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. 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).
5. Building a biomimetic nuclear pore complex with DNA origami
DNA origami nanotechnology has enabled us to design and build specific shapes and structures on the nanoscale. We use this approach to build a minimalistic version of the nuclear pore complex (NPC) by grafting the disordered proteins of its central channel to the insides of a hollow octagonal DNA origami with a diameter of 35 nm. This approach gives us precise control over the stoichiometry and the positioning of individual proteins. We utilize this platform to study the arrangement and structural dynamics of the FG-nucleoporins in the pore on the single-molecule level by a variety of methods, including TEM, AFM, mass photometry and single-molecule FRET.
We are looking for enthusiastic Bachelor or Master students that are interested in trying to build life from the bottom up. During the project, you will learn how to build a biomimetic nuclear pore complex, validate the correct assembly, and characterize its properties using state-of-the-art single molecule methods.
If you are interested in this challenging, interdisciplinary project, please contact Anders Barth at a.barth@[REMOVE THIS]tudelft.nl.
6. 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).
7. 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
8. Single-molecule investigation of RNA polymerase interaction with supercoiled DNA
Cells store their genetic information in DNA. Typical lengths of genomic DNA lie in the range of several millimeters to meters when stretched, which needs to be well-organized for still being able to reading genes of demand. Consequently, it is not an easy task to put this long polymer inside a micron sized cell in an organized fashion. Twisting and inter-winding of the DNA play a key role in spatial organization and packing the DNA, yet the perpetual access by the RNA polymerase (RNAP), which transcribes the genetic information from the DNA during the biosynthesis of vital proteins and enzymes, makes the structural status of DNA dynamic and challenging.
It is known that the activity of RNAP greatly depends on the supercoiled state of the DNA. For instance, while RNA is transcribed by RNAP, inter-winded DNA plectonemes are generated both downstream and upstream of transcription elongation complex consisting of DNA, RNAP, and RNA (see figure). This in turn can slow down RNA transcription by RNAP or even stop it. However, most in vitro experiments were performed on linear DNA that does not represent the conditions that RNAP encounters in vivo. Thus, we are interested in studying both the generation of DNA supercoils during transcription and the interaction of bacterial RNAP with supercoiled DNA using our newly developed high-throughput single-molecule technique (Ganji, Kim et al, Nano Let. 2016, DOI: 10.1021/acs.nanolett.6b02213).
A student with background in physics and/or biology will explore this interdisciplinary project and be involved in the experiment design, single-molecule fluorescence measurements, and data analysis. The outcome of this project will advance our understanding of the mechanisms underlying of how RNAP generates and interacts with transiently generated DNA plectonemes.
If this project sparks your curiosity, please contact Richard Janissen (r.janissen@[REMOVE THIS]tudelft.nl).