Student projects

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. BEP/MEP project: Study of Z-ring assembly dynamics inside artificial minimal cells

One of the most marvelous features in cell life is division, i.e. the creation of two daughter cells from a mother one. In most of bacteria, this beautiful process is orchestrated by FtsZ, a protein that assembles a ring at the cell mid-plane. Such structure serves as a scaffold to recruit and coordinate further proteins constituting the so-called Z-ring. Altogether, the ring constricts and divides the cell into two. The exact mechanisms underlying this process are still not entirely understood. In this project, we choose a bottom-up approach to assemble from scratch the minimal components required to assemble a Z-ring: FtsZ and its membrane anchor protein ZipA. By encapsulating this reduced machinery inside artificially made confinements that mimic the cell conditions, we aim to reveal the dynamics of this fascinating process. During the project, the student will acquire fundamental biophysics skills and a broad interdisciplinary knowledge.
For more information, please contact f.fanalista@[TUD].


Fluorescence image of a minimal Z-ring in unilamellar liposome.

2. 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]).

Figure 1: AFM image of condensin binding to DNA.

3. Single-Biomolecule Sensing in Graphene Tunnel Junctions

Nanogaps separating two electrodes are envisaged as the basis for the next generation of molecular fingerprinting technologies. The aim is to exploit quantum electron tunneling as the sensing principle, in which the electronic structure of the target molecule trapped in the nanogap is directly probed.
You will develop mechanically controlled break junctions (MCBJs) based on graphene electrodes to study the charge transport across graphene-biomolecule-graphene junctions. Particular strengths of graphene MCBJs are (i) the tunability of the gap size at the nanometer scale and (ii) the statistically significant datasets that can be obtained for a single junction (>2,000 conductance traces). The graphene edges will be functionalized to specifically bind biomolecules (e.g. amino acids and proteins) in order to extract their electronic fingerprint.
We are looking for a highly motivated master student to join the van der Zant and Dekker labs, working at the crossroads of quantum charge transport and single-molecule biophysics.
• You will acquire a diverse range of skills including nanofabrication and characterization (ebeam lithography, reactive ion etching, metal evaporation, graphene transfer, SEM, Raman spectroscopy etc.)
• You will be involved in transport measurements in mechanical break junction setups (room temperature, low temperature, in liquid) and in statistical analysis.
• This challenging project between the departments of Quantum and BioNanoscience has a large experimental character and scope for high impact publications.
Please contact Sabina Caneva (s.l.caneva@[TUD])

Figure 1: Top. Biomolecule trapping in a graphene nanogap. Bottom. MCBJ measurement scheme.

4. BEP/MEP: Exploring the interactions between proteins and DNA molecules using Atomic Force Microscopy

In our lab we explore the dynamics and physics of single DNA molecules (see other projects by Anthony Birnie and Eugene Kim). These projects often call for the use of DNA-binding proteins, which can alter both the local as well as the long-range structure of DNA (see figure). Although we can easily purify these proteins from cells such as E. coli, little is known about the mechanism of their interaction with DNA. Our aim is to understand this mechanism by using the single molecule technique of Atomic Force Microscopy (AFM). This technique enables us to visualize, with nanometer resolution, structural and dynamic shape-changes of the DNA upon binding of the proteins, such as bending, looping and stiffening.
For this challenging project, we are looking for an enthousiastic student who enjoys working independently and is not afraid of troubleshooting experimental challenges. You will be involved in all aspects of the project: experimental planning, data acquisition and of course the analysis. We offer an opportunity to learn in depth how to use AFM, and to gain knowledge about the physics and biology of DNA and the associated protein machinery.
Please contact Anthony Birnie via a.t.f.birnie@[TUD] for more information.

5. 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 [1]. 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@[TUD]

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Fig1. Time-lapse images of an E.coli cell lysis. DNA inside the cell is labelled with fellow fluorescent protein.

[1] World Health Organization. 2013. Mortality and global health estimates. Geneva, Switzerland: World Health Organization

6. BEP/MEP Genome-in-a-Box: transplanting the genome from a live cell into a synthetic cell.

The overarching goal of the Genome-in-a-Box project is to create a simple model system of the genome and its interaction with the cell wall and the various cellular components, such as the cytoplasm and DNA-binding proteins. This is not a computer model, but a “real” experimental synthetic cell containing artificial cytoplasm and a transplanted genome. We hope to re-create inside the synthetic cell the structure and dynamics of the genome observed in real live cells: essentially Feynman’s mantra of “what I cannot create, I do not understand”.

For this BEP/MEP project, we are looking for a creative student to help establish this model system, which would be a world-first! You will need to come up with a way to re-create the crowded conditions of the live-cell cytoplasm inside a synthetic cell. Secondly, the genome of the live-cell needs to be transplanted into the synthetic cell. Finally, we aim to let the synthetic cell expand and contract inside microfabricated structures, to see how the cell size and shape might influence the genome and its dynamics.

These are certainly non-trivial questions: what molecules do we use to make the artificial cytoplasm, what DNA-binding proteins do we add to alter the genome structure, what synthetic cell system do we use, how can we change the shape and size of a synthetic cell?

For this project, you will work independently and you will also be closely involved in the design of experiments. You will learn about microfluidics, cleanroom fabrication, synthetic cells, polymer physics, genome organization and many aspects of cell culture and DNA labeling.

Anthony Birnie
E-mail : a.t.f.birnie@[TUD]
Phone : +31-(0) 15 2789299

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7. BEP/MEP: Biomimetic Nuclear Pore Complexes

Life is a dynamic process composed of various complex and regulated sub-processes. One of the key questions is to understand the nuclear pore complexes (NPC) that regulate the selective exchange of RNA and proteins across the nuclear envelope in eukaryotic cells. To tackle this, we developed a minimalistic artificial version of the NPC (see Figure), by combining solid-state nanopores and purified nuclear pore proteins (FG-Nups). We can nicely detect single-molecule transport events of proteins through the Nup-coated pore and test its selective behaviour under different conditions. Ultimately, we aim to unravel the underlying fundamental mechanism of transport through the NPC. Besides, we take advantage of Transmission Electron Microscope (TEM) and Quartz Crystal Microbalance (QCM-D) as complementary techniques.

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We are looking for a motivated and creative Master / Bachelor student with a multidisciplinary background. Depending on the duration of your project, you will get trained on engineering nanopores with FG-Nups, performing nanopore experiments and data processing. Besides, you will get hands-on experience in using a TEM machine and detecting protein binding with a QCM-D.
If interested, please feel free contact Alessio at A.fragasso@[TUD] or drop by office (F0.170).
Further suggested Reading:
*Stefan W. Kowalczyk et al., TRENDS BIOTECHNOL.,29, 607–614 (2011).
** Stefan W. Kowalczyk et al., Nat. Nanotechnol., 6, 433–438 (2011).

8. BEP/MEP: Plasmonics assisted dielectric breakdown for nanopore production

Solid-state nanopores are a promising technique for rapid, low cost and accurate sequencing of DNA. A huge challenge in this technology is the high DNA translocation speed through the nanopore which are currently too quick for measurement resolution. A way to arrest or stall this motion is to induce large optical gradients to slow down the DNA. By coupling graphene with a plasmonic bowtie structures, we demonstrate through FDTD simulations that we are able to create highly confined field intensities at the edge of the nanopore for interaction with the DNA. The next step is to perform DNA translocation experiments in order to experimentally verify trapping of the DNA. The student will learn to work with graphene devices , perform plasmonic nanopore measurements and data processing.

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Fig. 1 Graphene Plasmonics Nanopore. a) TEM image of fabricated graphene plasmonics nanopore. b) Side view of Lumerical simulation of optical hotspots.

Wayne Yang
Email: w.w.w.yang@[TUD]

9. BEP/MEP: Atomic force microscope imaging on condensin-mediated dna condensation.

Condensin is a key molecular machinery to compact 2-meter DNA into μm-sized chromosomes. Condensin is an ATPase with a ring-structure of 5 subunits. It is still a mystery how this large amount of DNA content is packaged into such small structure. Our goal is to shed light on the molecular mechanism of condensin using a new, state of the art, single-molecule technique called high speed atomic force microscopy (HS AFM). HS AFM is an excellent tool for the understanding of protein’s structure and function because we can directly visualize real-time protein’s structure changes using a high spatial and temporal resolution (< 1nm, < 10 ms) in a liquid phase (in nearly physiological condition). If we can directly visualize both the conformational changes of condensin via ATP hydrolysis and DNA topological changes, we will be able to answer one of the most fundamental question about the molecular mechanism of condensin.
We are looking for enthusiastic and diligent master students to study the mechanism of condensing-mediated DNA compaction. The student will learn valuable skills in the operation of HS-liquid AFM as well as other biochemical experiences.

For more information, please contact Je-Kuyung Ryu (J.Ryu@[TUD]) or drop by office (F0170).

Real-time HS AFM images on two of the five subunits (the SMC dimers) of condensin. The dynamical conformational changes show that the coiled-coils of SMC dimers are flexible and show extensive fluctuations in time (Eeftens et al., 2016, Cell Reports 14, 1813–1818, 2016).

10. 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).

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@tudelft.nl).

11. 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@[TUD]
dnadynamics

12. 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@tudelft.nl).

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13. BEP/MEP: Towards membrane-less synthetic cells: growth and division of liquid droplets

This project (BEP/MEP) will explore synthetic construction and manipulation of membrane-less containers such that they will exhibit two fundamental characteristics of living systems: growth and division. Along with membrane-bound organelles, living cells contain numerous well-defined, membrane-less structures. Commonly known as coacervates or liquid droplets, these structures are formed by a liquid–liquid demixing process, through attractive electrostatic interactions between two or more oppositely charged polyelectrolytes or small multivalent molecules. Recent theoretical work has demonstrated the possibility of repetitive cycles of growth and division of coacervate-like systems, by maintaining them in a chemically driven non-equilibrium state. This project will aim towards achieving such a growth-division cycle experimentally.

You will be involved in an exciting and explorative research project, with plenty of scope for your own crazy ideas. Primary aim will be to come up with the right ingredients to make coacervates in a controlled way. The next step will be to maintain them out-of-equilibrium by feeding them with individual components and at the same time triggering a decomposition reaction within them. This degradation of components will be explored through various protein systems, mainly enzymes that will catalyse a specific reaction. A right balance of growth and decomposition will induce a shape instability, leading to a grown coacervate dividing into two/more daughter coacervates!

Video legend: coacervate formation via enzyme-catalyzed reaction

For further information please contact me,  Siddharth Deshpande (S.R.Deshpande@[TUD])  or just drop by my office (F0.190).

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14. Investigation of protein-induced division of liquid droplets

This student project will explore synthetic construction and manipulation of membrane-less containers that will exhibit two fundamental characteristics of living systems: growth and division. Along with membrane-bound organelles, living cells contain numerous well-defined, membrane-less structures. Commonly known as coacervates or liquid droplets, these structures are usually formed by a liquid–liquid demixing process, through attractive electrostatic interactions between two or more oppositely charged polyelectrolytes (polypeptides, polynucleotides, polysaccharides), or small multivalent molecules. Recent theoretical work has demonstrated the possibility of repetitive cycles of growth and division of coacervate-like systems, by maintaining them in a chemically driven non-equilibrium state. This project will aim towards achieving such a growth-division cycle experimentally.
The student will be involved in standardising a production method to make coacervates in a controlled way, preferably using microfluidics. Once a production method is established, the aim will be to maintain the coacervates out-of-equilibrium. Growth will be achieved by constantly feeding the liquid droplets with individual components. At the same time, a shape instability will be induced by triggering a decomposition reaction within the coacervates. This degradation of components will be explored through various protein systems, mainly enzymes that will catalyse a specific reaction. Several different enzymes will be tried to achieve the desirable result of a grown coacervate dividing into two/more daughter coacervates.

siddhuagain

Siddharth Deshpande (S.R.Deshpande@[TUD])

15. 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@[TUD]
Contact Number: 015 2788780

crispr

16. 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.
Sonja Schmid
s.schmid@[TUD]
015-278 7915
protein pore

17. Double nanopores for electrical tweezing of DNA molecules

DNA is the most essential molecule of life as it is the principal information carrier for all living systems. Studying the physical and chemical properties of DNA is not only fundamentally attractive, but also crucial for solving major biomedical challenges such as treating cancer or degenerative diseases.
Solid-state nanopores hold great promise for becoming a high-throughput analytical tool for studying DNA properties, particularly DNA sequencing. The beauty of the nanopore platform relies in its simplicity: the passage of biomolecules through a nanopore can be detected by a temporary modulation of the nanopore conductance that they induce. However, as compared to their biological counterparts, solid-state nanopores yet suffer from the immense speed at which biomolecules pass the sensor, which severely compromises the read-out precision. In this project we are going to overcome limitations of conventional solid-state nanopores using system of two separately-addressable nanopores to manipulate single DNA molecules. The key idea is manipulating an individual DNA molecule by stretching it in a nanoscale tug-of-war between solid-state nanopores and controlling it displacement by changing electric field in each of the nanopores (See animation) If you are eager to try on-chip electrical tweezing of single molecules, please contact Sergii Pud (s.pud@[TUD])

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18. Plasmonic nanopores

Biomolecules, the little molecular machines that make up life’s complexity, are at the center of disease and treatment. Understanding their properties on the single molecule level at high-throughput is essential for drug development.
Towards this end, we are developing a universal single-molecule biosensor based on a plasmonic gold nanoantenna; a nanoaperture in a freely suspended gold film. When light is impinged on this antenna, electrons in the antenna help focusing the light down to size of the metal nanostructure. This tight electromagnetic field concentration allows us to optically trap biomolecules and provides us read-out from the light scattered by the nanoantenna, providing us information about the physical properties of the biomolecules. Moreover the thin film allows us to actively attract molecules towards the sensor and act electrokinetic forces on them to calibrate our nanoplasmonic trap.
We encourage highly motivated bachelor and master students to join us promptly
for a bachelor (8-12 weeks) or master (6-12 months) project. This challenging project will have a largely experimental character and a decent background in optics, electronics and nanofluidics is welcomed. You will be involved in biomolecular trapping experiments and data analysis (MATLAB), and can be, depending on your interests, involved in device fabrication, modeling, data analysis (MATLAB), simulations (FDTD), building device electronics and control software.

Please contact Daniel Verschueren (d.v.verschueren@[TUD])

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