An intein-mediated control system for thyroid homeostatic regulation
Need of automatizing the treatment of hormonal imbalances
Hypothyroidism is a condition characterized by the lack of thyroid hormones, usually raised by dysregulations of the thyroid feedback. Despite it affects 5% of the overall population, its treatment is not well optimized . Patients are prescribed levothyroxine tablets, which are not effective in one-third of the cases, and their doses, besides acting in a narrow range without adverse effects, are adjusted by trial-and-error . Moreover, the tracking methodology to adjust the doses has evidenced to not properly adapt to the requirements of the current COVID pandemic situation, interrupting the treatment of the patients .
All in all, it is clear that a new therapy approach that considers the whole feedback system and that allows a telematic treatment is needed.
Chiovato, L., Magri, F. & Carlé, A. (2019) Hypothyroidism in Context: Where We’ve Been and Where We’re Going. Adv Ther, 36, 47–58. DOI:10.1007/s12325-019-01080-8
Fliers, E., Demeneix, B., Bhaseen, A., & Brix, T. H. (2018) European Thyroid Association (ETA) and Thyroid Federation International (TFI) Joint Position Statement on the Interchangeability of Levothyroxine Products in EU Countries. European thyroid journal, 7(5), 238–242
 Gorgojo Martínez, J., Zugasti Murillo, A., Rubio Herrera, M. and Bretón Lesmes, I. (2020) TELECONSULTA En Endocrinología Y Nutrición En Tiempos De La Pandemia COVID-19 Y Más Allá. [online] Seen.es. Available at: https://www.seen.es/ModulGEX/workspace/publico/modulos/web/docs/apartados/1433/160620_105727_7128864936.pdf [Accessed 22 October 2020]
 Taylor, P. N., Albrecht, D., Scholz, A., Gutierrez-Buey, G., Lazarus, J. H., Dayan, C. M., & Okosieme, O. E. (2018). Global epidemiology of hyperthyroidism and hypothyroidism. Nature Reviews Endocrinology, 14(5), 301–316. doi:10.1038/nrendo.2018.18
Sensing T3 hormone levels through an intein-mediated bacteria
- T3 Ligand Binding domain (from the Thyroid hormone receptor β1): to recognize T3 present in the media .
- Splicing intein domains (from the Mycobacterium tuberculosis RecA protein): to join the two superfoldingGFP (sfGFP) halves when activated by the ligand domain .
- Split superfolder GFP: to report the T3 concentration as fluorescence .
We expressed our IMT3 biosensor in E. coli and exposed them to different T3 concentrations in the media. Proportional fluorescence with T3 concentration was reported.
FURTHER STEPSThese IMT3 biosensors can be further improved to increase its precision, widening dynamic range or lowering detection limit. Different approaches have been explored such as more ligand sites  or directed evolution .
 Skretas, G. (2005). Regulation of protein activity with small-molecule-controlled inteins. Protein Science, 14(2), 523–532. DOI:10.1110/ps.04996905
 Yen, P. M. (2001). Physiological and Molecular Basis of Thyroid Hormone Action. Physiological Reviews, 81(3), 1097–1142. DOI:10.1152/physrev.2001.81.3.1097
 Wood, D. W., Wu, W., Belfort, G., Derbyshire, V., & Belfort, M. (1999). A genetic system yields self-cleaving inteins for bioseparations. Nature Biotechnology, 17(9), 889–892. DOI:10.1038/12879
 Gangopadhyay, J. P., Jiang, S., & Paulus, H. (2003). An in Vitro Screening System for Protein Splicing Inhibitors Based on Green Fluorescent Protein as an Indicator. Analytical Chemistry, 75(10), 2456–2462. DOI:10.1021/ac020756b
 Gonzalez-Flo, E., Alaball, M. E., Macia, J. (2020).Two-Component Biosensors: Unveiling the Mechanisms of Predictable Tunability. ACS Synthetic Biology, 9(6), 1328-1335. DOI: 10.1021/acssynbio.0c00010
 Badran, A., Liu, D. (2015). Development of potent in vivo mutagenesis plasmids with broad mutational spectra. Nat Commun, 6, 8425. https://doi.org/10.1038/ncomms9425
Goals and achievements
Hormonic is designed to reestablish the hormonal feedback system thanks to an external PID controller coupled to an Intein-Mediated T3 (IMT3) biosensor. But beyond that, it also aims to bring synthetic biology to the society. For this purpose, in the UPF_Barcelona team we have:
Designed a groundbreaking therapy approach.
Successfully engineered the IMT3 biosensor.
Used a lactone circuit as a proof of concept.
Engaged with the scientific & educational community.
Explored society needs & received experts advice.
Controlled administration of levothyroxine
This correction action (levothyroxine injection) will compensate for the error depending on proportional, integrative and derivative constants. They can be tuned with the aim of preventing hyperthyroidism (reduce overshoot), while maintaining the levothyroxine injection rate under its maximum (enlarge rise-time) and within the drug’s effective range (eliminate steady-state error). To do so, a mathematical model of subcutaneous injection of levothyroxine must be developed and characterized .
 Chen, S., Zhao, J., & Qian, J. (2007). A Design Method of Bang-Bang and PID Integrated Controller Based on Rough Set. Fourth International Conference on Fuzzy Systems and Knowledge Discovery (FSKD 2007). doi:10.1109/fskd.2007.17
 Li, J., & Johnson, J. D. (2009). Mathemical models of subcutaneous injection of insulin analogues: a mini-review. Discrete and continuous dynamical systems. Series B, 12(2), 401–414. https://doi.org/10.3934/dcdsb.2009.12.401
Lactone circuit as an analogy to MINIMAL HORMONAL REGULATION MODEL
The producer cells were induced by arabinose and inhibited by glucose, and were provided by the lab. In a hypothyroidism model, these cells would mimic the peripheral tissue cells, responsible for metabolizing levothyroxine into T3 hormones, lactone in our case.
The sensor cells produced superfolder GFP (sfGFP) when lactone was present in the media. In these cells, LuxR was expressed constitutively and sfGFP was induced by a pLux promoter, dependent on the binding of LuxR and lactone.
All these biological behaviours were characterized and demonstrated using Plate-Reader analysis.
 Ballestero, M. C., Duran-Nebreda, S., Monta, R., Solé, R., Macía, J., Rodríguez-Caso, C. A bottom-up characterization of transfer functions for synthetic biology designs: lessons from enzymology. Nucleic Acids Research, 2014, Vol. 42, No. 22
 Garcia-Ojalvo, J., Elowitz, M.B., Strogatz, S.H. Modeling a synthetic multicellular clock: repressilators coupled by quorum sensing. Proc. Natl Acad. Sci. U.S.A., 101, 10955–10960
 Brent G. A. (2012). Mechanisms of thyroid hormone action. The Journal of clinical investigation, 122(9), 3035–3043. https://doi.org/10.1172/JCI60047
Turbidostat, our continuous microbiological culture device
In order to perform a long-lasting in vitro demonstration of our feedback system, the turbidostat can maintain a user-fixed optical density for any kind of cell culture (producer + reporter cells in our proof of concept). This ensures constant conditions during the whole experiment avoiding thus interferences in the sensing process.
 Wong BG, Mancuso CP, Kiriakov S, Bashor CJ, Khalil AS. Precise, automated control of conditions for high-throughput growth of yeast and bacteria with eVOLVER. Nat Biotechnol, 2018 Aug;36(7):614-623. doi: 10.1038/nbt.4151. Epub 2018 Jun 11. PMID: 29889214; PMCID: PMC6035058.
Two ordinary differential equation (ODE) models have been developed to characterize and understand the behaviour of the T3 intein-mediated biosensor and the lactone proof of concept system. These models allow us to predict specific hormone concentration, and therefore, to regulate the wanted feedback loops. They were fitted with experimental data and were able to successfully reproduce it. Furthermore, since we are developing biosensors, we computed an analysis on the sensitivity and other parameters that affect the ability of the cell to sense, thus showing a balance between robustness and hypersensitivity. The T3 biosensor model sets a precedent for any kind of future intein-mediated biosensor.
On top of that, we have designed a model to characterize the ASV tag  and to mathematically demonstrate its ability to increase the protein degradation rate, thus reducing sensing time.
T3 intein-mediated biosensor
 Andersen, J.,Sternberg, C., Poulsen, L. K., Bjørn, S. P., Givskov, M. New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria. Applied and environmental microbiology, June 1998, p. 2240–2246
Integrated Human Practices
Feedback from multiple stakeholders has been gathered throughout all the stages of the project as to pursue a human-centered solution search process.
Doctor’s physiological knowledge was complimented with the demands of national and international endocrine associations, which arose biosafety and bioethical concerns that were also tackled. The defined scientific approach helped us design the future implementation that was later used for the entrepreneurship planification.
Bringing synthetic biology to the world is a very important task, but how can it be done in a year like 2020?
How can we deliver Hormonic to the world?
Hormonic’s final prototype was envisioned: a microneedle array coupled to a cell-free T3 sensor and to a Levothyroxine reservoir, whose administration is controlled by a PID. Also the biosafety and biosecurity was taken into account in order to make Hormonic safe for everyone.
A rigorous study of the potential users was also performed, including patients during the Covid-19 context, the ones who still exhibit symptoms after treatment or pregnant people among others.
According to EU regulation, Hormonic was classified as a III class device.
Business models are key to bring projects to the final customer. To make Hormonic a reality we elaborated a business plan, using a business model canvas, and the costs and revenues were studied in order to design a financial plan.
Our instructors: Marc Güell, Avencia Sánchez-Mejías, Dimitrije Ivančić & Sira Mogas
Other researchers that have helped us: Javier Macia, Guillermo Nevot, Nuria Rafel, Guillem Lopez-Grado, Nastassia Knödlseder, Jessica Jaraba, Maria Pallarès, Carlos Toscano, Jordi Garcia Ojalvo & Òscar Fornas
HARDWARE DESIGN SUPPORT
Laura Becerra, Aracelys Garcia & Mario Gomez
SPECIAL THANKS TO
Dr. Ricard Torras, Dr. Albert Goday Arno, Dr. Eric Fliers, Professor Vanesa Daza, Carles Taulé Flores & all the entities that have helped us: