The modelling team has simulated the 3-D structures of our protein and the aptamer and conducted preliminary rounds of docking to isolate the correct aptamer in silico. The Human Practices Team has highlighted details about UTIs that are often overlooked by conducting seminars and talks. The BioBricks and the Wet-Lab Teams have added basic and composite parts to the registry related to the FimH protein and have expressed our protein in strains available in the laboratory. We have also worked tirelessly to find sponsors for our project.
The director of our institute offered his invaluable advice to reach out to the local villages and get a different perspective on our project. Our professors and the administrative officials of our institute were instrumental in our survey in the villages. Our friends and family members have helped us by promoting our project in order to reach out to a larger population.
Our project focuses on the development of an aptamer-based solution to detect uropathogenic bacteria at the point-of-care. Urinary Tract Infections (UTIs) are the third most prevalent bacterial infection in India and the second most diagnosed infectious disease worldwide. Lack of sanitary hygiene, lack of awareness of UTI and unavailability of easy-to-use diagnosis methods at point of care leads to high prevalence of UTI particularly in the rural set-up. Several villages located near our institute, as per our survey, have shown a frequent occurrence of UTIs, which often go ignored and then develop into complications like kidney infections and sepsis. These infections, misdiagnosed as common fevers, are treated with a common group of antibiotics and paracetamol, leading to antibiotic resistance. These chronic infections are known to be highly fatal if not treated in time. In addition, the stigma regarding UTIs and other complications have prevented people from approaching doctors regarding treatments.
We found that Team iGEM13_NYMU-Taipei devised a fluorescence based methodology to test the functionality of the plac+RFP-FimH protein by expressing a in fusion protein of RFP-FimH and then checking the Fim H binding to the mannose .Later From our literature surveys (Schembri et al Expression and purification of the mannose recognition domain of the FimH adhesin; 01 July 2000; FEMS Microbiology Letters )related to the testing of FimH protein, we have found another in vitro methodology that can serve as a functionality check of purified FimH binding to mannose which used alpha-D mannosylated BSA (mimicking the mannose receptor) as a matrix for FimH binding .The technique is termed as Receptor Blot.
We have designed the modelling workflow for aptamer screening for our target ligand FimH protein which is more common in uropathogenic bacteria. Our modelling protocol goes as follows:
We had collaboration meetings with IISER Mohali, IISER Tirupati, IISER Thiruvananthapuram, NU Kazhakshtan, Rochester University and ICT Mumbai. We had insightful discussions on the drylab aspects of the projects. We brainstormed on methods to improve the existing molecular simulation protocols for better results and quicker workflow cycle. Our meeting with IISER Thiruvananthapuram also extended to Human Practices and how we can reach out to larger populations and educate them in the form of fun games and presentations regarding the upcoming developments in Synthetic Biology. We are also collaborating with them and a few other teams to form a Talk Series on Women’s Health and Hygiene, bound to start from the first week of October, 2022. The other teams we had meetings with were instrumental in helping us shape our projects to be more efficient and we benefited greatly from their advice.
Though the prevalence of UTI is very high, knowledge about its symptoms, causes, and treatment is still not widely known in rural settings of developing countries like India. To get more in-field information about the prevalence of this disease, people from different backgrounds, age groups, and genders were asked questions regarding the sanitation conditions of their restrooms and the frequency of the symptoms pertaining to urinary tract infections.
From this initiative we discovered the stigma that the rural folks have about, there is this fear that disclosing any information about their sanitary hygiene would make them a social outcast. Therefore we decided to develop an POC , a rapid and easy to use tool kit for the detection of UTI, so that they can be assured of their privacy while also properly getting diagnosed and treated.
Our project has the potential to reach out to a large sector of the population since the affected populations are people from the rural area, people with uncontrolled diabetes, and women, in general. We intend to continue this project beyond the boundaries of iGEM, the resulting prototype of which will be sent to our industry partners for scale-up . We are in constant contact with research labs and industries from the healthcare sector, focusing on aptamer selection as well as tagging protocols, who can help us fine-tune our project. We have also pitched our project in start-up initiatives like ‘Start-up Odisha Yatra 2.0’ and have been qualified to discuss our project with the Chief Minister of the state of Odisha. Succeeding in this initiative can help us fund projects to develop into a large-scale solution for the diagnosis of urinary tract infections.
Our collaboration meetings with IISER Mohali and IISER Tirupati gave us clarity regarding our choice between Whole-Cell and Ligand-Based SELEX. After choosing Ligand-based SELEX, our next issue was to choose the ligand. Dr. Blessymol Varghese was instrumental in helping us finalise our ligand as FimH, taking its virulence properties into consideration. Our next hurdle was choosing the right tagging procedures. After discussions with fellow iGEM teams and Dr. Swapnil Sinha, we were able to shift from our original idea of gold nanoparticles to fluorescence and colorimetric tags for our tagging. Dr. R. M. Anjana helped us widen our target population to include diabetic patients receiving certain medications like Empagliflocin and helped us reform how we approach diabetologists. Our initiative in the local villages helped emphasize on the stigma surrounding UTIs and reform our project to respect the privacy of the users and to make it more user-friendly.
Through modelling, we intend to screen and simulate aptamers that dock with the highest affinity to our target protein, FimH. For this purpose, we generated a random aptamer library, and using softwares like RNA Fold and RNA Composer, we generated three dimensional structures of the oligonucleotide sequences. Twist Biosciences also provided us with the gene sequence for FimH, which we translated into a 3-D structure using AlphaFold2. Our modelling pipeline enable us to run multiple molecular docking simulations in parallel inorder to screen a large aptamer library. Using AutoDock and visualization softwares like PyMOL we have begun our preliminary screening by individually performing molecular docking between the target protein and the aptamer. Our Modelling team has also helped generate the program to visualize global data regarding the prevalence, incidence and mortality rates of urinary tract infections in a normalized manner using heat maps.
We partnered with ICT Mumbai on improving the dynamic layouts of our websites and regarding the screening and docking of aptamers. They helped us gain insights into the tools for the aptamer structure prediction. They offered to help spread our survey to areas local to their institute to garner responses from a larger demographic. We offered to spread their questionnaire regarding the 5 'UTR libraries amongst our faculty and research fellows.
In collaboration with the IISER TVM team, our team plans to conduct a small discussion about Synthetic Biology with bright high schoolers from various parts of our country. In collaboration with IISER Thiruvananthapuram, IISER Tirupati, and IIT Roorkee, we are conducting 'We-talk', a women's health talk series, a three-day event in which the speakers are women from various fields of health sciences educating young women about the various health issues related to their gender.
We partnered with iGEM Team ICT Mumbai on improving the presentation of our project on our website and in the modelling aspects, regarding the screening and docking of aptamers with our target protein. Our partnership with the team during the project helped us finish the milestones we planned. We helped them with developing their wiki. We had brainstorming sessions on how to design responsive layouts for the wiki page and include dynamic elements, along with better loading animation of the content for the web pages. They helped us gain insights into new modelling techniques and tools for the structure prediction of DNA aptamers. They offered to help spread our survey to areas local to their institute and help us garner responses from a larger demographic. We offered to spread their questionnaire regarding the 5 'UTR libraries amongst our faculty and research fellows.
On the basis of literature survey our team made an improvement of an Existing Part in the registry. fimH HIS Tag (BBa_K3953004) is a DNA part that was developed by iGEM21_Pittsburgh (2021-10-21) which consists of the histidine tag sequence that codes for consecutive histidine amino acid residues within the fimH gene. From our literature survey (His tagged Proteins; Creative biostrucrure) we studied that a N teminal or a C terminal His tag is usually more desirable to enable easy purification of His tagged Fim H without interfering with its conformation. For this we devoloped two constructs Apt4UTI C1(BBa_K4449009) and Apt4UTI C2(BBa_K4449010) for expressing C terminal and N terminal 6X His tagged Fim H protein respectively. We have also devised some methodologies to test our His tagged proteins from our constructs.