Student Research Projects
Each year we welcome more than 10 students from the Netherlands and abroad for their Bachelor or Master internships. Please check out the projects of the PhD students below. If you are interested in one of the projects, e-mail the researcher with your motivation and your CV. Please note that projects shown here do not always have a vacancy. Therefore, please contact the researcher well ahead of your intended starting date!
Adrian Kopf obtained BSc and MSc degrees in Bio-Pharmaceutical Sciences from Leiden University. He started his PhD in 2017 at Membrane Biochemistry and Biophysics (MBB) in the group of Antoinette Killian. The research is focused on unravelling the mechanism of membrane solubilization by styrene-maleic acid (SMA) copolymers to form native nanodiscs. Currently he is investigating the role of polymer length and comonomer sequence on the solubilization efficiency of SMA for the formation of SMA-lipid particles (SMALPs).
Styrene-maleic acid (SMA) copolymers are able to solubilize lipid membranes into nanodiscs and thereby trap membrane proteins in their native lipid environment in a water-soluble form. With this method it is then possible to study not only properties of the protein itself, but also lipid-protein, protein-protein, and protein-ligand interactions. Membrane proteins make up a significant fraction of gene products and an even larger percentage of targets for pharmaceuticals. This highlights the importance of SMA as novel tool to study these highly sensitive and poorly soluble proteins.
Commercially available SMA polymers have the disadvantage that they have a very heterogeneous molecular weight distribution, as well as a non-uniform sequence distribution. Through collaboration with prof. Bert Klumperman from Stellenbosch University (South Africa, Department of Chemistry and Polymer Science) we have acquired for the first time well-defined and homogeneous polymers. Using these novel polymers it is being investigated what role polymer length and comonomer sequence have on the solubilization efficiency. The aim is to gain a deeper understanding of the mechanism of lipid membrane solubilisation by SMA to form nanodiscs. This will enable a more targeted approach when performing specific membrane protein solubilization studies for further downstream applications and it will help the development of new applications. To reach this goal the solubilization of lipid model membrane systems (liposomes) as well as biological membranes (E.coli) are being investigated. This is done by looking at various properties such as the kinetics, efficiency, and extent of solubilization. In addition, we aim to further characterize the resulting nanodiscs.
The SMALP field is rapidly growing and interest therein expanding.
The technique has the potential to revolutionize the study of membrane proteins and you may be part of this!
Depending on your individual interest and your precise project the techniques used may include:- Electron Microscopy (EM)
- Dynamic Light Scattering (DLS)
- UV/Vis spectroscopy
- Infrared (IR) Spectroscopy
- Differential Scanning Calorimetry (DSC)
- Size-Exclusion Chromatography (SEC)
- Gas Chromatography (GC)
- Thin Layer Chromatography (TLC)
- Cell Culture
- Gel Electrophoresis (SDS-PAGE)
- Nuclear Magnetic Resonance (NMR)
- Organic Synthesis
For further reading see the following review:Dörr JM, Scheidelaar S, Koorengevel MC, et al.
The styrene–maleic acid copolymer: a versatile tool in membrane research.
European Biophysics Journal. 2016;45:3-21. doi:10.1007/s00249-015-1093-y.
Mike Renne started his PhD in 2014 in the lab of Toon de Kroon and he uses the yeast Saccharomyces cerevisiae (baker’s yeast) as a model organism to study the regulation of membrane fluidity, which is biophysical parameter important for proper membrane function. Previous studies in this lab have shown that besides the activity of the acyl-CoA desaturase, which converts saturated into unsaturated acyl chains, also certain acyltransferases have a role in determining membrane lipid unsaturation.
In previous research, we have genetically manipulated membrane lipid composition by loading the membrane with saturated fatty acids (from ±20% saturated acyl chains to ±35%), which is achieved by overexpressing the glycerol-3-phosphate acyltransferase gene SCT1. The overexpression of SCT1 causes a growth defect, which we can use as a simple readout to screen for synthetic genetic interactions that alleviate this effect and thus may be involved in regulation of lipid metabolism and/or stress responses.
Currently, Mike is expanding this research and working on elucidating novel regulatory mechanisms in lipid acyl chain homeostasis. Several projects are available for students to work on.
The mitogen-activated protein (MAP) kinase signaling pathways are highly conserved in evolution and the presence of MAP kinases is shared between fungi, plants, animals and other eukaryotes. In general, a receptor or sensor is activated, which induces a phosphorylation-based signalling cascade, involving various kinases, resulting in the activation of transcription factors. In yeast, MAP kinase signaling regulates various physiological processes such as cellular integrity in response to stress, adaptation to changing osmolarity and adaptation to changing temperature.
Recently, the cell wall integrity MAP kinase signaling cascade has been implicated in the regulation of membrane fluidity homeostasis (Lockshon et.al. – Plos ONE 2012). Furthermore, the high osmolarity MAP kinase signaling cascade was proposed as a novel candidate for the regulation of sphingolipid homeostasis (da Selveira dos Santos et.al. – MBoC 2014).
In this project, we will focus on the MAP kinase signaling cascades from the cell wall integrity pathway (MAP kinase: Slt2p) and the high osmolarity glucose response pathway (MAP kinase: Hog1p). We will combine classic yeast genetic-, biochemical and molecular biological approaches with state of the art lipid analysis techniques to elucidate the role of these signalling cascades in the regulation of lipid metabolism.
Are you interested in doing an internship with Mike, please email him your motivation and CV.
Xiaoqi Wang did the Bachelor and Master Degree at China with biochemistry background. She got the 2015 China Scholarship Council – Utrecht University PhD Programme and started the Phd program in March 2016 to study the MOA of Lantibiotics under the supervision of Eefjan Breukink .
Lantibiotics, a group of peptides belonging to Class I bacteriocins, exhibit great antibacterial activity against a broad spectrum of gram-positive bacteria including several antibiotic-resistant pathogens. Studying the MOA of lantibiotics may help us to understand how to tackle the current resistance problem.
Epilancin 15X is, like nisin, bactercidal and displays activities that point to a pore-formation mechanism, as it causes depolarization of the membrane potential in intact cells at concentrations around the MIC value. We used this membrane potential depolarization assay to test several potential antagonists that may provide insight into the nature of its target. These results point to a possible interaction of epilancin with Lipid II. However the interaction of epilancin 15X with Lipid II seems not of a sufficiently high affinity to explain the low MIC of the bacteria. In addition, we could not detect any Lipid II-dependent leakage caused by epilancin in model membrane vesicles. Attempts to identify the actual target of epilancin 15X are underway.
Techniques used in our search for the target include: Protein purification (making different types of purification (open column, HPLC etc.), Agar diffusion assay for determination the activity of lantibiotics, Model membrane vesicles for testing the interaction between epilancin 15X and potential targets. Biochemical and molecular biological approaches for studying the MOA of lantibiotics.
In further research, we will use both a genetic and chemical biology-based approach. The genome of resistant mutant strains will be compared with the wild-type to uncover the key genes in his mode of action. While in chemical biology-based approach, the lantibiotics will be labeled with report groups to determine the biological targets.
Are you interested in doing an internship with Xiaoqi, please email her and include your motivation.
Yang Xu obtained his master degree in Shanghai Jiao Tong University in March, 2015 with a pharmaceutical Chemistry Background. After he did some research about industrialization of artemisinin as a research assistant in the same University for one year, he got the 2016 China Scholarship Council – Utrecht University PhD Scholarship and then started his research on labeling lipopolysaccharide and peptidoglycan by using synthetic biomolecules under the supervision of Dr. Eefjan Breukink. This research should lead to a screening format to find potential antibiotics in fungal extracts. In his project, both synthetic chemistry and biological chemistry are involved.
Compared to Gram-positive bacteria, gram-negative bacteria are becoming more of a threat to man-kind in view of their increased resistance to antibiotics. One essential reason is that gram-negative bacteria possess an outer membrane. Lipopolysaccharide (LPS) is a main and essential component of this outer membrane. We are currently trying to specifically label the LPS of a gram-negative bacterium in order to follow its biosynthesis in real-time. When successful we will use this for screening fungal extracts that are active towards this biosynthesis pathway.
Peptidoglycan which is an important component of gram-negative bacteria cell wall can be labeled via by supplying certain precursors, which allows the specific labeling of the PG-layer in later stages of growth. . We will use this labeling in order to screen for compounds that are affecting the permeability barrier of the outermembrane, which would result in making gram-negative bacteria more sensitive to antibiotics.
The research in these programs involves both synthetic chemistry and biological chemistry. Some techniques that are used are: chemical synthesis, enzyme catalysis, column chromatography, NMR spectroscopy, Mass-spectroscopy, TLC, SDS-PAGE, plasmid isolation and transformation, fluorescence spectroscopy, fluorescence microscopy, solid phase synthesis ( using peptide synthesizer), LPS isolation and peptidoglycan isolation…
If you are interested in doing an internship with Yang, please feel free to contact him using the "Contact Yang Xu" button providing also a motivation. Students with a background in chemistry and/or biochemistry are welcome!
Xue Bao obtained her bachelor degree at the Shandong Normal University in 2009 and her master degree at the Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences (中国科学院青岛生物能源与过程研究所 ) in 2012. Subsequently, she obtained a scholarship of the 2012 China-Utrecht University PhD programme and continued her studies as a PhD candidate in the group of Toon de Kroon. After she obtained her PhD degree in March 2018, she is continuing her studies as Post Doc togther with Toon de Kroon.
Phosphatidylcholine (PC) is the most abundant membrane lipid in most eukaryotes and considered essential. The yeast double deletion mutant cho2opi3 lacks the methyltransferases which convert phosphatidylethanolamine (PE) to PC. Consequently, the cho2opi3 mutant is a choline auxotroph that relies on supplementation with choline for the synthesis of PC by the CDP-choline route. However, recently we isolated cho2opi3 suppressor (cho2opi3S) clones that suppress the auxotrophy for choline, and show robust growth in the absence of choline or choline substitute. Preliminary analysis of the lipidome of cho2opi3S clones showed that PC is below the detection limit after culture without choline; instead PE has become the most abundant phospholipid. The neutral lipid triacylglycerol strongly accumulates in the PC-free cho2opi3 suppressors. Importantly, the lipidome of the PC-free cho2opi3S reveals an overall shift to shorter average acyl chain length, which is thought to play a key role in maintaining membrane physical properties. Whole genome sequencing of a subset of suppressor mutants suggested 2N-1 aneuploidy as mechanism underlying the adaptation, which was proven by engineering a corresponding 2N-1 cho2opi3 mutant. Objectives
To solve the mechanism that enables yeast to live without PC, we want to address the following questions:
(1) How does cho2opi3S shorten the average acyl chain length?
(2) What are the roles of PE and TAG metabolism in PC-free cho2opi3S?
(3) What is the molecular genetic mechanism of PC-free cho2opi3S?
(4) How is aneuploidy induced?
Lipid analysis: Thin layer chromatography (TLC), Gas chromatography (GC), Mass spectrometry (MS), Isotope labeling
Yeast molecular biology: Gene deletion by homologous recombination, CRISPR-Cas9, DNA and RNA isolation
Are you interested in doing an internship with me, feel free to send a e-mail with your motivation and CV.