Integrated Human Practices
Promoting AMR Awareness Within Our Community
Human Practices
Overview
Antimicrobial resistance (AMR) occurs when bacteria, viruses, fungi, and parasites evolve to become resistant to antimicrobial medications (WHO, 2023). In 2021, AMR caused approximately 1.14 million deaths – a number projected to reach 2-10 million per year making it one of the leading causes of death by the year 2050 (Murray et al., 2022; Naghavi et al., 2024). To understand this issue, Lambert iGEM began researching potential approaches to address AMR by contacting scientists and experts in synthetic biology and AMR. Throughout the development of our project, Lambert iGEM has been influenced by a diverse array of stakeholders, including contagious disease researchers, educational administrators, university doctors, and our local community. Their contributions ranged from wet lab protocols to strategies for addressing gaps in AMR awareness and assessing the feasibility of our initiatives. By integrating their feedback, we’ve refined aspects of our project like CRISPRi gene regulation, Toehold detection methods, and AMR awareness education, and created a map that highlights AMR prevalence in our community. This process of iterative feedback has ensured that SHIELD effectively addresses AMR by creating a system that quickly creates and tests new AMR therapies, combating the rapid evolution of antibiotic-resistant strains of bacteria. Lambert iGEM aimed to create a lasting impact on our community by raising awareness about antimicrobial resistance to prevent antibiotic misuse and ensuring our project relevance and safety by constantly communicating with diverse stakeholders. To accomplish these goals we used a multifaceted approach including:
Scientific Collaboration: We deliberated every decision in designing our CRISPRi/Toehold system, frugal liquid handler hardware device, and toehold design software program through virtual calls, interviews, emails, and meetings with leading researchers, field experts, and doctors.
Educational Outreach: By collaborating with elementary, middle, and high schools in our home state of Georgia, we successfully raised AMR awareness across various demographics, fostering a community-wide understanding of this critical issue. Locally, Lambert iGEM taught the neurodivergent community about AMR and synthetic biology through multiple workshops and lessons. We also hosted a district-wide middle school workshop covering AMR and its agricultural/healthcare usages, while also publishing a children’s book that would allow elementary students to gain exposure to this critical issue. To further our impact, Lambert iGEM hosted nationwide webinars and created shadowing opportunities for high schoolers and teachers all over Georgia through the Frugal Science Academy. Extending our efforts internationally, we developed an AMR/synthetic biology curriculum for female adolescents in Afghanistan; therefore breaking through cultural and societal barriers towards scientific and AMR education.
Citizen Science Initiative: Our soil AMR prevalence map has benefited greatly from community-contributed soil samples. Their participation has enabled us to conduct more comprehensive testing of tetracycline and class 1 & 2 integrons to create a detailed local map of Antimicrobial Resistance, providing valuable data for our research.
How Our Journey Began
Antimicrobial Resistance Solution Exploration with Dr. Christopher LaRock
A professor and faculty member of the Antimicrobial Resistance Training Program at Emory University, Dr. LaRock leads a lab studying factors of virulence in bacteria (LaRock Lab). As one of the first researchers we talked to, he gave us insight into the world of antimicrobial resistance as a whole, as well as his outlook on future solutions. Dr. LaRock highlighted the pressing need for novel methods in combating bacterial infections, as resistance can’t be completely prevented. He recommended finding a way to eliminate resistant bacteria while also minimizing the residual threats of antimicrobial resistance. Using his advice, Lambert iGEM identified the challenge that our project would need to address.
Evaluating Current Methods of Combating AMR with Dr. Natasha Mavengere
To validate Lambert iGEM’s focus on producing alternatives to antibiotics we spoke with Dr. Mavengere, a Fulbright Research Scholar from the Wisconsin Institute of Discovery. Her research focuses on microorganisms that produce antibiotics, and exploring how resistance develops in bacteria to develop more effective treatments. She told us about the historical and current impacts of antibiotics; specifically, highlighting overuse in parts of the world where second-line drugs aren’t affordable leading to increasingly higher rates of resistant infections. This emphasizes the need for our project as an alternative to traditional antibiotics. Ultimately, supporting the usage of CRISPR interference to kill resistant bacteria and make it accessible for those who can’t afford standard medicine. In addition, she emphasized the lack of education in this field, reassuring our approach to improving education.
Global To Local
Our home state of Georgia is one of the leaders in the United States in the agricultural and farming industries. During our research into the global issue of AMR, we discovered that it greatly impacted Georgia in particular, due to the widespread misuse of antibiotics in agricultural practices. Livestock can consequently release antibiotics into the environment contaminating local water sources, and eventually making their way into our drinking water (Ghimpețeanu et al., 2022).
In response to this challenge, Lambert iGEM established a wet lab initiative focused on soil-based research. By testing soil samples from various locations within the state of Georgia, we mapped the relationship of AMR prevalence through agriculture. The creation of our soil wet lab committee has been crucial in identifying and analyzing pathways of antimicrobial resistance locally (see Agriculture).
The Formation of Project SHIELD
To address AMR, we proposed SHIELD: a versatile toolbox for developing novel antimicrobials to combat infections, while mitigating the effects of resistance through its rapidly adaptable nature. SHIELD utilizes a CRISPR interference (CRISPRi) to suppress critical genes, alongside a toehold biosensor testing pipeline to validate its efficacy. This approach employs a cell-free system with linear DNA, bypassing the time-consuming cloning process. To further enhance SHIELD’s capabilities we developed machine learning-based software to optimize toehold design and a frugal automated liquid handler to aid experimentation.
Wetlab
CRISPRi
Exploring CRISPRi with Mrs. Janet Standeven
Lambert iGEM was mentored by Mrs. Janet Standeven, the director of the Frugal Science Academy at the Georgia Institute of Technology. In the initial phase of ideation, we had the opportunity to receive feedback on the wet lab portion of our project. Initially, we considered utilizing RNA interference to knock out critical genes in bacteria. However, Mrs. Standeven advised the use of CRISPR interference as a better-suited approach for our needs – providing a safer method of gene regulation and increased accuracy in terms of binding and on-target effects.
Continuation of CRISPRi & Bacteria with Mrs. Taylor Blackburn
In our investigation of CRISPRi, we had the opportunity to speak with Ms. Blackburn, a graduate student at Emory University. She provided valuable insights into bacterial survival mechanisms against antimicrobials, focusing on toxin-antitoxin systems and efflux pumps. Her explanation underscored the urgent need for novel compounds to combat bacterial infections, validating our approach of using CRISPR interference to address antimicrobial resistance (AMR). Furthermore, Ms. Blackburn shed light on a critical issue in the field: the diminishing investment in AMR research by major pharmaceutical companies due to limited profit potential. This revelation highlighted a significant gap in current research efforts, emphasizing the importance of our work in this crucial area of study.
Feasibility Input & Validation of Tuberculosis with Dr. Kiatichai Faksri
While researching model organisms for CRISPRi, we found that Mycobacterium tuberculosis has a high mortality rate and is notably susceptible to antimicrobial resistance and noted that the inHa gene was frequently used in literature(WHO, 2023). To validate our findings, Lambert iGEM met with Dr. Kiatichai Faksri, the dean of Khon Kaen University and an expert on Tuberculosis (TB). Dr. Faksri confirmed the relevance of our TB focus, citing the global prevalence of multidrug-resistant strains. He also endorsed our interest in the inhA gene, noting its widespread use in both clinical practice and research. Dr. Faksri emphasized that the inhA gene has a robust foundation in TB experiments, further supporting our decision to incorporate it into our study.
Verifying Experimental Details with Dr. Vincent Noireaux
Dr. Vincent Noireaux is the Professor of Synthetic Biology and Biological Physics at the University of Minnesota. His lab specializes in the development of cell-free systems, and he has conducted extensive research on cell-free CRISPRi. When our initial experiments with the myTXTL Sigma70 cell-free system and known deGFP plasmids unexpectedly yielded low fluorescence values, we reached out to Dr. Noireaux for guidance on the system’s efficiency and our protocol. He provided valuable feedback regarding our initial experimentation details. We learned that our plasmids should be grown at 30°C to optimize their amplification. He also shared the plate of working dCas9 and deGFP plasmids, enabling us to begin testing earlier than expected.
FSA CRISPRi Guidance from Daeun (Esther) Lee
Esther Lee is currently an undergraduate at the Georgia Institute of Technology with a focus in Biomedical Engineering. She was also a member of the 2022 Lambert iGEM team. Due to her extensive studies with CRISPRi with Dr. Noireaux, Ms. Lee was able to assist us throughout our early testing process. In her role as a mentor during the Frugal Science Academy, she played a crucial part in helping us determine the optimal reagent concentrations for our GFP testing phase.
Confirmation of CRISPRi’s Advantages with Dr. Scot Oullette
To discuss the qualities of CRISPRi as a therapeutic application, we reached out to Dr. Scot Ouellette, a professor and postdoctoral researcher at the Department of Pathology, Microbiology, and Immunology at the University of Nebraska Medical Center. Dr. Ouellette corroborated our findings on the CRISPRi system, confirming that it poses a lower risk of chromosomal mutations compared to conventional antibiotics, which supports our assumption regarding its precision. Furthermore, he agreed that a successful CRISPRi system will be able to outpace AMR, thus validating our solution as a whole and allowing us to move forward.
CRISPRi Discussion with Dr. Jeremy Rock & Ms. Kathryn Eckartt
Dr. Jeremy Rock is an Assistant Professor at Rockefeller University and Principal Investigator of the Rock Laboratory, with a primary focus on mycobacterial pathogenesis. To answer our questions, he introduced us to Kathryn Eckartt, a PhD student at the Rock Lab and a former iGEM member. Ms. Eckartt provided great insight into how the CRISPRi system could be affected by mutations, bringing the concept of gene vulnerability to our attention. In our situation, this means that a critical gene, such as inhA, is not required to be fully repressed to kill the bacteria. After further analysis of research done by the Rock Lab, we were able to determine that anything above 50% repression of inhA will inhibit the growth of the bacteria, and we were able to compare this value to the repression that we obtained. Furthermore, she highlighted the benefits of using CRISPRi over traditional therapeutics. For example, she mentioned traditional tuberculosis treatments have harsh side effects because they are taken orally and affect the whole body. However, CRISPRi has a specific target that only matches the bacteria. Also, Ms. Eckartt states that CRISPRi guides are significantly easier alternatives to design and use. This information enhanced Lambert iGEM’s understanding of CRISPRi’s benefits in healthcare, enabling the team to more effectively advocate for its adoption.
Discussing CRIPSRi screening with Amy Enright
Amy Enright is a Microbiology PhD student in the Jason-Peters Lab at the University of Wisconsin-Madison and has extensive experience using CRISPRi screens. We discussed various details of the system’s application. She agrees that an essential gene such as inhA will need relatively little knockdown to kill the bacteria, and that partial knockdown would be enough to see a significant effect. She also suggested the use of a multiplexed CRISPRi system in the future, as it would be much more effective in overcoming the possibility of mutation, and make the system a more viable therapy.
Toehold
Exploring toehold switches and verifying experimental design with Dr. Megan McSweeney
Dr. Megan McSweeney, a postdoctoral researcher in the School of Chemical and Biomolecular Engineering at the Georgia Institute of Technology, works in the Styczynski Lab. Her extensive research background in toeholds and cell-free systems made her an ideal consultant for Lambert iGEM. After our first GFP toehold reaction, we sought her assistance in troubleshooting. During our initial meeting, Dr. McSweeney recommended running PCR and PCR cleanup on our constructs to increase linear DNA purity. She also advised changing the molar ratio of our toehold to trigger from 5:1 nM to 1:5 nM. Given the low binding efficiency of individual constructs, she suggested expanding our testing pool to 6 different constructs created from NUPACK, improving our chances of identifying successful candidates. In a follow-up meeting, Dr. McSweeney aided our experimental design by providing resources to determine the binding efficiency of our toehold and trigger pairs. She also recommended a range of concentrations to generate a concentration curve, allowing us to identify optimal conditions for testing our toeholds.
Discussing toeholds with Sachintha Ashok
Sachintha Ashok, an undergraduate student at the University of Georgia and one of our mentors at the Frugal Science Academy (FSA), provided valuable guidance on our experiments and project. As a former member of Lambert iGEM’s 2021 Team, which also researched toehold switches, Sachintha was an excellent resource for troubleshooting. During our time with Sachintha, we gained insights into experimental design, including reagent concentrations and overall conditions. She recommended concentrations she had used during her time on the team and reviewed our constructs designed at FSA to prevent potential issues. She also connected us with Dr. Megan McSweeney as a resource leveraging her past relationship with her from the iGEM team, providing a valuable resource for knowledge about experimental design that greatly contributed to the success of our project.
Soil
Introducing Integrons with Dr. Kevin Forsberg
Dr. Forsberg is a professor at UT Southern Medical Center who earned his PhD studying antibiotic resistance in soil. With his research and work with AMR in soil, Dr. Forburg advised us to look into integrons. Although first we were testing using individual Tetracycline primers, Dr. Forsberg suggested the use of integrons. Further, recommending the use of integron primers in PCR to test for the presence of AMR instead of trying to test for specific resistance genes like Tetracycline. After further research, we incorporated class 1 and class 2 integron primers in our tests because of their efficiency in detection, and association with agricultural-based antibiotics. This method is far more efficient than using tetracycline primers as it allows for testing and detection of AMR as a whole and not just tetracycline-related genes.
PARE Database Discussion with Dr. Carol Bascom-Slack
Dr. Bascom-Slack is a research assistant professor at Tufts University School of Medicine and has developed a course-based undergraduate research program to shed light on the prevalence of antimicrobial-resistant genes. She has guided us in inputting our soil data into Tufts University’s PARE Database, which is a global database that tracks the prevalence of Tetracycline resistance. Dr. Carol Bascom-Slack explained how selective pressures in the soil contribute to antimicrobial resistance (AMR). Antibiotic-resistant bacteria are selected for in soil, especially when antibiotics are overused in farming, creating environments where resistant bacteria thrive.
Drylab
Software
Analyzing the Challenges of Current Toehold Design with Dr. Megan McSweeney
Dr. Megan McSweeney, a postdoctoral researcher at the School of Chemical and Biomolecular Engineering at Georgia Institute of Technology, currently works in the Styczynski Lab. Given her extensive expertise in toeholds and cell-free systems, Lambert iGEM sought her insights on the current challenges of toehold design and its effectiveness. Through her feedback, we learned that scientists often have to test toeholds 3-6 times to identify a functional one, highlighting a significant inefficiency in the design process. This insight led us to recognize the potential value of developing a software program that could streamline and automate toehold design, making the process both more efficient and reliable. Using this information, we built SWORD, a software program to help generate and predict the effectiveness of toeholds.
Modeling
Researching various mathematical equations with Pratyusha K.R.
Pratyusha K.R. is a post-doc currently working on the use of computational tools to study the dynamics of soft & active matter systems at the Bhamla lab and an expert in mapping biochemical reactions using ordinary differential equations. During our meeting, we discussed various mathematical equations available and as well as which would suit our reactions best. Following the discussion, we realized that to utilize Mass Action & Michaelis-Menten Kinetics – the equations that Pratyusha recommended – we needed to go through a deep dive into the literature and find reaction constants to run our MATLAB simulation.
Discussing potential modes of calculations and parameters with Srirag Tatavarti
Srirag Tatavarti is an alumnus of Lambert iGEM 2022 currently studying as a math major at Columbia University. Srirag taught our team the finer differences between three key equations: Mass Action laws, Michaelis-Menten enzyme kinetics, and Hill equations. He led us to realize how each of these unique modes of calculation could be used, as well as how to create parameters based on our experimentation. After we met with Srirag, we began the process of creating a GFP calibration curve for the plate readers used in our CRISPRi and toehold experiments.
Exploring the validity of models for toehold reactions and parameters with Dr. Mark Styczynski
After making some preliminary adjustments to our model, we reached out to Dr. Mark Styczynski – an associate professor at the Georgia Institute of Technology’s School of Chemical and Biomolecular Engineering. We contacted Dr. Styczynski to confirm the validity of our model for the toehold reaction pathway, especially regarding the leakage rate of the binding between toehold triggers and their corresponding switch regions. Dr. Styczynski pointed us to various resources that allowed us to obtain parameters that we weren’t able to derive from our wet lab. Following Dr. Styczynski’s recommendations, we refined our model by altering our reaction pathway and contacted Dr. Vincent Noireaux, an expert in CRISPR gene editing technology, for his assistance in finding obscure parameters for CRISPRi.
Reviewing future potential equations and models with Yue Han
Alongside Dr. Styczynski, we came into contact with PhD candidate Yue Han, who has worked extensively with mathematical modeling and data integration for synthetic biology. We met Ms. Han over Zoom as well as in person at the Frugal Science Academy poster session, where she advised us on potential future directions we could pursue. Ms. Han introduced us to the idea of using partial differential equations and stochastic models, which would give us a better understanding of the expected results from our experiments. We took her advice and began research into new avenues of modeling, which we will continue throughout the next season.
Evaluating parameters and reaction rate constants with Dr. Vincent Noireaux
Dr. Vincent Noireaux is a professor at the University of Minnesota’s School of Physics and Astronomy who specializes in biophysics and implementation of cell-free transcription-translation technology. After our previous feedback from Dr. Styczynski, we decided to reach out to Dr. Noireaux, who we have been working with regarding our CRISPRi experimentation. Dr. Noireaux was able to direct us to one of his previous projects that quantified various reaction rate constants based on data from wet lab research. This paper was crucial for our modeling committee to find parameters regarding rare components of our CRISPRi reaction and complete our mathematical model.
iHP Structure
“There is nothing permanent except change. The world is in constant flux, and true wisdom lies in recognizing the impermanence of all things and adapting with grace and resilience.” - Greek Philosopher Heraclitus
This year, Lambert iGEM focused on collaboration and engaging with a diverse cohort of stakeholders. Through this communication, we were able to explore the iterative scientific process, adjuting our project along the way, while also involving the community in our initiatives.
Introduction to Scientific Storytelling and Science Communication with Dr. Jacob Harrison
To tackle this problem, Lambert iGEM reached out to Dr. Jacob Harrison, a postdoctoral researcher in the Bhamla Lab at Georgia Tech who specializes in scientific storytelling and science communication. At our meeting, Dr. Jacob Harrison taught us the art of scientific storytelling and how it is essential in bridging the gap between scientific experts and the general public. We applied this same concept to our Integrated Human Practices structure to develop a framework that maximizes the integration and impact of the feedback we got from our stakeholders. After he introduced us to a variety of powerful storytelling techniques, we were able to utilize the Hero’s Journey (a classic storytelling structure) to create a narrative of our integrated Human Practices journey. By mapping our project onto the Hero’s Journey framework, we created a structured yet flexible system to thoughtfully analyze and reflect on every one of our interactions with our stakeholders - helping us evaluate the outcomes of our initiatives and see how our project and goals changed over time
Summary
SHIELD empowers our community through targeted healthcare and agriculture initiatives, providing an innovative approach to combat antimicrobial resistance and securing a sustainable future.
Safety
Participants in all surveys provided signed consent for the release of their responses from themselves or a legal guardian. Additionally, participants in all events hosted by Lambert iGEM provided consent for photo and video release. Proper safety instructions and procedures were given during the in-person and virtual camp activities.