We welcome students from a variety of backgrounds to participate in our research. Our group currently has the following opportunities for carrying out a bachelors or masters research project.
1. Understanding Palladium Zero-Mode Waveguides
Zero-Mode waveguides are sub-wavelength nanopores in metals films. They have the property to block incident light, whereas molecules can still diffuse into them and light up in there. This makes them a perfect tool for background supression in fluorescence microscopy. Typically ZMWs made from Aluminium or Gold are used, but we recently showed that it can be advantageous to use Palladium as a substrate material. Thus we would like to get a deeper understanding of this system by characterising it. We have a set up a protocol to make ZMWs of different diameters and of different depth with which it is possible to study effects on fluorescence lifetime, brightness etc.. For instance, it is known that ZMWs can lead to a fluorescence enhancement due to plasmonic effects, but it is not clear to what extent this plays a role for our structures.
Additionally we see that our fluorescent bursts are shorter than we would expect. These are only two of many questions we would like to address during this project.
In this project, it would be possible for you to acquire your own data at a high-end optical microscope, get to learn how to analise single-molecule fluorescence data and also simulate the system in your computer in order to gather a deeper understanding.
If you find this interesting, please send an email to n.klughammer@tudelft_remove_this_.nl
2. AFM-Fluorescence characterization of the plasmid partition protein ParB
3. Shining Light on the Nuclear Pore Complex using a Zero-Mode Waveguide
Selective transport of the Nuclear Pore Complex (NPC) can be reconstituted in minimalistic systems that consist of a solid-state nanopore functionalized with FG-nucleoporins. However, such systems present limitations in terms of biological relevance (due to applied electric field), and scalability to investigate more complex scenarios.
In this project, we tackle all these issues by with a method based on Zero-Mode waveguides (ZMW), sub-wavelength apertures in metal films (Fig.1), which we engineer with FG-nucleoporins. Even though light can not pass through the metal layer, molecules can still diffuse through the small aperture. As they reach the other side, fluorescent molecules are illuminated by the laser, one by one, resulting in single spikes in the detected signal.
With this technique we can for the first time study:
– Selective transport at the single-molecule level in free diffusion (no applied electric field)
– Parallel detection and discrimination of two, or more, proteins that translocate simultaneously
This approach will yield new mechanistic insights about transport through the real nuclear pore complex.
During this inter-disciplinary project you will learn how to work with ZMWs and operating a high end microscope, in order to do single molecule fluorescence experiments. Additionally, you will gather experience in
handling biological samples, functionalizing ZMWs with purified proteins, protein labelling, and learn about the nuclear pore complex.
If this triggers you, please contact Nils Klughammer (n.klughammer@[REMOVE THIS]tudelft.nl).
4. Building nanomachines
In Eukaryotes, cell division is accomplished by constriction of the actomyosin ring. In this wonderful biological machine, the concerted action of molecular motors, actin filaments and cross-linkers is able to achieve membrane constriction at a scale that is several orders of magnitude larger than the proteins themselves. Inspired by the architecture of the actomyosin ring, this project aims at engineering a synthetic constriction machinery by using DNA and RNA origami nanotechnology. This machine will be able to assemble on the surface of giant vesicles and induce membrane constriction, thus matching the complexity of its biological counterpart. This will constitute an invaluable a tool to study the molecular mechanism of constriction with an unprecedented level of control; it will also provide new engineering principles to develop future origami nanorobots.
We are looking for enthusiastic students who are interested in bottom-up synthetic biology. During this project you will learn how to design and assemble DNA/RNA origami and how to work with biological membranes, in particular Giant Unilamellar Vesicles and Supported Lipid Bilayers. You will image the constriction machinery in action by using a combination of fluorescence microscopy, Atomic Force Microscopy (AFM), High-Speed AFM and Electron Microscopy.
5. mRNA NEXT
Compartimentalization of DNA in the nucleus is one of the central features that distinguishes eukaryotic cells from simpler organisms. Like a door connecting two rooms, a huge multiprotein complex, the nuclear pore complex (NPC), enables communication and controlled passage of molecules between the nucleus and the cytoplasm. Fundamental cellular functions, like protein production, require the messenger molecule RNA to exit the nucleus through the NPC door; yet, how this transport actually occurs is still unclear.
The final goal of the mRNA NEXT project is to recreate a minimal nuclear export system that is able to drive transport of RNA in a functional mimicking of NPC. Recreating the functionality of the NPC de novo will allow us to unravel the nature of the minimal transport complex.
We use a current-base reading that enables us to distinguish single transport events of proteins and RNA through the biomimetic NPC. Since the proteins involved perform other tasks in the cell, our unique bottom-up approach is vital to address these questions. By joining this project, you will contribute to the first-ever successful reconstruction of mRNA export in a completely in vitro system!
You will be directly involved in experimental design, trained on protein production, molecular biology techniques, single molecule nanopore technology and QCM-D, in a unique combination of interdisciplinary methods at the merge of cell biology, biochemistry and bioengineering.
We are looking for independent and enthusiast Master/Bachelor students with Physics, Biology, or interdisciplinary background. International students are especially encouraged to apply.
For more info, contact Paola De Magistris (P.Demagistris@[REMOVE THIS]tudelft.nl) or visit our labs in Applied Sciences (office 58.F0.150).
6. DNA-origami scaffold for NPC mimics
With the help of DNA-origami nanotechnology, we built a minimalistic version of the NPC, where FG-Nups are anchored to the inner walls of a ~30 nm large DNA-origami pore, with predefined stoichiometry and positioning. We employ this platform to study the arrangement of FG-Nups within the pore volume using AFM and TEM imaging techniques.
We are looking for an enthusiastic Master/Bachelor student with an interest in synthetic bottom-up biology. Depending on the duration of your project, you will get trained on building NPC mimics using DNA-origami and FG-Nups, imaging them with TEM and AFM, and processing the data (Matlab/Image J/Gwyddion).
If interested, please contact Alessio at A.fragasso@[REMOVE THIS]tudelft.nl or drop by office (F0.170).
7. Mimicking selectivity of the Nuclear Pore Complex with Designer FG-Nups
In this project, we attempt at reconstituting NPC selective transport in biomimetic nanopores using bottom-up designed artificial FG-Nups. By testing the impact of systematic variations of artificial FG-Nups on selectivity, we aim at unraveling what are the minimal features for having an efficient and selective transport. We believe this novel approach will finally shed light on some fundamental physical principles that govern the NPC behavior.
We are looking for an enthusiastic Master/Bachelor student with a multidisciplinary background or interest. Depending on the duration of your project, you will get trained on engineering nanopores with FG-Nups, testing selective behavior of biomimetic NPCs and processing data (Matlab). Besides, you will get hands-on experience in using a TEM machine and detecting protein binding with a QCM-D.
If you feel thrilled, please contact Alessio at A.fragasso@[REMOVE THIS]tudelft.nl or drop by office (F0.170).
8. Characterisation of Zero-mode waveguides for Protein Sequencing
The possibility to measure a protein’s abundance in a very accurate way, will have giant impact on fields such as, cell biology cancer research and proteomics. However, the identification of proteins on a single molecule basis is still a great challenge. In this project we are setting up a system for the identification of individual proteins by protein fingerprinting. With this technique only the sequence of few aminoacids needs to be detected, in order to identify a protein. Our approach is based on Zero-mode waveguides, which are sub-wavelength pores drilled in a metal film.
Zero-mode waveguides have the property to block light, that shines on them. Still molecules can traverse through, either by pure diffusion or driven by a force. Whereas the analyte is not excited by the laser light on one side, it starts fluorescing and gets detected, when it has travelled through. If fluorophores travel through the pore with a spatial separation, this should be visible in a temporal sequence of fluorescence intensity bursts, the signal needed for the protein fingerprinting.
In the current state, we are able to detect fluorophores that are freely diffusing through our Zero-mode waveguides, but the system is not well characterised yet. The aim of this project is to use single fluorophores bound to DNA to find out about temporal and spatial resolution of our setup. The project is extremely interdisciplinary on the interface between Biology and Physics. During the project you will learn how to work with nanopores and how to operate a high end microscope, in order to do single molecule fluorescence experiments. Additionally you will get trained in handling biological samples and how to analyse fluorescence data.
If you find this interesting, please contact Nils Klughammer (n.klughammer@[REMOVE THIS]tudelft.nl
9. BEP/MEP: single-molecule investigation of RNA-polymerase interaction with supercoiled DNA
Cells store their genetic information in DNA. Typical length of the genomic DNA lies in a range of several millimeters to meters which needs to be well-organized for reading genes of demand. Consequently, it is not an easy task to put this long polymer inside the micron sized cell in a very organized fashion. Inter-winding and twisting of the DNA play a key role in packing the DNA into the cell, yet the perpetual access by the proteins, which reads the genetic information from the DNA and convert it into the vital chemical processes, makes the structural status of DNA dynamic and challenging. One such vital process is RNA-transcription where the DNA is read by a protein called RNA-polymerase (RNAP).
It is known that the activity of RNA-polymerase greatly depends on the supercoiled state of the DNA. For instance, while RNA is transcribed by RNAP, plectonemes are generated both downstream and upstream of RNAP/RNA/DNA complex (see figure). This in turn slows down RNA transcription by RNAP. However, most in vitro experiments were performed on linear DNA which does not represent the conditions that RNAP encounters in vivo. Thus, we are interested in studying the interaction of RNAP with supercoiled DNA using our newly developed high-throughput single-molecule technique (see Ganji and Kim et al, Nano Let. 2016). The outcome of this project will improve our understanding of mechanism of RNA transcription by providing detailed mechanism of how RNAP interacts with transiently generated plectonemes. A student with background in physics and/or biology will explore this interdisciplinary project and study how the supercoiled state of the DNA modulates the activity of RNA-polymerase.
For more information, please contact Eugene Kim (e.kim@[REMOVE THIS]tudelft.nl).
10. Single-molecule study of condensin-induced loop extrusion dynamics
It has been hypothesized that Structural Maintenance of Chromosomes (SMC) protein complexes such as condensin and cohesin spatially organize chromosomes by extruding DNA into large loops. We have recently provided experimental evidence for loop extrusion by directly visualizing the formation and progressive extension of DNA loops by yeast condensin in real-time (See Ganji et. al. Science 2018). We aim to further extend this study to investigate loop extrusion mechanism in supercoiled DNA (i.e. over- or under-wound DNA strands) as well as in the presence of RNA polymerase. This project is highly interdisciplinary and the student will be able to gain in-depth knowledge on single-molecule biophysics and experimental skills on HiLO/TIRF/wide-field microscopy and flow cell preparation.
For more information, please contact Eugene Kim (e.kim@[REMOVE THIS]tudelft.nl).