Group: Membrane Enzymology
Supervisors: Laura Heinen and Bert Poolman
Project 1: Real-time monitoring of metabolic energy conservation in synthetic cells – a comparative study of proteinogenic ATP sensors.
This student project lies at the heart of synthetic biology. It is embedded into the BaSyC research programme, where scientists from six Dutch research institutes jointly aim to build a synthetic cell. Following a modular bottom-up approach, our task is to energize the synthetic cell, i.e. providing modular enzymatic reaction networks for the provision, recycling, and homeostasis of cellular energy. Recently, we could publish a system for the regeneration of adenosine triphosphate (ATP), the prime energy currency of a cell.1
Now, to better understand the energy demand and spatiotemporal energy fluxes of a synthetic cell, we want to monitor in-situ the dynamics of the ATP levels upon integration with other functional modules, i.e. energy-consuming reactions. However, real-time monitoring of ATP dynamics by means of ATP-sensing molecular probes is often affected by other environmental parameters: varying pH, ionic strength or other chemically similar metabolites and nucleotides can influence the read-out drastically. Hence, a robust, reliable, and specific molecular ATP sensor is needed.
The aim of this research project is to screen different proteinogenic, synthetic and hybrid fluorescent ATP sensors to probe ATP levels selectively and reliably under a broad range of conditions.2–4 This comparative study of different ATP sensors will benefit the understanding of many of our systems and also find broad applicability outside our group for the study of ATP-driven metabolic processes.
The research project comprises the expression of the genetically encoded fluorescent protein sensors and the systematic and in-depth in-vitro characterization of the sensors properties (e.g. sensitivity, selectivity, dynamic range) under different ambient conditions, both in solution and in vesicular systems, including the calibration of the fluorescent signal. Finally, the sensors will find application in our synthetic cell to gain deeper insight into the dynamics of metabolic processes and energy fluxes.
The focus of this project will be adapted according to the background and interest of the student.
We are happy to discuss the project further with you and welcome you in our synthetic cell group!
(1) Pols, T.; Sikkema, H. R.; Gaastra, B. F.; Frallicciardi, J.; Śmigiel, W. M.; Singh, S.; Poolman, B. A Synthetic Metabolic Network for Physicochemical Homeostasis. Nat. Commun. 2019, 10 (1), 4239. https://doi.org/10.1038/s41467-019-12287-2.
(2) Kitajima, N.; Takikawa, K.; Sekiya, H.; Satoh, K.; Asanuma, D.; Sakamoto, H.; Takahashi, S.; Hanaoka, K.; Urano, Y.; Namiki, S.; Iino, M.; Hirose, K. Real-Time in Vivo Imaging of Extracellular ATP in the Brain with a Hybrid-Type Fluorescent Sensor. eLife 2020, 9, e57544. https://doi.org/10.7554/eLife.57544.
(3) Botman, D.; van Heerden, J. H.; Teusink, B. An Improved ATP FRET Sensor For Yeast Shows Heterogeneity During Nutrient Transitions. ACS Sens. 2020, 5 (3), 814–822. https://doi.org/10.1021/acssensors.9b02475.
(4) Tantama, M.; Martínez-François, J. R.; Mongeon, R.; Yellen, G. Imaging Energy Status in Live Cells with a Fluorescent Biosensor of the Intracellular ATP-to-ADP Ratio. Nat. Commun. 2013, 4 (1), 2550. https://doi.org/10.1038/ncomms3550.