Team:Rochester/Description
UteRus
Index
Our project
Subteams
Clear and Concise Description of Project
Application of Synthetic Biology
Goals
Inspiration for Our Project
Impact of COVID-19 on Our Project
Description
We sought to create a novel, noninvasive diagnostic for endometriosis using menstrual effluent. Endometriosis is a chronic disease that causes aberrant endometrial-like tissue growth outside of the uterine cavity. It affects more than 200 million women worldwide and can lead to severe symptoms impacting reproductive health (Mutter, 2014). Currently, there are diagnostic methods available except for exploratory surgery (Biacchardi et al., 2011). Our wet lab team selected biomarkers for endometriosis in menstrual effluent and collaborated with expert physicians and researchers in the field of endometriosis to create lateral flow assays that can both qualitatively and quantitatively measure the presence of these biomarkers in menstrual effluent. This work was supported by the efforts of the modeling team, who created a predictive model to predict the likelihood that a patient has endometriosis based on demographic data and clinical symptoms. We incorporated this model into a software that clinicians can use to assess patient endometriosis risks. Additionally, our hardware team created a menstrual cup best suited for the collection of menstrual effluent and the comfort of endometriosis patients, and designed inexpensive laboratory equipment for clinics without easy laboratory access. Together, we were able to create a simple diagnostic for endometriosis that can be employed in a variety of clinical settings and used to resolve the gap of knowledge and raise awareness for female reproductive healthcare.
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Wet Lab
The goal our project was to create a rapid, inexpensive diagnostic for endometriosis using principles of synthetic biology. In alignment with these goals, we designed a lateral flow immunoassay using antibodies that we designed to be produced in E. coli. We hope that the marketing of this diagnostic to physicians will raise awareness and education about this chronic disease and foster better physician-patient relationships.
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Modeling
To aid wet lab in their lateral flow assay design and hardware in their sensor design, we created a model that describes how the output signal responds to various design parameters, choosing the design optimized to meet wet lab and hardware requirements. Additionally, we also focused on therapeutic biobrick development by creating a model that describes reporter production under various T7 promoters, estrogen response elements, and ribosome binding sequences choosing the combination of these elements that met desired outcomes. We also created a predictive model to predict the likelihood that a patient has endometriosis based on demographic data and clinical symptoms.
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Hardware
We aimed to provide an easier and more accessible experience for both the patient and the physician. We designed a menstrual cup and collection vial that prioritized ease of sample transportation and comfort of endometriosis patients. We also created a UV sterilizer to thoroughly clean the surface of menstrual cups in order to prevent infection. By creating a “Do It Yourself” centrifuge and an incubator, we were able to provide an alternative for cheaper and more accessible equipment for clinics. Lastly, we developed an image processing software for a simplistic way to analyze the lateral flow assay results.
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Policy and Practice
To address the under-diagnosis of endometriosis, it is important to raise awareness about what endometriosis is and how it affects individuals worldwide. We worked with physicians and endometriosis foundations to create endometriosis educational material for clinics that target both patients and physicians. Additionally, to successfully implement our project in society, we investigated the feasibility, safety, and ethics of our project through literature review and by connecting with our stakeholders. We also worked with physicians and endometriosis foundations after extensive literature review to create endometriosis educational materials and collaborated on how to make our product sustainable and safe to use. Finally, we explored the ethics surrounding accessibility of our diagnostic approach in clinics with limited resources and treatment options once the patient is diagnosed with endometriosis, and feasibility of implementation in these areas.
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Software
Our goal was to create user interfaces that connect the public to our project. We designed a software tool that integrates the modeling team’s endometriosis risk calculator algorithm. This allows physicians to enter a patient’s demographic and clinical variables and calculate their risk of having endometriosis. This tool also explains how each variable is related to endometriosis. Additionally, we developed a software application containing our endometriosis educational program to provide the public with the general information about women’s reproductive health and endometriosis.
We sought to create a novel, noninvasive diagnostic for endometriosis using menstrual effluent. Endometriosis is a chronic disease that causes endometrial-like tissue growth outside of the uterine cavity. It affects more than 200 million women worldwide and can lead to severe symptoms impacting reproductive health (Mutter, 2014). Currently, there are no diagnostic methods available except for exploratory surgery (Biacchardi et al., 2011). Our team selected biomarkers for endometriosis in menstrual effluent and collaborated with expert physicians and researchers in the field of endometriosis to create lateral flow assays that can both qualitatively and quantitatively measure the presence of these biomarkers in menstrual effluent. We supported this work by creating an ordinary differential equations model to determine lateral flow assay parameters as well as a predictive model to determine if a patient has endometriosis based clinical symptoms. We incorporated the clinical predictive model into a software that clinicians can use to assess patients’ endometriosis risk. Additionally, we created a menstrual cup best suited for the collection of menstrual effluent and the comfort of endometriosis patients, and designed inexpensive laboratory equipment for clinics without easy laboratory access. Together, we were able to create a simple diagnostic for endometriosis that can be employed in a variety of clinical settings and used to resolve the gap of knowledge and raise awareness for female reproductive healthcare.
Producing Antibodies in Escherichia coli
Our project makes use of synthetic biology by synthesizing proteins and antibodies through the creation of genetically modified organisms. The use of synthetic biology in producing materials is valuable because of its customizability and ease-of-use, which are principles that are integral to our project design. When creating our test, we found several important biomarkers for endometriosis that we believe to be critical in accurately predicting and diagnosing the disease. Immunoassays provided the best approach for the sensitive detection of our desired biomarkers, but as a first year team, we were worried about the high cost of using antibodies. As such, genetically engineering organisms to produce our required antibodies would help lower the cost of an immunoassay design for both ourselves and future iGEM teams. Previous literature research has shown the efficacy of using a specially engineered E. coli strain, SHuffle, for the production of antibodies. This strain has an oxidative cytoplasm and additional chaperone proteins that allows for proper folding and disulfide bond formation in full length immunoglobulin proteins (Lobstein et al., 2012). Our team created BioBricks for the variable and constant regions of a therapeutic antibody used in our design, Siltuximab, including a mutated constant chain for efficient use in E. coli SHuffle. This mutated constant region will be available for use by future iGEM teams who wish to produce their own antibody by combining our constant region BioBrick with their desired variable chain region via standard assembly for efficient production in E. coli SHuffle. This rapid, inexpensive method of antibody production will allow not only for immunoassay development for iGEM teams but also lays a foundation for the production of promising immunotherapy methods for the treatment of endometriosis and other chronic conditions.
Therapeutic Estrogen Response Elements
Our team hopes to create a therapeutic sensor for estrogen levels in endometriosis patients. This goal will be completed by designing a gene circuit that becomes active at elevated levels of estrogen to produce a reporter protein which can indicate to the patient the recommended dosage of hormone-targeting drugs. This gene circuit will build off of the work of the 2016 MIT Team, who worked on creating a promoter that could be induced by the binding of an estrogen receptor to estrogen response elements (EREs). EREs are short sequences of DNA preceding promoters which are activated when bound to by estrogen receptors (which translocate to the nucleus to promote transcription when activated by estrogen). We hope to improve the design of their circuit by altering the promoter to only respond to clinically elevated levels of estrogen and using E. coli as a chassis organism rather than human cells. The use of E. coli will make this gene circuit more accessible to other iGEM teams and will also avoid the ethical issues of designing a system for therapeutic use in human cells. While we do not intend to create a hardware device for this gene circuit, we postulate that in the future, this system could be integrated with hardware to provide rapid results of elevated estrogen levels that recommend a medication dosage for management. This could be beneficial if used as a personal device for patients undergoing hormone therapy compared to standard laboratory blood tests.
Create reliable diagnostic tools for endometriosis
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Wet Lab
Perform in-depth literature review.
Connect with experts in endometriosis diagnostics and research.
Determine simplest and most cost efficient method for a point of care diagnostic.
Select threshold values for assays.
Work with the modeling team to determine assay parameters.
Design BioBricks using Snapgene to create a sequence optimized for our chassis organisms of choice.
Develop protocols for the transformation of our target chassis organisms and method of isolation.
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Modeling
Obtain patient data sets from endometriosis researchers.
Work with the Institutional Review Board at University of Rochester to ensure the proper protocols are in place to protect patient information.
Use various classification methods to create algorithms then determine which model has the highest diagnostic accuracy.
Use various classification methods to determine the importance of each clinical variable in diagnosing endometriosis.
Find sensitivities and specificities of various biomarkers from literature.
Create a script to determine which combination of biomarkers has the highest sensitivity and specificity through combining the log odds ratios.
Confirm independent antibody-biomarker-receptor binding:
Predict 3D structure of antibodies with Repertoire Builder.
Find epitopes on biomarker with Rosetta SnugDock.
Antibody pairs with non-overlapping epitopes are chosen as detector-receptor pairs.
Behaviors of molecules on an LFA test strip are described by a system of partial differential equations (PDE), which are solved in MATLAB. The solution shows how the test line signal develops over time for a given LFA design. The model is used to choose the LFA design whose signal meets wet lab and hardware requirements.
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Hardware
Research current methods of signal detection for lateral flow assays and gold nanoparticles
Investigate and design more affordable and accessible options for signal detection
Connect with experts in molecular recognition and biosensing
Decide on the method of analysis, we chose to use a smartphone camera to provide a semi quantitative analysis of the gold nanoparticles presence
Test a dilutions series for gold nanoparticles and then use a phone camera to see if it is possible to differentiate between them
Find the threshold that indicates whether the patient has endometriosis or not
Design the 3D model for the phone holder and lateral flow assay chamber
Connect with experts in optical biosensing
Look into finding the appropriate optical fibre bundle, and which LED color and nanowave are most appropriate for the used size of gold nanoparticles
Model the proper LED light angle for best reflection angle on the assay to get the clearest image
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Software
Use Xcode (Swift) to integrate our risk calculator model that allows users to enter their clinical variables and predict their risk of having endometriosis.
Integrate endometriosis educational program and menstrual tracking program into a software application.
Let potential users try the software application and then edit the application based on their feedback.
Utilize designs that are compatible with endometriosis symptoms
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Modeling
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Hardware
Model of menstrual cup in vaginal canal
After speaking to endometriosis specialists and menstrual cup companies, we learnt that the menstrual cup designs in the current market are unsuitable for endometriosis patients, because their vaginal muscles are stiffer than non-patients. We wanted to design a new menstrual cup that would put the least amount of pressure on the vaginal canal. We did this by creating a model of a cup in a vaginal canal in FEBio and exploring various cup shapes. We started with designs of available menstrual cups that are said to be more comfortable in online user reviews (non-protruding rim, toggle stem, soft material, etc.) and made tweaks to the design to reduce the pressure.
Pesign 3D model on Onshape
Design a syringe or vial for the purpose of transporting the menstrual affluent to the clinics
Further research into endometriosis therapeutics
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Wet Lab
Perform literature review into the feasibility of circuit design.
Collaborate with modeling to determine the best promoter and RBS to use according to desired levels of estrogen.
Put together information from past literature and modeling to design a new estrogen responsive BioBrick.
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Modeling
Perform literature review into modeling gene expression
Create model that utilizes ordinary differential equations
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Hardware
Connect with pelvic floor physiotherapists
Utilize the data from the vaginal canal pressure modeling
Look into vaginal dilator designs and method of improvement for endo patients
Design an improved vaginal dilator that has muscle relaxant properties
Design affordable and easy to use laboratory equipment
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Hardware
Design centrifuge using recycled components
Submit safety proposal
Sketching the circuits and components diagram
Build 3D model for the sample holder
Build the centrifuge prototype
Coding the motors, buttons, LCD screen and the 4 digit 7 segment display
Test runs and improve the code based on the results
Add safety features such as the plexiglass chamber surrounding the rotation center
Write a comprehensive guideline for future teams to be able to build the centrifuge
Look into different substitutes for the 3D parts to make the building of centrifuge more accessible
Send model to Lambert High school for testing using their speed sensors, and send the guidelines and pieces to Linkoping to check the comprehensibility of steps
Further look into methods of testing using speed sensors to prove functionality by University of Rochester staff members
Spread awareness of endometriosis and women’s reproductive health
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Public Relations
Create content that informs the general public on endometriosis and issues surrounding endometriosis
Connect with endometriosis activists, researchers, and organizations through social media
Share content that informs the general public of our project and how our project aims to address issues surrounding endometriosis
Utilize press releases in local newspapers and news outlets to share our project
Create an engaging promotional video for our project
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Outreach
Collect information about endometriosis and menstrual health from scientific papers.
Connect with endometriosis specialists to receive feedback and determine what content to include in the educational materials.
Collaborate with endometriosis organizations and health clinics to distribute the materials.
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Policy and Practice
Conduct a literature review.
Develop a survey regarding individuals’ experiences with reproductive health care and endometriosis.
Work with the Institutional Review Board at University of Rochester to ensure the proper protocols are in place to protect participants’ information.
Analyze the data obtained to identify relationships between different countries and these experiences.
Incorporate feasibility, ethics and sustainability into project design
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Policy and Practice
Conduct a cost analysis of our project including the direct material cost for both hardware and wet lab, fixed cost and both direct and indirect labor cost
Perform research on current competitors, including price and advantages
Research on market trend and potential gain of our project
Conclusion on our project’s feasibility
Perform SWOT analysis (pro and con of our products compared with competitors)
Develop short and long term plan of action for doing business with our project with patent, research, marketing, distribution in consideration.
Perform research on typical ethics concern and sustainability problem with regarding the use of non-invasive diagnostic, antibody test, menstrual cup and UV Sterilizer
Work with stakeholder to discuss the potential to bypass the ethical and sustainable concern
Finalize our research on past igem documentations
Lab safety, product safety, ethics (Sustainability)
Make our project, and other iGEM projects, accessible across languages and cultures
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Collaboration
Collaborate with iGEM teams to translate each others’ promotional videos and social media content into a multitude of languages.
Present our project in poster sessions and workshops.
Attend other teams’ poster sessions and workshops to learn what other current teams are doing and gain insight into syn-bio techniques they plan to apply.
Utilize the feedback from these meetups to improve our project design.
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Pubic Relations
Translate materials into as many languages as possible.
Connect with leaders from the local Deaf community to learn how to make our content accessible to the deaf and hard of hearing.
Create audio descriptions for videos to make our content accessible to the visually impaired.
Increase knowledge about synthetic biology and resources for iGEM teams
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Software
Work with the wet lab and modeling team to develop the content for a biomarker database.
Create the biomarker database using MySQL on the University of Rochester server.
Create a website that links and presents the database.
Collaborate with other iGEM teams to add more biomarkers to the database.
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Outreach
Connect with experts in pedagogy to help design the course.
Select topics in general biology, genetics, and synthetic biology.
Design presentations and activities for each module.
Receive feedback from high school students and teachers to improve the modules.
Connect with elementary teachers to help design the experiments.
Create simple science activities that introduce basic concepts in biology, genetics, and synthetic biology.
Receive feedback from students for each activity and use the feedback to improve the following weeks’ activities.
Courses developed for school 19 are improved after.
Improve science activities based on feedback from Sonshine Camp and deliver improved activities to RCSD School 19.
The major inspiration for our project came from a recent research publication detailing the presence of biomarkers for endometriosis in menstrual effluent (Warren et al., 2018). Having a team member who had been diagnosed with endometriosis, we were shocked to learn about the lack of research and funding that were allocated to this chronic disease and wanted to improve the quality of care provided to endometriosis patients. Warren et al. not only exposed our team to endometriosis research and unique diagnostics methods, but also inspired us to approach this problem with synthetic biology principles.
We were also inspired by a previous iGEM team, MIT 2016, who tried to detect endometriosis using newly characterized parts from mammalian synthetic biology. Their work demonstrated how synthetic gene circuits can be utilized to detect endometriosis biomarkers and inspired our team to not only build upon this work, but find new ways to utilize synthetic biology in endometriosis diagnostics.
COVID-19 forced our team to perform the work for the majority of our project online. With team members from different areas of the world, communication and organization became difficult. We also had to heavily rely on past research and published literature for developing our project rather than hands on lab research.
However, despite the difficulties, the switch to online research provided new opportunities for collaboration between our sub-teams. Our modeling team provided critical information to our wet lab team regarding the selection of materials and set up of our lateral flow assay. Our hardware team was also able to recognize the difficulties of having limited access to materials and were inspired to create build-it-yourself centrifuge to resolve this issue for teams, clinics, and laboratories across the world. Furthermore, all in person activities were cancelled but we were still able to connect and partner with organizations and foundations that support and actively engage in endometriosis. While our team faced setbacks due to COVID-19, we also took advantage of unexpected opportunities that shaped our project goals.
