Human Practices
Human Practices
Overview
Last year, Lambert iGEM identified patients as the end goal of CADlock’s implementation. In order to accomplish this, we interviewed multiple leading microRNA researchers and cardiologists, who contributed to identifying the direction of our project as a point-of-care diagnostic screening tool to track the progression of coronary artery disease (CAD). Inspired to expand last year’s project with a focus on inclusivity, we improved and implemented wet lab mechanisms, including exponential rolling circle amplification (eRCA) from RCA see RCA. We also used protein purification to create our own phi29 DNA polymerase to minimize reaction costs. In addition, we expanded upon MicroQ, implementing an adaptation called MicroQ Pro. Furthermore, after talking with a graduate student, we developed LabPilot, an automated pipetter. We created a podcast and spoke to multiple doctors and professionals about the direction of our project. Drawing on the expertise of exceptional stakeholders, our journey transformed from conceptualization to implementation (see Fig. 1).
Doctors
Dr. Gentry
We spoke to Dr. Mindy B. Gentry, a cardiologist in Marietta, Georgia, about sex inclusivity and CAD. We originally planned to use a gene called SVEP1, a gene that influences atherosclerosis. Specifically, SVEP1 expresses a protein that stimulates the development of plaque in arteries. This gene has a negative correlation with a miRNA named miR-1269b. Instead, she guided us to look at CAD correlation with an emphasis on estrogen-induced pathways. With her help, we were able to find a correlation between CAD and an estrogen-specific miRNA. Dr. Gentry, who runs a female cardiac health center, expressed the need for proactive measures to be taken to preserve heart health for females. The incorporation of Lambert iGEM’s estrogen initiative, guided by her aid, strengthens the proactive approach by making a diagnostic for a specialized risk group.
Dr. Fairweather
Dr. DeLisa Fairweather is a director of research at the Department of Cardiovascular Diseases at Mayo Clinic’s Florida campus. She conducts research on individualized therapies and improved diagnosis in the Cardiovascular field. Due to her specialized and intensive research on estrogen in cardiovascular disease, we spoke to her about our initiatives in trying to incorporate sex inclusivity in our project. She informed us that estrogen increases autoimmune disease in women, and inflammation associated with it leads to CAD in women. Initially, we had the understanding that miR-20b should protect women from CAD, but she clarified when estrogen drops with menopause, the Estrogen Receptors Alpha pathway would be so low that miR-20b would have no effect. On the other hand, miR-20b is a viable indicator for CAD in men. She guided us to expand our focus to this miR to encapsulate men, as well as have a specialized risk group for women who have symptoms of inflammation. Thus, we were able to not only target our proactive initiative towards women but men as well.
Researches
Dr. Bhamla
Dr. Saad Bhamla, head of the Bhamla Lab at the Georgia Institute of Technology, has been an invaluable collaborator and supporter of Lambert iGEM. Through projects like the Frugal Science Academy (done in partnership with Lambert iGEM advisor Janet Standeven), Dr. Bhamla has provided funding, feedback, and opportunities to test designs in real-world settings. Last year, his input enabled Lambert iGEM to develop Micro-Q, a low-cost fluorometer, and test it in Thailand. This year, Dr. Bhamla’s insights allowed further iteration and adaptation of Micro-Q Pro to better its accuracy and facilitate the real-world impact of many Lambert iGEM projects.
Rajas Poorna, (a grad student at GT)
We spoke to Rajas Poorna, a graduate student working for the Bhamla Lab at the Georgia Institute of Technology, regarding alternatives to our existing RCA methodology to increase its accessibility in the field. Rajas informed us about existing capillary-tube-based diagnostics methods that used PCR and suggested modifying RCA to use capillary tubes. During our testing, we encountered problems with our experiments, such as capping the tubes and drawing up liquid. Some of his suggestions included using a biocompatible wax sealant to effectively seal the tubes and maintain the sample’s integrity by tilting the PCR tubes sideways to counteract the force of gravity on the liquid as it rose in the tube. Our conversations with Rajas gave us insight into developing a novel method of quantifying that does not require a fluorometer, enabling greater inclusivity and accessibility of our diagnostic tool.
Dr. Sliverman
This year, Lambert iGEM wanted to produce phi29 DNA Polymerase, a protein involved in the amplification process of RCA, by utilizing the Immobilized Metal Affinity Chromatography (IMAC) mechanism see Protein Purification. However, after talking to professors at Georgia Tech, they informed us to consider obtaining it through the cell-free system, as the cells will be able to intake the plasmid better than in the in vivo system while creating the optimal amount of phi29 DNA Polymerase needed in our lab. We then spoke to Dr. Adam Sliverman, a Scientist at Sherlock Biosciences, who guided us in choosing either cellfree or IMAC. He informed us about the inability to scale large amounts of protein in vitro. Therefore, he suggested our team grow a liter of E. coli with phi29 DNA Polymerase in a pET expression vector, inducing it with IPTG and outgrowing it overnight. He also assured us that the purification of the protein from that point is relatively straightforward. With this information, we continued our research on the IMAC mechanism to selectively extract the his-tagged phi29 DNA polymerase from the lysate to obtain the purified phi29 DNA polymerase for the RCA mechanism. Aside from protein purification, we also wanted to pursue exponential rolling circle amplification (eRCA). Dr. Silverman has previously experimented with RCA and believed eRCA to be a sufficient mechanism for our sensor’s sensitivity. As eRCA exponentially increases output, he advised us to include additional controls to ensure we are not amplifying errors. He suggested using an ON-state reporter mechanism, as it would reduce error/noise by promoting signal gain. While we had been experimenting with the ON-state lettuce DNA aptamers, Dr. Silverman also proposed using G-quadruplexes or SYBR Green instead, as DNA aptamers are not very fluorescent. Although we considered these mechanisms, we found that the efficiency and cost of linear probes — an OFF-state reporter — was more suitable for our project. We also considered multiplexed detection of miRNAs using RCA, however, Dr. Silverman advised us against this due to the low efficiency of SplintR Ligase. He suggested attempting multiplexing with pre-ligated padlock probes first to eliminate the potential error created by the enzyme, but we plan to test this idea in the future. Dr. Silverman’s feedback in improving the affordability and accuracy of our biosensor enabled us to create a higher-quality assay that makes preliminary care more accessible for all patients.
Dr. Charles Searles
Dr. Charles Searles is an associate professor of medicine at Emory Healthcare Hospital and a cardiologist specializing in biomarkers correlated with CAD. Last year, he provided us with hsa-miR-451a — unrelated to the pathophysiology of CAD — as a control for our proof-of-concept experimentation. Dr. Searles has tested our biosensors in his lab and offered to test them with actual patient serum samples in the future. He also gave us helpful insights on what would be useful in a miRNA database for CAD and validated the need for a database like CADmir. After we built CADmir, we shared it with Dr. Searles, and he gave us valuable feedback on improvements we could make to the platform. We iterated on his input and modified CADmir with filtration and testing on our publications. With his help, we were able to develop a miRNA database for CAD that helps miRNA researchers find information faster.
Kimberly Ann Rooney
Kimberly Ann Rooney, a lab technician at the Searles Cardiovascular Lab led by Dr. Charles Searles, ran RCA reactions at Emory utilizing our protocol to test whether our biosensor could be practical and applicable as a diagnostic tool. Ms. Rooney gave us feedback on our methodology, advising that we reduce the number of variables used as controls and use serial dilutions to obtain more precise miRNA concentrations. She guided us towards optimizing the overall time of our assay, allowing us to further emphasize proactive care by decreasing the time it takes for hospitals to communicate results to patients.
Dr. Mark Styczynski
Dr. Mark Styczynski, a researcher from the Georgia Institute of Technology, told us that eRCA is a plausible mechanism. If the error with the RCA assay is from the technological aspect, eRCA should perform as expected. However, it could potentially amplify the discrepancy in our data if the error is due to the biological process of RCA. Dr. Styczinski also pointed out that using an OFF-state reporter, such as linear DNA probes, has a limited dynamic range as it is losing signal. He suggested using an ON-state sensor instead, as it would address this fundamental limitation. While we had been working with split lettuce DNA aptamers, Dr. Styczynski urged us away from this mechanism as they are more unreliable than other reporters. We then proposed producing whole aptamers through RCA. He was against this idea as the secondary structures of multiple aptamers’ would likely interact poorly on a single strand of DNA. Therefore, we utilized eRCA to produce whole lettuce aptamers, as this mechanism produces separate, isolated strands of DNA rather than a single, long strand.
Megan McSweeney
Megan McSweeney is a graduate student at the Georgia Institute of Technology and part of the Styczynski lab. She stated that the efficiency of eRCA would depend on the source of the error, paralleling Dr. Styczynski’s input. eRCA could be more accurate than RCA if it does not amplify the error. She was also concerned that our discrepancy could be due to contamination, leading us to take extra precautions to ensure an aseptic and RNase-free environment. Her feedback helped us increase the accuracy of our biosensor, therefore increasing its proactiveness and applicability in point-of-care testing.
Priya Soneji
Priya Soneji is an undergraduate researcher at the Georgia Institute of Technology and part of the Bhamla Lab. She designed and built a device called Trackoscope, a frugal tracking microscope for tracking the movement of microorganisms. It has a similar frame and structural design we intended to use for LabPilot, which is the inexpensive, automated micropipetter we are creating to aid labwork. We proposed our design on Fusion 360, a computer-aided design (CAD) software tool, to Priya, who verified that this software was the best option for CAD design. She also recommended that we try aluminum supports in case the 3D printed parts were time-consuming and imprecise issues. Furthermore, Priya uses the Nema 17 motors in her Trackoscope design, which includes a micro-stepping feature that enables precise motions. We then asked her if using Nema 17 motors seemed appropriate for our build, and she said they are very reliable and accurate motors. She confirmed the validity of our overall plan for controlling each motor axis on the XYZ plane and methods for motor movement. Priya greatly improved our understanding of the accuracy of our pipette mechanism and also allowed us to look into the affordability of some elements.
Institutions/Broader Community
Dr. Shana Scott
We also spoke to Dr. Shana Scott, one of the leading members of the American Heart Association (AHA). She helped us establish and co-hosted the September Heart Health Month. She also guided us towards initiating an updated CERP (Cardiac Emergency Response Plan) at our school in order to cater to a more inclusive community. Dr. Shana Scott was also integral in promoting and increasing our outreach countywide. Lambert iGEM originally planned school-wide September Heart Health webinars and only planned CPR/First Aid education. With Dr. Scott’s guidance, we were able to increase our outreach and expand our CPR program to a policy.
Mrs. Michelle Routhenstein
Mrs. Routhenstein, a nutritionist, inspired our initiative to shift towards a more proactive approach. As a guest on our podcast, she detailed the importance of maintaining a balanced diet and implementing exercise in a daily routine as crucial practices in preventing heart disease altogether. She discussed general trends she’d seen in her patients with CAD, such as a poor diet filled with saturated fats, and a lack of an active lifestyle. She explained how as a nutritionist, her minor changes to these patients’ diets improved their condition tremendously. She even inspired us to conduct our September Heart Health webinar to encourage the message of the importance of diet and nutrition in preventing CAD to a broad and diverse audience.
