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. 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
2. Genome-in-a-box: transplanting the genome from a live cell into a synthetic cell (BEP/MEP)
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? Do liquid-liquid phase transitions have a role to play in genome organization? How can we change the shape and size of a synthetic cell? How does the local action of, for example, a single loop-extruding protein lead to emergent features on the global scale of the genome?
Specifically, we look for a self-motivated student who would like to experience working as an independent scientist. You will be closely involved in the design of experiments and you will learn about microfluidics, cleanroom fabrication, synthetic cells, polymer physics, genome organization and many aspects of cell culture and DNA labeling.
If this sounds like you, please contact me at:
E-mail : a.t.f.birnie@[REMOVE THIS]tudelft.nl
Phone : +31-(0) 15 2789299
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. BEP/MEP project: mRNA NEXT
Compartimentalization of DNA in the nucleus is one of the central features that distinguishes eukaryotic cells from less evolved organisms. This separation allows reaching on unprecedented regulation of cellular functions in order to achieve higher complexity, for purpose of adaptive benefits. Communication between nucleus and cytoplasm is achieved through a huge multiprotein complex, the nuclear pore complex (NPC). These are unique among biological gates, regulating the selective exchange of RNA and proteins across the nuclear envelope through their central channel, rich in special nuclear proteins (nucleoporins or nups).
The final goal of the mRNA NEXT project is to recreate a minimal nuclear export system that is able to drive transport of mRNPs in a biologically relevant mimicking of NPC. Reducing the complexity of the yeast mRNA export system to its basic components in a bottom-up approach will allow us to unravel the nature of the minimal transport complex and describe its mechanism in details. By joining this project, you will contribute to the first-ever successful reconstruction of mRNA export in a completely in vitrosystem!
To achieve this, you will learn the biomimetic nanopore technology (see Figures).
By coating solid-state nanopores with nucleoporins produced in-house, we use a current-base reading of single transport events of macromolecules through the biomimetic NPC. In addition to this, 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.
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. BEP/MEP: high speed atomic force microscope (HS AFM) imaging on condensin-mediated DNA-loop extrusion
Condensin belongs to the Structural Maintenance Complex (SMC) protein family that is involved in a higher order chromosome formation. Recently, our group showed condensin extruded the DNA loop with an ATP hydrolysis manner using a single-molecule fluorescence microscope. The key feature of this SMC protein complex is the ring-shaped structure consisting of 3 major components – two structural maintenance complex (SMC) subunits and one kleisin that together form a ring with two co-factors. Here, we want to show the video imaging of condensin-mediated DNA loop using a high-speed atomic force microscope (HS AFM) imaging technique. HS AFM is an excellent tool for the understanding of protein’s structure and function because we can directly visualize a protein’s structure with high spatial and temporal resolution (< 1 nm, < 100 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 loop extrusion, we will be able to answer how the conformational changes of condensin extrude the DNA loop. We are looking for enthusiastic and diligent master students to study the mechanism of condensin-mediated DNA compaction. The student will learn valuable skills in the operation of HS-liquid AFM as well as other biochemical experiences.
Contact information: Je-Kyung Ryu, workrjk@[REMOVE THIS]gmail.com, 0638703809
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. An artificial cell cortex made of DNA
In Eukaryotic cells, the actin cortex underlying the plasma membrane fulfills several important functions. These include providing mechanical resistance, regulating diffusion of membrane components, and inducing constriction during cytokinesis. In particular, active remodeling and force generation by the cortex is achieved by the concerted action of both the motor activity of Myosin-II and a number of different proteins acting as cross-linkers.
In a bottom-up approach, actin cortex has been reconstituted inside giant vesicles by several groups. One limitation of this approach is the intrinsic complexity of the actin system, in which the interplay between polymerization, depolymerization, different kinds of cross-linkers and myosin motor activity is relatively poorly understood.
We have developed a minimalistic system based on DNA tiles reconstituted inside giant vesicles, which recapitulates the properties of an actin cortex, including regulation of diffusion and vesicle constriction. Such design allows a better control over the properties of the system, and can be further developed to encode new functions, such as spatial localization of constriction.
We are looking for creative and enthusiast Master/Bachelor students of any background. By joining this project, you will be able to develop and test your own ideas; you will learn how to produce and manipulate giant vesicles, how to work with DNA assemblies and how to interface the two systems. Such expertise are essential components of the toolbox in bottom-up synthetic biology.
Please contact Nicola De Franceschi