Fulfillment of Medal Requirements
We have satisfied the Bronze Medal Requirements as detailed below:
Competition Deliverables: We completed the wiki, presentation video, and judging form.
Attributions: We have detailed our attributions on our attributions page.
Project Description: We have described our project inspiration, motivation, and goals on our project description page.
Contribution: We have contributed to the field of synthetic biology by raising awareness of the need for orthogonality assessment, developing a toolkit for orthogonality assessment including both a model and sensor circuits, producing an RNA-seq data set, creating a pamphlet and presentation for retirement homes, designing a guide for using Google Drawings, and writing an review of orthogonality assessment in the field.
We have satisfied the silver medal requirements as detailed below:
Engineering Success: We have demonstrated engineering success by describing in detail the iterative design, build, and test cycle for our project. We extensively detailed how we constructed our circuits, developed experiments to test the circuits, evaluated the outcome of our experiments, dealt with unexpected results, repeated the process, and planned further steps.
Collaboration: Our team collaborated with specific individual teams and participated in, as well as hosted, collaborations with many teams. We collaborated with several iGEM teams by organizing and hosting this year’s Mid-Atlantic Meetup, which seven teams participated in. We participated in the Dusseldorf Postcard Project and the SDG Impact Challenge. In addition to broad collaborations, we collaborated specifically with OSU iGEM’s modeling team and Gaston Day School iGEM. Our team also collaborated with Virginia iGEM to test one of their genetic parts using our orthogonality toolkit. Finally, we completed surveys for several other teams.
Human Practices: Our team engaged in extensive Human Practices, interviewing stakeholders and experts that guided, informed, and changed the design and direction of our project on multiple levels.
Proposed Implementation: We presented a proposal for the implementation of our project, detailing the distribution of our sensor circuits and model, as well as how teams would use our project to assess the orthogonality of their own circuits.
We have satisfied the gold medal requirements as detailed below:
Integrated Human Practices: We conducted in depth interview sessions with 7 experts and stakeholders: Mr. John Marken, Mr. Aqib Hasnain, Dr. Jeffrey Barrick, Dr. Gale Smith, Dr. Brian Renda, Mr. Luis Ortiz, and Mr. Paul Maschhoff. We demonstrate how they informed our project and how our project direction and design changed substantially based on their input.
Improvement of a Part: Our team was able to improve a part designed by the iGEM12_Tokyo-NoKoGen iGEM team.
Project Modeling: We employed modeling to generate a standardizable metric for orthogonality. Our data-driven model has two separate parts: a system of ordinary differential equations describing increased burden on a host and a comparison of marker gene expression in circuits with and without a host. We created a model page detailing every aspect of our model including its assumptions, data, parameters, and results.
Proof of Concept: We have written a proof of concept analysis of our circuit.
Education and Communication: We provided educational experiences to those in our community through presentations at a local festival and at local retirement communities in our area. These presentations provided basic information about biology, genetic engineering, synthetic biology, and applications of synthetic biology in the real world. Our team created a pamphlet and powerpoint to help other teams reach out to individuals in their local retirement communities.
Excellence in Another Area: We exhibited excellence in another area through several ways: writing an in depth guide to the plastic degradation protein, PETase, developing a graphic guide teaching individuals how to use Google Drawings, producing RNA-seq data of our own, and creating a critical in depth literature review of orthogonality assessment in synthetic biology.
Additionally, we would like to be considered for the following prizes:
Education: In addition to our K-12 outreach at community events, this year our team’s education activities focused on an underserved group in synthetic biology education: residents of retirement homes. Our team visited and spoke with local retirement homes about synthetic biology. The residents were extremely engaged in our presentations and demonstrated a clear interest in learning more about synthetic biology. As a result, we wanted to encourage and support other iGEM teams to engage with this community. To accomplish this, we developed a pamphlet with information on presenting to retirement homes, including what information to cover, how to best convey that information, and presentation strategies that allow for effective communication. Our pamphlet is informed by feedback we received from both residents and the administration of retirement homes on the most effective ways to deliver introductory information on synthetic biology. Our pamphlet is accessible to all future iGEM teams on our wiki.
Inclusivity: Our team demonstrated inclusivity through team diversity, project inclusivity, and education outreach. Our team itself is inclusive, demonstrated by the racial and gender diversity of our members. 50% of our team are students of color and 70% of our team, including our two team captains, are female. In addition, we designed our project to be as accessible as possible. Our model is able to incorporate inputs with varying levels of complexity ranging from straightforward measurement of fluorescence values from our circuits to RNA-sequencing data. For our circuits, our team developed a system to make orthogonality assessment more accessible and affordable. Finally, inclusivity was an important component of our education. Our education outreach centered around a highly overlooked group of individuals in terms of synthetic biology: retirement communities. Our team’s goal was to educate these individuals, who may not otherwise have this opportunity, about the field of synthetic biology.
Integrated Human Practices: Every aspect of our project was continually modified and extensively improved by feedback from both experts and stakeholders in the field including representatives from academia and industry. All experts agreed on the pressing need of such assessment, but there was considerable disagreement on the specific delivery system. Dr. Barrick and Dr. Smith highlighted the accessibility of TXTL systems, they expressed concerns with removing cell barriers. Mr. Marken and Mr. Ortiz both noted the technical challenges of using TXTL systems, and Mr. Ortiz suggested using a plasmid system for ease of use. Given the consensus for using an in vivo system, we constructed and tested a plasmid system while designing protocols for genome integration and TXTL systems for future use. Additionally, experts suggested specific gene markers to include; Drs. Barrick and Smith suggested construction of a sensor for negative feedback within the cell, prompting us to design our AG43 sensor.
Measurement: Measurement was a central driving force for our project since its inception and impacted every phase of project development. In order to determine which genes should be assessed by our orthogonality sensor, our team performed an extensive quantitative analysis of existing RNA-seq data collected by previous researchers, creating Venn diagrams to determine which genes were most affected by the presence of a circuit. Once our team identified these genes, we conducted an analysis to ascertain which parts would best fit for the design of our circuits. During circuit testing, we used a plate reader to take measurements at six time points for each sample in order to collect dynamic data for our model and ensure its accuracy. While independent calebrants were not available to us (we read Dr. Jake Beal’s papers), we performed multiple positive and negative controls and also conducted RNA-seq to complement other measurements.
Model: This year, our team developed two discrete but integrable approaches to quantifying a circuit’s orthogonality to its host; a mechanistic model of ODEs coupling classical models of metabolic burden in E. coli and markers of orthogonality derived from analyzing RNA-sequencing data, as well as a purely data-driven framework that directly processes RNA-seq datasets and outputs a quantitative evaluation of orthogonality. The former simulates different conditions of an E. coli cell, either untransformed or acting as a host to a circuit, and applies dynamical systems analysis to examine differences in their steady states, with parameters for the model derived from fluorescence measurements from our sensor circuits. The latter uses principal component analysis to determine correlations between individual genes and overall differential gene expression, and uses those genes as proxies for measuring overall orthogonality. Together, these models offer an accessible approach to holistically evaluate orthogonality with regard to and beyond metabolic burden.
Safety: A primary goal of our entire project was to help ensure the safety of all synthetic biology circuits. We accomplished this by developing an accessible system for assessing orthogonality, defined as the lack of unintended interactions between the circuit and the host. Not only do unintended interactions compromise circuit efficacy, but they raise serious safety issues. Orthogonality is crucial to ensuring the safe “fieldable” application of all genetic circuits. By inputting measurements from our circuit system into our model, teams can assess orthogonality of their circuits and use their results to improve its efficacy and safety. Additionally, our team employed extensive safety practices in our lab including adherence to CDC, NIH, and W&M Safety Office best practices including an iGEM IBC approved protocol developed by the team. Additionally we engaged in extensive training from the University, iGEM, and our PI, taking advantage of every safety training opportunity.