Our Original Plan:
We aim to develop an aptamer-based diagnostic kit to detect uropathogenic bacteria; both gram-negative (E.coli, Klebsiella, E.faecalis) and gram positive (Staphylococcus saprophyticus, S.agalactiae, E.faecium) that cause UTIs using a common platform. The aptamers will be selected from random ssDNAs sequence libraries using SELEX technique, where they can together detect multiple UTI-allied bacteria.
- The focus is on developing complete cell SELEX-based aptamers for the detection of UTIs as our target is a wide range of bacteria pathogens.
- We would screen aptamers that could specifically bind and detect whole cells of pathogenic bacteria with nanomolar affinity.
- The iterative binding with the ssDNA library, followed by washing off of the nonspecific oligonucleotides will be performed.
- The aptamers with high specificity and affinity will be further sequenced with next-generation sequencing (NGS).
- ML and AI algorithms will be applied to identify the top 4-5 aptamer sequences to experimentally validate for the range of the target bacterial pathogens.
- Urine samples from UTI patients will be obtained from the clinical sites to validate aptamers identified from the screening library.
- The 5’end of the aptamer will be conjugated with gold nanoparticles to serve as a biosensor for detection.
- Final prototype will be an easy-to-use microfluidic/capillary based APtamer kit for UTI (Apt4UTI) for translation at the POCs.
Paradigm shift: Whole-Cell to Ligand-Based SELEX:
In our brainstorming sessions with iGEM Teams IISER Mohali and IISER Tirupati, we came to realise that whole-cell SELEX would require brute forcing the docking procedures for every single aptamer, since we the whole surface of the UPEC was our target. This would be very time-consuming and cumbersome given the time constraints of this competition and the technical expertise it would require to perform such protocols. This prompted us to consider shifting our mode of operation to something less time consuming, like Ligand-Based SELEX.
Choosing our target ligand:
The input to focus towards virulence factors and the pili and fimbriae was given to us by Dr. Blessy. She reviewed our survey, and her reviews prompted us to reform our survey to make it more accessible and informative to the public. She guided us through the basics of UTIs and forced us to look at the pathogenesis and how the fimbriae react with the uroepithelium. After our collaboration meetings with IISER Mohali and IISER Tirupati, we realised that we would have to focus on Ligand-based SELEX, prompting us to choose FimH as our target ligand.
Detection of Pathogens:
We were given the great opportunity to converse with Dr. Swapnil Sinha, the founder of Altanostics Pvt. Ltd. She is an expert in working with aptamers and their selection by SELEX. It gave us a sense of the kind of approach we would be pursuing with respect to using SELEX technique and our tagging methodologies. Her project involved the development of a nanosensor using aptamers for the detection of Urinary Tract Infections. Her expertise helped us focus on using fluorescence and colorimetric tags for the detection technique.
Diabetes medications and UTIs:
Dr. Anjana introduced us to the strong link between Diabetes and UTI. Her expertise in the field of Diabetes has helped us focus on how diabetic medications can trigger urinary tract infections and how kidney abscesses can also be traced back to UTIs. She also instructed on the importance of asymptomatic UTIs and how to trace and track them. Her advice has helped us shape how we approach diabetic patients regarding our project and how to modify our questions when we reach out to diabetologists for a statistical perspective.
Empathizing to the cause:
Our trip to the local villages, as suggested by our honorable director Dr. K. V. R. Chary, was insightful and instrumental in shifting our focus point from the urban to the rural set-ups. From this initiative we discovered the stigma that the rural folks have- they are fearful that disclosing any information about their sanitary hygiene would make them a social outcast. This helped us emphasize on the urgency to develop a user-friendly, efficient kit that gives results personal to the users, allowing them to preserve their privacy.
How Human Practices modified our project:
- Shift from Whole-Cell SELEX to Ligand-Based SELEX
- Selection of FimH protein as a target ligand
- Using fluorescence and colorimetric tags for detection, instead of gold nanoparticles
- Shaping survey questions to diabetologists
- Development of a more user-friendly kit keeping the privacy of users in mind
