Overview

Healthcare is broken down into one of two categories: reactive and proactive. The reactive approach utilizes medical intervention to treat already present symptoms and conditions (Waldman, 2019). Unfortunately, the United States healthcare system focuses strictly on reactive treatment, accounting for more than 75% of healthcare spending in the U.S. (Mercury Healthcare, 2018). These reactive practices occur in coronary artery disease (CAD) diagnosis; patients undergo invasive tests after showing observable symptoms (Skelly, Hashimoto, & Buckley et al., 2016). Although there is a shift from reactive to proactive healthcare regarding age-related diseases, patient retention remains an issue (Mercury Healthcare, 2018). While people visit facilities for early testing, the delayed process of receiving lab results- whether it be from outsourcing samples to different labs, scheduling, etc.- has the effect of fewer people returning to healthcare facilities for preventative care (Mercury Healthcare, 2018) . Furthermore, four out of seven studies reported missed diagnoses of malignancy, hypothyroidism, hyperthyroidism, osteoporosis, and microbiological results (Callen et al., 2011). CADlock aims to increase the accessibility to preventative healthcare by emphasizing affordable screening, early diagnostics, and accessibility for all. This approach will ensure care for individuals of all demographics and socioeconomic statuses. Therefore, our team’s goal was a multifaceted approach using synthetic biology to develop diagnostic solutions, to educate our community about proactive healthcare, and to include underrepresented populations within our initiatives (see Fig. 1).

Figure 1. The expansion of our inclusivity focus this year, combining accessibility, affordability, and accuracy under proactivity.

Our inclusive preventative approach begins by diagnosing CAD via microRNAs. We expanded Rolling Circle Amplification (RCA) to exponential RCA (eRCA) which increases signal strength and decreases time to detection from picomolar concentrations of miRNA in serum samples. This approach directly targets the issue of patient retention by increasing the accessibility of knowledge patients have about their condition. This early diagnosis and consultation brings patients back in since they know they may be susceptible to a disease, and thus may prevent it before their condition worsens. In addition to using eRCA, we also demonstrate using capillary tubes to perform RCA reactions with the goal of incorporating a future all-in-one reaction/visualization device for point-of-care testing. To address the high cost of phi29 polymerase we experimented with cloning and purifying our own proteins. Under the advice of Dr. Charles Searles, in conjuction to changing the RNA detection methods, we also expanded our library of miRNAs to include multiple novel sequences shown to be indicative of CAD in African American populations in Atlanta, GA (Dluzen et al., 2016). We envision an eventual library of miRNA padlocks available to clinicians to provide individualized medical diagnostics, thus enabling clinicians to directly provide individualized early diagnostics to their patient populations. Essentially, the increase in the rate at which patients receive their test results is positively correlated with proactive care because patients are more willing to take precautionary measures when they are given optimal accessibility to care (Mercury Healthcare, 2018).

During our research into miRNAs, it was evident that a lack of a database for miRNAs specifically indicated in coronary diseases was needed. To address the gap, we developed CADmir, a large language model (LLM) powered web app that synthesizes scientific information backed up by open-source, peer reviewed miRNA research (see Fig. 2). This will allow anyone with computer access to quickly conduct a literature review on heart disease related miRNA research, increasing accessibility to scientific knowledge.

Figure 2. Image of the CADmir user interface

Pipetting multiple reactions for trials in triplicate can be tedious and lead to errors that interfere with data analysis or worse, having to repeat costly experiments. We developed LabPilot, an automated pipette system to increase pipet reliability and increase the researcher’s time efficiency. According to multiple interviews with current Ph.D. and Postdoctoral researchers, pipetting is time consuming, error prone, and seen as a hindrance to their “real” work LabPilot is a low-cost alternative to commercial liquid handling systems that cost $10,000+, limiting their use to well funded labs (see Fig. 3).

Figure 3. LabPilot pipeline

In our current use, RCA reactions output a fluorescent signal leading to the need for a method of quantifying the output. Last year, we developed Micro-Q, a low-cost fluorometer that utilized a photoresistor, cloud-based quantification algorithms, and an intuitive mobile app to quantify biotechnology reactions. This year, we improved Micro-Q to quantify most fluorescent materials by utilizing a 5000k LED strip and expanded the capabilities to quantify a strip of PCR tubes with a camera-based quantification method. Ultimately, this device increases accessibility to fluorescence based experiments and diagnostics worldwide.

Preventative care is two-fold, relying on early detection and educating patients. By implementing different RNA detection methods, software, and novel hardware, we aim to increase early detection. Educating local and dispersed populations was another major initiative this year. We held a local Heart Healthy Awareness month and contributed to a state-wide teacher professional development. To reach a broader audience, we created a podcast called SynbioOutLoud, and collaborated with Flowers for the Future, an international organization dedicated to supplying resources in order to promote STEM education for girls in Kabul, Afghanistan.

Wetlab

RCA

Furthering last year’s approach with accessibility, affordability, and accuracy, we continued utilizing and improving RCA to assess CAD risk groups while also addressing underrepresented demographics of people. Improvements to last year’s RCA mechanism include testing the biosensor’s specificity, comparing reporter mechanisms, and optimizing reaction times. In addition to RCA modifications, Lambert iGEM pursued a new wet-lab initiative called exponential rolling circle amplification (eRCA). eRCA provides a more sensitive reading of microRNAs with a faster reading time. The emphasis on additional accuracy through eRCA and affordability to end users enables CADlock to be accessible to a wide range of demographics, not limited to race, sex, and economic status See RCA: eRCA.

Capillary

Lambert iGEM explored optimizing the reaction time and resources by running the reaction in capillary tubes instead of PCR tubes for DNA amplification and imaging. This quicker response time allows for more patients to be tested in a point-of-care setting. This increase in healthcare accessibility and thus patient retention time allows for an emphasis to be placed on proactive care. MicroRNAs (miRNAs) can easily diffuse through the capillary tube due to the tube’s small diameter. This results in high amounts of DNA in isolated locations (see Fig. 4). When exposed to a specific wavelength of light, individual specks of DNA can be seen by the naked eye. DNA can then be counted without the use of complex fluorescence quantification devices such as fluorometers or plate readers, which makes the test more accessible and lowers the technology barrier. Overall, capillary amplification is a reliable, efficient, and user-friendly assay. See Wetlab: Capillary.

Figure 4. A representation of the difference between the RCA reaction in the PCR tube vs. the capillary tube.

Protein Purification

In addition to frugal technologies, Lambert iGEM aimed to decrease the cost of Rolling Circle Amplification (RCA) reagents by producing the enzymes required through protein purification. Phi29 DNA polymerase, priced at $251.00 for 1,250 units from New England Biolabs, not only covers the majority of the cost of RCA reagents but also possesses invaluable qualities such as strand displacement and processive synthesis properties that are necessary for RCA (New England Biolabs, n.d). By producing phi29 DNA polymerase through protein purification, combined with the accuracy of exponential RCA, Lambert iGEM aims to increase the affordability and in turn accessibility, both of which are contributing factors to inclusive care. This allows for more tests because the proteins are much more affordable, increasing the preventative aspect of healthcare. See Wetlab: Protein Purification.

Estrogen

While researching CAD risk factors and statistics, we came across literature that informed us about disparities among the sexes concerning early CAD diagnosis. We first conferred with Dr. Mindy Gentry, a women’s cardiologist, to get her opinion on the sex differences in cardiovascular health. She confirmed our insights about estrogen being a factor in increased susceptibility to CAD in women. To further our scientific research, we spoke to Dr. DeLisa Fairweather, a director of translational research in the Department of Cardiovascular Medicine at Mayo Clinic’s Florida campus. She educated us on estrogen and its role as a CAD suppressor, and explained how women after menopause are more likely to develop heart disease because estrogen levels drastically decrease after menopause, causing women to be more likely to develop inflammation. Dr. Fairweather detailed that women who have inflammation in the heart are more likely to develop autoimmune disease and CAD. Additionally, she also guided us in the further development of our inclusivity by discussing how the hormone affects men as well. She told us that since men have a low estrogen level that also remains stable, they would be an ideal demographic to test for consistency in results, unlike women who have fluctuating levels of the hormone. Under the guidance of Dr. Fairweather, we tested miRNA-20b using Rolling Circle Amplification. In the presence of estrogen, CAD suppressors are activated and miRNA-20b is produced. miR-20b causes a negative feedback loop and suppresses estrogen, which will in turn inhibit the CAD suppressors. Dr. Fairweather explained how in men, a high level of miR-20b means that they are likely to develop CAD, however, in premenopausal women with inflammation symptoms, miR-20b protects them from developing CAD. She explained how if there is a high level of 20b, it will inhibit the estrogen from causing a catalyst between the developing autoimmune disease and CAD. We tested miR-20b with the RCA mechanism and ran a 1% gel to validate the presence of our product (see Fig. 5). Ultimately, increasing the factors that can be tested to signify CAD correlation encompasses a wider yet specified group, allowing for quality proactive care to be implemented. See Wetlab: Inclusivity.

Figure 5. The working gel, two wells on the right of the ladder contains the estrogen RCP.

Software

CADmir

Lambert iGEM developed CADmir as a centralized microRNA (miRNA) database to enhance researchers’ access to functional information about miRNAs more efficiently. (see Fig. 6) Currently, valuable insights on miRNAs are dispersed throughout academic literature, posing a challenge for researchers seeking detailed information about specific miRNAs. CADmir aims to address these issues by offering a cohesive and easily accessible platform that organizes all of the current scientific literature about miRNAs, facilitating quick and efficient searches. Lambert iGEM hopes that with CADmir, more researchers will explore miRNA research and incorporate miRNAs into more projects without requiring substantial amounts of time dedicated to reading through papers. CADmir aims to advance the field of miRNA research by improving information accessibility and reducing the time barrier associated with obtaining high-quality scientific information on these topics. See Drylab: Software.

Figure 6. Timed research trial for the research through CADmir, approximately four minutes to find the estrogen miRNA and resources.

LabPilot

LabPilot was targeted towards underfunded laboratories and independent researchers to increase accessibility to this crucial equipment. The leading retailing liquid handler costs around $10,000, prohibiting less-privileged laboratories from purchasing liquid handlers and instead manually pipetting (OT-2 Robot, 2023). LabPilot is primarily made with 3D printed parts, making it affordable and easily replicable by any community with a 3D printer at a build cost of $250 and a pipette cost of $409 (Eppendorf Research Plus Adjustable Volume, Single Channel Pipette, 2 - 20 uL, Yellow)(See Parts List), and about 6.5% the cost of a commercial device (OT-2 Robot). LabPilot upholds the standard of accuracy found in other liquid handlers by having a significant difference when comparing the volume of liquid pipetted by LabPilot and a human (See Proof of Concept). LabPilot’s intuitive software utilizes critical user experience (UX) principles: a user can quickly transfer liquid from one location to another with just three taps. Additionally, LabPilot’s software conforms to the Web Content Accessibility Guidelines 2.1 standards, ensuring a quality user experience for people with disabilities. See Drylab: Hardware.

Micro-Q

Many medical diagnostics utilize fluorescence reporter mechanisms with mathematical models to quantify substances. However, commercial fluorometers are often bulky and cost over $10,000, making these diagnostics inaccessible. Last year, Micro-Q exhibited single-tube green fluorescence quantification for $16 but was limited to one sample. This year, Micro-Q Plus allows multi-sample, multi-spectral quantification with camera based fluorescence detection enabling automatic generation of fluorescence/protein expression curves. All these capabilities fit in a pocket-sized portable box, increasing accessibility to underfunded labs and the public. See Drylab: Measurement.

Education

Summer camps

Lambert iGEM held a summer Biotechnology Bootcamp for students between the ages of 12-14 where students participated in hands-on experiments while learning fundamental scientific concepts and skills needed in preparation for high school. The students learned about DNA structure and functions, acids and bases, proteins and enzymes, and more through a biotechnology application lens. The students were given pre and post surveys every day to gauge their learning progress. For example, we presented statements like “I can describe the function and purpose of a gel electrophoresis.” and the campers would respond with how confident they were. After speaking with our advisor, Dr. Brittney Cantrell, she provided us with a standards alignment chart that is used by the Forsyth County Board of Education. We reorganized the chart to include a link to all presentations, activities, and pre-planned labs that Lambert iGEM has made so that teachers are able to follow the new biotechnology curriculum and find easily accessible materials. Alongside the teaching materials, we linked competitions and events that younger students who are passionate about science can participate in.

Teacher training

To supplement the Standards Alignment teacher resources, Lambert iGEM attended the Georgia Teacher Training Conference at Athens Technical College. Teachers from around the state participated in learning how the new introductory synthetic biology curriculum ( which was created by following the Science Georgia Standards of Excellence) will embed itself within the current science curriculum, and specifically how teachers can make these new concepts and applications of science in the real-world engaging and understandable to their students. We led and assisted teachers through a lab that they could implement in their classrooms. These lessons will particularly be implemented at the middle school level, where students have enough background knowledge of cellular functions and will be able to understand synthetic biology. Additionally, we were able to conduct pre and post-surveys at the teacher training, which helped us evaluate the level of impact that students and teachers will have upon implementation of the synthetic biology curriculum using the Standards Alignment resources (see Fig. 7)

Figure 7. Images from the data collected from surveys at the teacher training workshop.

Podcast

Human Practices involves speaking with various professionals, doctors, and researchers to gain insight into the direction and impact of our project. This year, Lambert iGEM made the knowledge available to anyone interested by creating a podcast on Spotify called SynbioOutLoud (see Fig. 8). In this podcast, we invited a diverse set of several guests, including cardiologists and nutritionists, to discuss barriers they have experienced in the STEM field and how they handled prospective prejudice. We took their primary accounts and feedback to inspire our project direction for this year (see Fig. 9). Dr. Mindy Gentry, a women’s cardiac care specialist, expressed the lack of heart health awareness for women, enabling us to expand sex inclusivity to promote our ongoing research on estrogen’s correlation to CAD (see Estrogen, Inclusivity). Mrs. Saru Bhardwaj, Lambert High School’s AP chemistry teacher, expressed the lack of education regarding gender disparities, and the changes she encountered when she was a student. In addressing this, we conducted a meta-analysis on gender distributions spanning six years of iGEM teams and compiled the findings into a comprehensive spreadsheet. In the study, we found that although the demographic distributions on teams are equal, the specific occupations on the team are very segregated. For example, the females on the teams are primarily centered around human practices, while the majority of males work with hardware. We noticed this trend in our teams too, and so combatting this trend was an integral inspiration for our inclusivity this season. Dr. Cantrell, a member of the Forsyth County Schools Secondary Science Specialist, iGEM advisor, and guest on our podcast informed us about a lack of community involvement she has observed while trying to promote inclusivity in science education. She expressed the need for female role models in the current society. In order to synthesize and express what Lambert iGEM has learned from these conversations, we developed a final episode of our podcast where we talk about our personal account of being students and women, and what we have noted from this experience. We discussed the “why” of our initiatives this year and the ideas behind their implementations. Overall, the SynbioOutLoud podcast enabled Lambert iGEM to create products catering to the community’s education and awareness (see Fig. 10).

Figure 8. SynbioOutLoud podcast cover image.

Figure 9. The number of plays and published episodes of our podcast.

Figure 10. The data per person who spoke on our podcast per listener interest.

Heart Health Awareness month

Lambert iGEM planned to expand upon the SynbioOutLoud podcast by hosting an audience consisting of Lambert High School’s HOSA chapter, Science Olympiad, and Women in STEM club members to listen to highlighted guest speakers and professionals in the field. However, we wanted to expand the outreach by increasing the number of people who had access to this opportunity, so we converted the event to virtual webinars that focus on all aspects of heart health, both proactive and reactive, for the entire month of September. As the name suggests, the “Science for All” purpose was to access a wider audience, thus further creating an inclusive approach to education. Lambert iGEM hosted webinars discussing the importance of nutrition and exercise to prevent heart disease, and the process of First Aid/CPR/AED as a response system for when heart emergencies occur. Lambert iGEM was also accompanied by the American Heart Association (AHA), who co-hosted the webinar, teaching the audience about emergency response and CPR. While the members were not certified due to safety concerns, they were equipped with the proper knowledge to be an upstander rather than a bystander in the event of a cardiac emergency (see Fig. 11). To conclude the event, we specifically presented our gender research of all iGEM teams, along with the analysis of the impact created and the perceived trend for future demographic distribution in science roles. By advertising the “Science for All” webinars through the county for greater demographic participation, Lambert iGEM was able to address a larger number of people through social media connections, the involvement of the AHA in promoting the webinar, and outreach towards other STEM clubs such as Women in STEM, Science Olympiad, and HOSA. This event attempts to spread the knowledge of synthetic biology and STEM. We are also currently in the process of updating a formal inclusive Cardiac Emergency Response Plan to be implemented and piloted at Lambert High School.

Figure 11. Webinar participants at our school receive prizes for answering questions correctly.

Flowers for the Future

After a discussion with some of our members who are part of the Lambert Women in STEM club, we decided to contact an organization called Flowers for the Future. They are a student-led organization that helps girls in Afghanistan by providing resources for STEM education for girls. Some of the topics covered include chemistry, biology, electricity, cellular pathways, computer science, coding, and mathematics. We noticed that their curriculum does not include resources for biotechnology concepts such as electrochemistry, application, lab equipment, basics of central dogma, DNA extraction, the purpose of pipetting, PCR, and Gel electrophoresis. After a discussion with the Flowers for the Future members, we agreed to send them multiple videos and presentations of these concepts with additional labs that they could do with household items, as they were not allowed access to many items due to Taliban restrictions. In order to maintain safety and confidentiality for our students and partners, Flowers For the Future makes no mention of any names, locations, and/or specificities anywhere on websites, social media, or the internet (e.g. “learning academy,” not “[name] school”) to avoid the potentially dangerous path to the academy. The organization also fundraises to provide the girls with laptops and wifi so they can learn from home and not be seen or have their safety undermined. They also do not publicize the organizations they work with, and make sure that any and all released video lessons are put on the Flowers for the Future YouTube channel, with no other information given than the instructional topics being presented.

References