University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2019-05-15 Novel split trehalase-based biosensors for the detection of biomarkers of infectious diseases Drikic, Marija Drikic, M. (2019). Novel split trehalase-based biosensors for the detection of biomarkers of infectious diseases (Unpublished doctoral thesis). University of Calgary, Calgary, AB. http://hdl.handle.net/1880/110360 doctoral thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY Novel split trehalase-based biosensors for the detection of biomarkers of infectious diseases by Marija Drikic A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRADUATE PROGRAM IN VETERINARY MEDICAL SCIENCES CALGARY, ALBERTA MAY, 2019 © Marija Drikic 2019 1 ABSTRACT Infectious diseases remain a serious public health challenge in the field of human and veterinary medicine. For instance, infectious diseases were among the leading causes of mortality worldwide in 2016 according to the WHO. In veterinary medicine, infectious diseases have a significant negative impact on animal productivity and welfare leading to decreased profits and increased health-associated costs. Furthermore, the WHO estimates that 61% of diseases affecting the human population are zoonotic. Therefore, programs to control and eradicate these diseases are of crucial importance. Correct diagnosis, supported by robust diagnostic tests, is an essential first step in achieving these goals. An ideal diagnostic test needs to be accurate and robust while being easily accessible and widely available. Although different studies over the years have aimed to develop such a diagnostic test, there have not been many that have managed to enter into clinical practice. The most successful class of biosensors currently on the market is the glucometer used to monitor blood glucose. Glucometer's sensitivity and specificity, as well as its performance in clinical samples, have been mainly perfected. Measuring devices have been miniaturized and their production costs optimized. In this thesis, we developed and characterized a novel biosensor able to detect antibodies and other infectious disease markers. The biosensor is based on the protein complementation principle and uses an E. coli glycolytic enzyme, trehalase (TreA), as a reporter. Trehalase converts trehalose to glucose, which can then be detected by a glucometer. TreA was split into two non-functional fragments, and each fragment was fused to the sensing element that was specific for the targeted analyte. In the presence of the analyte, the sensing elements bind to it and induce the dimerization of the TreA which then becomes active. In the second chapter, we demonstrated that conditional complementation of the trehalase fragments leading to trehalose hydrolysis and glucose production could be used to detect antibodies, bacterial cells, small molecule, protein-protein interaction, and protein aggregation. We also 2 demonstrated the retention of activity of split TreA in undiluted clinical samples like blood or milk. In the third chapter, we attempted to increase the sensitivity and shorten the time for signal detection of the TreA biosensor by introducing split inteins. We placed the inteins within the previously developed biosensor, but we were not successful in achieving analyte-mediated dimerization of the two biosensor components. In the fourth chapter, we applied this TreA biosensor to develop and validate a new diagnostic tool for the quantification of the total amount of immunoglobulins in bovine colostrum and serum (named STIGA). We demonstrated that STIGA performs more efficiently in quantifying total immunoglobulins in colostrum and calf serum than other diagnostic tools (Colostrometer and Brix) that are used on a farm. Therefore, it is a suitable detection assay to establish colostrum quality and calf immune status in the field. We also proposed a modified format of STIGA for this on-farm application and demonstrated that its performance remained high. In the fifth chapter, we explored the applicability of a modified STIGA with the anticipation to detect immunoglobulins in different animal species. We analyzed 29 animal species and proved that the same detection protocol could be applied with success to the majority of animal species. In conclusion, we engineered a trehalase-based biosensor that requires minimal sample preparation and can be integrated with existing glucometers or sensors, which offers a versatile and convenient method for point-of-care applications. 3 PREFACE This dissertation consists of four manuscripts and two appendixes. Two manuscripts have been already published while the third one is accepted for publication. The first author was involved in the design of all the studies included in the thesis, experimental data acquisition, analysis, and interpretation, drafting the manuscript and critical revision. This work was done under the guidance of the senior author and supervisor. All authors provided critical reviews of the manuscripts. After receiving written permission from the publishers and co-authors, all five manuscripts were reproduced in their entirety as chapters in this dissertation. Manuscript 1) Drikic M, De Buck J. Split trehalase as a versatile reporter for a wide range of biological analytes. Biotechnol Bioeng. 2018;115(5):1128-36. Epub 2018/02/08. Manuscript 2) Drikic M, De Buck J. Engineering split inteins to be activated by ligand-induced dimerization. Manuscript 3) Drikic M, Windeyer C, Olsen S, Fu Y, Doepel L, De Buck J. Determining the IgG concentrations in bovine colostrum and calf sera with a novel enzymatic assay. J Anim Sci Biotechnol. 2018;9:69. Epub 2018/09/15. Manuscript 4) Drikic M, Olsen S, De Buck J. Detecting total immunoglobulins in different animal species with a novel split enzymatic assay. BMC Veterinary Research. 2019; Appendix 1) Drikic M, and De Buck J. Directed evolution of TreA. Appendix 2) Drikic M, and De Buck J. Trehalase features. 4 ACKNOWLEDGMENTS I would like to acknowledge my supervisor and mentor Dr. Jeroen De Buck for his doctorate guidance, moderate patience, and scientific challenges. He always believed in me and encouraged me to explore different paths and develop as a scientist. His approach taught me strength and resilience, his questions sparked my curiosity, and his enthusiasm inspired my persistence even when my experiments were failing. I would like to thank him for all the valuable lessons that will serve me well even outside of the lab. I would like to acknowledge my committee members Drs. Ray Turner, John Gilleard and Tao Dong for their support and guidance through this journey. Their advice and perspective were valuable for the completion of this thesis. I would particularly like to thank them for always having my best interest in mind. I would like to give my warm appreciation to three research assistants that were directly supporting my work, Evan Compton, Steven Olsen and especially Yongpeng Fu. Firstly, Steven, your hard work was appreciated; thanks. Secondly, I will always be grateful for Evan Compton for helping me settle into the lab and teaching me all the nuances of living and surviving in Canada; thank you. And finally, special recognition goes to Yongpeng Fu who was always ready to support my work, help me out with EndNote issues, patiently listen to my complaints and provide moral support even for non-lab related problems; You are a dear friend; thank you very much. Special recognition also goes to all my summer students Crystal Ma, Seung Won Choi, Jan Y Xue, and Andy YW Lam. Without them, this thesis and my Ph.D. experience would have been as great of an experience. Thank you for your life long friendship, dedication, patience, and curiosity. Thank you for all the long nights you spent in the lab with me, for all the long hikes we took together, for all the 5 questions you asked about science and life. Thank you for giving me the opportunity to learn how to be a supervisor. I became a better student and a better person thanks to you all. Thank you, Rakel Arrazuria Fernandez, for your warm friendship and your patience. It was a pleasure spending my lunch breaks with you enjoying all the stimulating and never-ending discussions. Thank you for always understanding. Ravi Holani and Juliette De Francesco, you shared this journey with me. Thank you for their friendship and moral support, for all the coffee's, late night dinners, parties and all the great adventures we went through together. I will never forget it. I would also like to thank Uliana Kanevets for her friendliness and laboratory experience and to James Wang for his patience and laboratory managing, who indirectly supported me through all these years. I would like to thank Kasia Wojdyla Judycki for having answers to all my questions even when they were not related to the veterinary medicine program. I would like to thank her of all the meaningful conversations we had over the years. I would also like to thank Diego Nobrega and Sohail Naushad for all their life advice, moral support, and warm friendships. Thanks to all the past and present members of the De Buck lab. Thank you all for your support, advice and constructive discussions in our lab meetings. Thanks to all other students from the lab groups Cobo, Orsel, Barkema, Kutz, and Gilleard which I had a chance to meet, share the office and take part in meaningful discussions. I would like to thank all my friends outside of academia, my Zouk friends, Canadian, Italian and Serbian friends. Many of you will not read these acknowledgments, but if you ever do, you should know that you kept my heart warm and my spirit alive.