptide, abbreviated as GBP, is the three-repeated of following 14 aminoacid sequences: [MHGKTQATSGTIQS], which was developed in an E. coli cell-surface display system. [4] The mechanism of the connection between GBP and gold metal plane is still 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. [5]
The utilize of gold chip in biosensor
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.
Reference
[1]Blood Tests and Biomarkers http://www.asbestos.com/mesothelioma/blood-test.php
[2] Biosensor surface chemistry for oriented protein immobilization and biochip patterning Linköping Studies in Science and Technology Licentiate Thesis No. 1573 (2013)
[3] 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)
[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)
[5] Assembly of Gold-Binding Proteins for Biomolecular Recognition, Zareie HM1,2* and Sarikaya M3, Austin Journal of Biosensors & Bioelectronics (2015)
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 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.
- 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.
- 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.
E.Cotectors Plus detected the antigens in the serum by dual-displaying scFv and gold binding peptides on their outer membranes:
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, 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 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 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.
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
Skype meetup
We had 4 engaging internet discussions with teams that were KAIT_ Japan, Amoy_ China, TU Delft, and Oxford. We always learned so much from each team’s amazing idea. After exchanging ideas, we were amazed by them and what’s more, as we talked to different people from different culture, culture shock and multicultural interaction experience were something we were excited about. With all these cross-cultural meet ups, our lives had been spiced up.
Skype meeting with Amoy on May 13
Skype meeting with KAIT_Japan on May 20
Skype meeting with TU_Delft on August 4
Mini Conference with Mingdao & HSNU
Sunday, June 7th We held mini conference in NCTU with Mindao and HSNU to share our projects.
Mini Conference with CGU
Sunday, June 14th
Biocamp
Wednesday, July 8th
In the summer, our department held a Biocamp for senior high students. Most of our team members participated in holding the event. The purpose of Biocamp is to let high school students learn about biology and have fun during the process. Three of our teammates designed a lecture to teach them synthetic biology with simple concept. What’s more, we promoted iGEM to them with introducing different iGEM projects, and conveying the essence and values of iGEM that we have realized during the processes. At last we also share our project with them specifically, the main idea of our biobricks and goals. After the lecture, we received some great response from them. All of them were fascinated by the idea of using enzymes and different assemblies of biobricks to create new functions, and interested in participating in iGEM. Hope all of them have the opportunity to become an iGEMer, enjoying the processes just like us
Doctors visit
Thursday, July 16th An oncologist visited our university, we grasped the opportunity and we talked to the doctor and elaborated our project. We were wondering how we will help patients with our project and if we really provide a better way to prediagnose. After a long discussion about the practical use of our project and the advantages. The doctors aid he was pretty amazed by our intelligent idea that we just use ecoli to prediagnose. That simplified the procedure and rose the efficiency. We were so excited that our project could also be applied well in practical use. It could really help people!
NCTU Asia iGEM Meetup
July 19th- 23rd
The big event that NCTU_FORMOSA had been preparing for so long started on 8/19, which lasted for 4 days. We had 27 teams present, about 210 attendees from China, Hong Kong and Taiwan. We even have teams from India and Kazakhstan join us during the meetup through the internet. So all the process of Asian conference is open to the whole world. Throughout the 4-day conference, each team had prepared presentation for their amazing projects and ideas. We learned about each team’s idea and the progress of their project so After all the presentations, our professors also as judges will give pieces of advice to the teams. Afterwards, Q&A time was a chance to interact with each team. Besides presentation, poster time during every meal not only fed us with exquisitely prepared food but also knowledge. All these activities enable us to rethink our project with different angles, improving our 27 projects into a new level. At the end of the conference, each team were given special gifts, embraceD bunch of friends, and feel happy and proud to be igemers. After the conference, we planned a day trip in Taipei, visiting National Palace Museum and Taipei 101. Both are the must-go attraction that nobody would miss when coming to Taiwan.
Meeting with a previous iGEM team supervisor
Thursday, July 30th
Professor Westenberg from Missouri University of Science and Technology came to NCTU. He gave us some valuable advice on how we can alter our experiment. We had a really great time-sharing each other's project, and show him around our campus.
Achievement and Value
What have we done?
In the project
◆We created the fluorescent E.Cotectors with the single chain variable fragment (scFv) of monoclonal antibody used on targeted therapies on its outer membrane.
◆We created our E.Cotectors with the gold binding peptide (GBP) on its outer membrane.
◆We created the different colors of fluorescent E.Cotectors with different scFv of monoclonal antibodies.
◆We proved that the scFv have binding specificity and our E.Cotectors can bind to antigens to distinguish antigens overexpressed in cells.
◆We proved that our E.Cotectors can apply on different techniques of cell staining.
◆We proved that the GBP can let our E.Cotectors adhere to the gold surface.
◆We combined the Quartz crystal microbalance (QCM) technique with our E.Cotectors to measure the concentration of antigens.
More than the project
◆We constructed 25 biobricks and conducted a series of experiments to verify their functions.
◆We constructed the models to calculate the optimized environment for the highest binding affinity.
◆We designed the mechanism to guarantee the safety on the gene level and the microorganism level.
◆We promoted the communication and the cooperation among various professional fields such as synthetic biology, measurement technology, immunology, and medical science via the participation in iGEM and the accomplishment of the project.
Value on the measurement technology
◆Simultaneously detected the multiple antigens.
◆Enlarged the signal of targeted antigens.
◆Combined the antigen measurement technique with others technique, enhanced the precision, and expanded its application.
◆Enhanced the process yield in immobilization of antibodies on the medium gold surface.
◆The cost of using our E.cotectors is lower than using monoclonal antibodies targeted drugs directly on measuring antigens.
◆Value on medical diagnosis
◆Offer a new way to let doctors have more selections to make diagnosis.
◆Directly used the antibody fragment of monoclonal antibody used on targeted therapy to distinguish antigens overexpressed in cells. Provided prescription of combination, personalized, and effective targeted drugs.
Prepared for the new era of medical digitalization.
Judging Criteria
Gold medal
◆ Expand on your silver medal Human Practices activity by demonstrating how you have integrated the investigated issues into the design and/or execution of your project.
◆Improve the function of an existing BioBrick Part or Device, enter this information in the registry.
◆Help any registered iGEM team from a high-school, different track, another university, or institution in a significant way by.
Silver medal
◆Experimentally validated our parts
◆Demonstrate how your team has identified, investigated and addressed one or more of these issues in the context of your project.
◆Submitted new standard biobrick parts to the iGEM Parts Registry
◆Our project may have implications for the environment, security, safety and ethics and/or ownership and sharing. Describe one or more ways in which these or other broader implications have been taken into consideration in the design and execution of your project.
Bronze medal
◆Registered the team, had a great summer, and planned to have fun at the Jamboree.
◆Completed and submitted the Judging form.
◆A Team Wiki was designed and constructed to share a description of our project.
◆Plan to present a Poster and talk at the iGEM Jamboree.
◆Attributed all work to the proper persons.
◆Submitted at least one new standard biobrick part to the registry.