We are always looking for talented bachelor and master students who are interested in origins of life, synthetic cells, coacervates, physical biology, big data and machine learning or multi-omics analysis methods, who like to work in an interdisciplinary cutting-edge environment. If you are interested in joining, please contact us here, or approach one of the members of the group directly.
Immobilization of enzymes on polyelectrolytes
In Enzymatic subgroup we aim to design artificial enzymatic networks that are responsive to stimuli and can exhibit controlled complex behaviour, for final goals of creating smart materials and biomolecular information processing units. To keep the enzymes enclosed in a reactor or on a surface, we covalently immobilize them on polyacrylamide (PAAm) gel beads. It has been recently shown, that the pH optimum of an enzyme urease can be shifted from 7.5 to 4, with sufficient activity even at pH 3, via immobilization on a polycationic amino resin. We conducted initial studies and observed similar effects for a broader range of enzymes upon immobilization on polyacrylamide-co-polyelectrolyte beads. We would like to study this effect of pH optima shifts in greater detail now and pay additional attention to pH-dependent swelling/deswelling of the gel beads. The tuning of the pH activity curves will allow us ultimately to finely tune enzyme response parameters and create more effective networks. It is also of fundamental interest to understand what factors influence pH shifts upon immobilization. The phenomena connected to pH-dependant swelling-deswelling of the gel beads can be a foundation to construct systems with controlled complex behaviour, such as chemomechanical feedback or an oscillator.
The project will include work with microfluidic chips to produce the beads, optimizations of immobilization reactions, and analysis of activity of various enzymes with use of flow chemistry, fluorescent and UV-vis spectrometry, and pH-metry. The swelling/deswelling phenomena and activation of autocatalytic urease beads will be studied via fluorescent microscopy on OLYMPUS IX-83 and the images will be analysed in FIJI software. Some co-monomers or linkers will be synthesized.
The ideal candidate has experience in protein chemistry or polymer chemistry, and interest to learn new techniques. Some experience with organic synthesis is an advantage.
- N.M. Ivanov, M.G. Baltussen, C.L. Fernández Regueiro, M.T.G.M. Derks, W.T.S. Huck. Computing Arithmetic Functions Using Immobilised Enzymatic Reaction Networks. Angew. Chem. Int. Ed. 2023. 62(7). E202215759. DOI: 10.1002/anie.202215759
- D. Yanga, J. Fana, F. Caoa, Z. Denga, J.A. Pojman, L. Ji. Immobilization adjusted clock reaction in the urea–urease–H+ reaction system. RSC Adv., 2019, 9, 3514-3519. DOI: 10.1039/C8RA09244C
Keywords: Enzyme Assays, Polymer synthesis, Microfluidic devices, Enzymatic networks
Supervisor Nikita Ivanov
New approaches for photoswitchable inhibitors
Artificial enzymatic reaction networks are an excellent tool for designing responsive materials and coupling controlled dynamics to chemical functions. To tune the enzymatic activity by a light input, one can use photoswitchable inhibitors. Ideally, one of the photoswitchable isomers serves as a strong inhibitor and another is weak (measured by switching ratio: SR = Ki, form 1/Ki, form 2.)
Until now, most of the developed photoswitchable inhibitors were based on azobenzene photoswitches. In many cases, achieving a high switching ratio was difficult or not possible, because of too small difference in molecular properties of E and Z forms of azobenzene.
Previously, we synthesized a series of photoswitchable inhibitors of an enzyme urease (UrPI’s), based on azobenzenes. These compounds possessed moderate half-lives and switching ratios. As one part of the current project, we would like to further develop the azobenzene-based UrPI’s by introducing a range of substituents, most probably via cross-coupling chemistry.
A novel class of photoswitchable molecules, alternative to azobenzenes, is acylhydrazones. While acylhydrazone photoswitches are well described in literature for various uses, they were not yet applied to design photoswitchable inhibitors. Some properties of acylhydrazones make them favorable for design of photoinhibitors, such as facile synthesis, excellent photophysical tunability, and high water solubility. Most importantly, the molecular structure of acylhydrazones allows to introduce the inhibitory warhead right at the switchable double bond, and the drastic change of configuration of the inhibitory warhead upon switching must be beneficial for obtaining higher switching ratios. While we have broad hands-on experience in synthesis of azobenzenes, the acylhydrazone direction will be new to our lab and requires creative approach.
The techniques applied in the project will include organic synthesis, UV/vis spectrometry, various modifications of NMR (including LED-NMR for characterization of the photoswitchable forms), and measurements of enzymatic activities. Fluorescence microscopy and flow chemistry will be used to investigate applications of successful photoinhibitors.
The ideal candidate has experience in organic synthesis and spectroscopic characterization, and some experience or interest in enzymes.
- Volarić J. et al. Molecular photoswitches in aqueous e Chem. Soc. Rev. 2021. 50. 12377-12449. DOI: 10.1039/D0CS00547A
- Teders M. et al. Reversible Photoswitchable Inhibitors Generate Ultrasensitivity in Out-of-Equilibrium Enzymatic Reactions. Am. Chem. Soc. 2021. 143. 15. 5709–5716. DOI: 10.1021/jacs.0c12956
- van Dijken D., Kovaříček P. et al. Acylhydrazones as Widely Tunable Photoswitches. Am. Chem. Soc. 2015. 137. 14982-14991. DOI: 10.1021/jacs.5b09519.
Keywords Photoswitching, Azobenzenes, Organic synthesis, Inhibitors, Enzymatic networks
Supervisor Nikita Ivanov
Cell-free expression systems based on minimal bacterial cells
In vitro systems that perform transcription and translation (cell-free expression systems) are necessary for building a synthetic cell. In partnership with the J. Craig Venter Institute (La Jolla, USA), we are developing cell-free expression systems based on the minimal bacterium JCVI-syn3A. We are using two different approaches: (i) cell lysate-based systems and (ii) PURE-based systems (protein synthesis using recombinant elements).
Keywords Mycoplasma, JCVI-syn3A, cell lysate, PURE, minimal cell, cell-free expression system.
Deadlines Master’s students should apply before 01-10-2023, no deadline for bachelor students.
Supervisor Andrei Sakai
Studying cellular signaling in dynamic environments with single cell/omics techniques
The aim of this project is to further our understanding of how biochemical information flows through cells upon external, dynamic stimulation. We study cellular signaling networks with phospho-specific flow cytometry or immunodetection by sequencing techniques. You will work on B-cell signaling activation upon different types of stimuli and inhibitors in both healthy and diseased cell models.
Keywords Signaling, Molecular Biology, Phospho-flow Cytometry, Immunodetection by sequencing, Omics, Data analysis
Supervisor Melde Witmond