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Janissen, R. (author), Barth, R. (author), Polinder, M.L. (author), van der Torre, J. (author), Dekker, C. (author)
Transcription-coupled supercoiling of DNA is a key factor in chromosome compaction and the regulation of genetic processes in all domains of life. It has become common knowledge that, during transcription, the DNA-dependent RNA polymerase (RNAP) induces positive supercoiling ahead of it (downstream) and negative supercoils in its wake ...
journal article 2024
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Palepšienė, Rūta (author), Muralidharan, A. (author), Maciulevičius, Martynas (author), Ruzgys, Paulius (author), Chopra, S. (author), Boukany, P. (author), Šatkauskas, Saulius (author)
RNA interference (RNAi) is a powerful and rapidly developing technology that enables precise silencing of genes of interest. However, the clinical development of RNAi is hampered by the limited cellular uptake and stability of the transferred molecules. Electroporation (EP) is an efficient and versatile technique for the transfer of both RNA...
journal article 2024
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Amini Hounejani, R. (author)
The past decades have seen the rapid development of many aspects of synthetic biology. For example, attempts to build synthetic cells under controlled conditions in the laboratory have led to significant achievements. Following a bottom-up approach, scientists aim at building a self-reproducing synthetic cell with a minimum number of biological...
doctoral thesis 2023
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Dogterom, A.M. (author)
journal article 2023
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Tišma, M. (author), Janissen, R. (author), Antar, Hammam (author), Martin Gonzalez, A. (author), Barth, R. (author), Beekman, T.G.T. (author), van der Torre, J. (author), Michieletto, Davide (author), Gruber, Stephan (author), Dekker, C. (author)
In most bacteria, chromosome segregation is driven by the ParABS system where the CTPase protein ParB loads at the parS site to trigger the formation of a large partition complex. Here, we present in vitro studies of the partition complex for Bacillus subtilis ParB, using single-molecule fluorescence microscopy and AFM imaging to show that...
journal article 2023
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Holub, M. (author), Agena, Ethan (author)
A biofoundry is a highly automated facility for processing of biological samples. In that capacity it has a major role in accelerating innovation and product development in engineering biology by implementing design, build, test and learn (DBTL) cycles. Biofoundries bring public and private stakeholders together to share resources, develop...
journal article 2023
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Godino, E. (author), Danelon, C.J.A. (author)
Mimicking bacterial cell division in well-defined cell-free systems has the potential to elucidate the minimal set of proteins required for cytoskeletal formation, membrane constriction, and final abscission. Membrane-anchored FtsZ polymers are often regarded as a sufficient system to realize this chain of events. By using purified FtsZ and...
journal article 2023
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Japaridze, A. (author), van Wee, R.G. (author), Gogou, C. (author), Kerssemakers, J.W.J. (author), van den Berg, D.F. (author), Dekker, C. (author)
The bacterial chromosome is spatially organized through protein-mediated compaction, supercoiling, and cell-boundary confinement. Structural Maintenance of Chromosomes (SMC) complexes are a major class of chromosome-organizing proteins present throughout all domains of life. Here, we study the role of the Escherichia coli SMC complex MukBEF...
journal article 2023
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Lewis, R.W. (author), Muralidharan, A. (author), Klemm, B. (author), Boukany, P. (author), Eelkema, R. (author)
Polycationic carriers promise low cost and scalable gene therapy treatments, however inefficient intracellular unpacking of the genetic cargo has limited transfection efficiency. Charge-reversing polycations, which transition from cationic to neutral or negative charge, can offer targeted intracellular DNA release. We describe a new class of...
journal article 2023
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Davidson, Iain F. (author), Barth, R. (author), Zaczek, Maciej (author), van der Torre, J. (author), Tang, Wen (author), Nagasaka, Kota (author), Janissen, R. (author), Kerssemakers, J.W.J. (author), Wutz, Gordana (author), Dekker, C. (author), Peters, Jan Michael (author)
In eukaryotes, genomic DNA is extruded into loops by cohesin<sup>1</sup>. By restraining this process, the DNA-binding protein CCCTC-binding factor (CTCF) generates topologically associating domains (TADs)<sup>2,3</sup> that have important roles in gene regulation and recombination during development and disease<sup>1,4–7</sup>. How CTCF...
journal article 2023
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van den Berg, Cyntha M. (author), Volkov, V. (author), Schnorrenberg, Sebastian (author), Huang, Ziqiang (author), Stecker, Kelly E. (author), Grigoriev, Ilya (author), Gilani, Sania (author), Frikstad, Kari Anne M. (author), Dogterom, A.M. (author)
Microtubules are dynamic cytoskeletal polymers, and their organization and stability are tightly regulated by numerous cellular factors. While regulatory proteins controlling the formation of interphase microtubule arrays and mitotic spindles have been extensively studied, the biochemical mechanisms responsible for generating stable...
journal article 2023
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Klein, M. (author), Bera, Subhas C. (author), Anderson, Thomas K. (author), Wang, Bing (author), Papini, Flavia S. (author), Arnold, Jamie J. (author), Cameron, Craig E. (author), Depken, S.M. (author), Dulin, D. (author)
journal article 2023
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Yang, Sora (author), Klughammer, N. (author), Barth, A. (author), Tanenbaum, M. (author), Dekker, C. (author)
Single-molecule fluorescence imaging experiments generally require sub-nanomolar protein concentrations to isolate single protein molecules, which makes such experiments challenging in live cells due to high intracellular protein concentrations. Here, we show that single-molecule observations can be achieved in live cells through a drastic...
journal article 2023
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Ramirez Montero, D.F. (author), Sanchez González, H. (author), van Veen, E.N.W. (author), van Laar, T. (author), Solano Hermosilla, B.P. (author), Diffley, John F.X. (author), Dekker, N.H. (author)
The eukaryotic replicative helicase CMG centrally orchestrates the replisome and leads the way at the front of replication forks. Understanding the motion of CMG on the DNA is therefore key to our understanding of DNA replication. In vivo, CMG is assembled and activated through a cell-cycle-regulated mechanism involving 36 polypeptides that...
journal article 2023
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Abil, Z. (author), Restrepo Sierra, A.M. (author), Danelon, C.J.A. (author)
In cell-free gene expression, low input DNA concentration severely limits the phenotypic output, which may impair in vitro protein evolution efforts. We address this challenge by developing CADGE, a strategy that is based on clonal isothermal amplification of a linear gene-encoding dsDNA template by the minimal Φ29 replication machinery and...
journal article 2023
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Nova, I.C. (author), Ritmejeris, J. (author), Brinkerhoff, H.D. (author), Koenig, T.J.R. (author), Gundlach, Jens H. (author), Dekker, C. (author)
Current methods to detect post-translational modifications of proteins, such as phosphate groups, cannot measure single molecules or differentiate between closely spaced phosphorylation sites. We detect post-translational modifications at the single-molecule level on immunopeptide sequences with cancer-associated phosphate variants by...
journal article 2023
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Klughammer, N. (author), Barth, A. (author), Dekker, Maurice (author), Fragasso, A. (author), Onck, Patrick R. (author)
The nuclear pore complex (NPC) regulates the selective transport of large biomolecules through the nuclear envelope. As a model system for nuclear transport, we construct NPC mimics by functionalizing the pore walls of freestanding palladium zero-mode waveguides with the FG-nucleoporin Nsp1. This approach enables the measurement of single...
journal article 2023
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Bruurs, Lucas J.M. (author), Müller, Micha (author), Schipper, Jelle G. (author), Rabouw, Huib H. (author), Boersma, Sanne (author), van Kuppeveld, Frank J.M. (author), Tanenbaum, M. (author)
Antiviral signalling, which can be activated in host cells upon virus infection, restricts virus replication and communicates infection status to neighbouring cells. The antiviral response is heterogeneous, both quantitatively (efficiency of response activation) and qualitatively (transcribed antiviral gene set). To investigate the basis of...
journal article 2023
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Godino, E. (author), Restrepo Sierra, A.M. (author), Danelon, C.J.A. (author)
The reconstitution of basic cellular functions in micrometer-sized liposomes has led to a surge of interest in the construction of synthetic cells. Microscopy and flow cytometry are powerful tools for characterizing biological processes in liposomes with fluorescence readouts. However, applying each method separately leads to a compromise...
journal article 2023
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Huber, S. (author), Terwiel, D. (author), Evers, W.H. (author), Maresca, D. (author), Jakobi, A. (author)
Gas vesicles are gas-filled nanocompartments that allow a diverse group of bacteria and archaea to control their buoyancy. The molecular basis of their properties and assembly remains unclear. Here, we report the 3.2 Å cryo-EM structure of the gas vesicle shell made from the structural protein GvpA that self-assembles into hollow helical...
journal article 2023
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