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. 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.
2. 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).
3. 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).
4. 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).
5. 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
6. BEP/MEP: Function and structure relationship of DNA-interacting proteins
In our lab, we have expertise in DNA and protein interactions, and in particular we study how proteins can organize the DNA into different conformations. Single-molecule techniques enable us to gain an understanding of the function and structural relationship of these proteins. Among the most important proteins which interact with DNA is the SMC (Structural Maintenance Complex) protein family. These proteins, including condensin, cohesin, or SMC5/6, are used for spatial organization of chromosomes and chromosome segregation. Using various single-molecule techniques, our group has shown the remarkable molecular mechanism of condensin. Our aim is to study how the structural changes of the SMC proteins are related to its function using complementary atomic force microscopy (AFM) and optical imaging methods. The students will learn valuable experimental skills such as dry AFM, liquid AFM, and optical microscopy.
For more information, please contact Je-Kyung Ryu (J.Ryu@[TUD]).
7. BEP/MEP: single-molecule investigtation 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).
8. Bacterial nucleoid project: study of nucleoid dynamics inside live E.coli
It is becoming clear that the spatial organisation of DNA is crucially important for its biological function. In living cells, DNA is highly confined in space with the help of condensing agents, high levels of supercoiling and numerous DNA binding proteins. Despite extensive research, the mechanical and geometric properties of the chromosomes, as well as their impact on DNA segregation are open to debate.
Here, through cell shape manipulation, quantitative imaging and numerical simulations, we unravel the intrinsic structure of E.coli chromosome. We use drugs and molecular genetics tricks to disrupt the cytoskeleton of bacteria, leading to cell sizes far larger than the rod-like wiltype cells. By releasing the lateral constraint on the chromosome in the bacterial cell we reveal an intrinsic donut topology of the chromosome composed of two dense bundles flanking the origin of replication, bridged by a thin Ter filament. The chromosome is highly dynamic and heterogeneous in density, showing morphological rearrangements as well as DNA-density redistribution at sub-minute timescales. We show that cellular crowding and entropic effect can lead to the spatial separation of chromosomes, which is reinforced by their weak associations with internal cell membrane.
For more information, feel free to pass by the office (F0.170) or drop an e-mail: a.japaridze@[REMOVE THIS]tudelft.nl
9. 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).
10. A bio-nano approach to point-of-care testing of parasitic DNA in resource limited settings
Background: Diagnostics are important for adequate health care. Current diagnostics are often expensive; require skilled personal, a stable source of electricity and a well-equipped lab to operate- requirements that are often not met in resource limited settings. There is a great need for cheap and simple point-of-care tests that do not require skilled users to probe for disease. Many treatable diseases remain untreated due to this lack of cheap and simple point-of-care tests.
Aim: The aim of this research project is to develop an innovative point-of-care diagnostic test that can be used for molecular diagnosis in resource-limited settings. We aim to probe for the presence of pathogenic DNA in body fluids using a cheap and simple, yet robust molecular test. The proposed method does not require electricity or skilled laboratory personal to operate. Furthermore, the test will function at a broad temperature range, yet remain highly sensitive and specific. Advantages over traditional methods of pathogen culturing and microscopy include the following: molecular diagnostics is rapid and enables results in real-time as opposed to culturing that takes weeks; it can be miniaturized in a lab-on-chip format and it is very specific- able to detect genetic factors such as drug resistance.
Detailed project description: The key scientific innovation is to use the CRISPR/Cas9 genome-engineering system as a DNA detection tool. The Cas9 protein and its sensing RNA have the unique capability to find any specific target DNA sequence in a given sample. We will exploit this capability and equip the Cas9 protein with guide RNA that enables detection of a DNA target sequence specific to a pathogen of interest. Furthermore, the cas9-guide RNA complex will be coupled to an enzymatic RNA molecule that will provide an amplified colorimetric readout in the presence of the specific pathogenic DNA sequence. A simple colour change, detectable to the naked eye, will be produced when the DNA sequence is present.
Short term research goals: To develop a molecular detection method directed at double stranded DNA by elucidating the molecular biology of the Cas9-guide RNA complex that will detect target DNA (Lambda DNA will be used as a model for proof of principal studies).
Long term research goals: To develop a microfluidic device. I will explore the use of paper fluidics and lateral flow assays to develop the microfluidic device. To develop a prototype of the molecular test and collaborate with the Royal Tropical Institute in Amsterdam on the evaluation and implementation of the test. Field-testing, in collaboration with various universities and hospitals, in disease endemic countries will be explored in the last phase of this research project.
Michel Bengtson (MSc)
Email: M.L.Bengtson@[REMOVE THIS]tudelft.nl
Contact Number: 015 2788780
11. Protein sequencing using nanopore technology
Proteins are the molecular machines in the human body. Therefore, understanding protein function is key to understand, detect, and cure diverse diseases. This function is tightly linked to the proteins’s 3D structure and ultimately its specific amino acid sequence.
Electric detection using single molecule nanopore technology has emerged as one of the most promising techniques to sequence proteins in a cheap and fast way: upon translocation through the pore, the protein of interest partially blocks the pore, thus changing the pore’s electric conductance. However, the higher complexity of proteins as compared to DNA (20 amino acids vs. 4 nucleotides) has remained a challenge.
In project 1, we address this complexity by attaching artificial marker molecules to specific amino acids, enabling their reliable recognition. The design and characterization of such chemical tags covers interdisciplinary concepts, such as the physics of nanopore technology, the chemistry of nanopore-tag interactions, and protein biochemisty.
Project 2, aims at controlling the notoriously fast translocation time of molecules through the pore that limits the observation time. Specifically, we exploit a motor protein to control translocation in an ATP-dependent manner. In this innovative approach, we combine the best of two worlds, namely physical single-molecule detection with the function of protein nano-machines. Experience in protein chemistry techniques is favourable for project 2.
Both projects are suitable for BEP and MEP. We look for highly motivated and independent students who seek to pursue interdisciplinary research at the nanoscale.
12. Influence of antibiotics on bacterial chromosomes
Diseases caused by various bacteria constitute one of the biggest public health problems worldwide, causing ~15 million deaths per year . Therefore improving our understanding of the antibiotic function as well as bacterial resistance mechanism is of paramount importance.
In our lab, we developed a novel method to manipulate bacterial cell shapes and by doing so we investigate the finer structure of E.coli chromosome. In the current framework we plan to study the influence of antibiotics on the chromosomes of live bacteria, by using fluorescence microscopy and quantitative data analysis (Fig.1).
We are looking for enthusiastic and hardworking students who are interested in multidisciplinary research. During this project you will learn how to work with bacterial cultures, how various types of antibiotics function and work with fluorescence microscopy to study the effects of drugs on bacterial chromosomes.
For more information pass by the office (F 0.170) or drop an e-mail : a.japaridze@[REMOVE THIS]tudelft.nl