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  • 1.
    Amselem, Elias
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Marklund, Emil
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Kipper, Kalle
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Johansson, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Deindl, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Real- Time Single Protein Tracking with Polarization Readout using a Confocal Microscope2017In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 112, no 3, p. 295A-295AArticle in journal (Other academic)
  • 2.
    Baltekin, Özden
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Boucharin, Alexis
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Tano, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infection medicine.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Elf, Johan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Antibiotic susceptibility testing in less than 30 min using direct single-cell imaging2017In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 34, p. 9170-9175Article in journal (Refereed)
    Abstract [en]

    The emergence and spread of antibiotic-resistant bacteria are aggravated by incorrect prescription and use of antibiotics. A core problem is that there is no sufficiently fast diagnostic test to guide correct antibiotic prescription at the point of care. Here, we investigate if it is possible to develop a point-of-care susceptibility test for urinary tract infection, a disease that 100 million women suffer from annually and that exhibits widespread antibiotic resistance. We capture bacterial cells directly from samples with low bacterial counts (10(4) cfu/mL) using a custom-designed microfluidic chip and monitor their individual growth rates using microscopy. By averaging the growth rate response to an antibiotic over many individual cells, we can push the detection time to the biological response time of the bacteria. We find that it is possible to detect changes in growth rate in response to each of nine antibiotics that are used to treat urinary tract infections in minutes. In a test of 49 clinical uropathogenic Escherichia coli (UPEC) isolates, all were correctly classified as susceptible or resistant to ciprofloxacin in less than 10 min. The total time for antibiotic susceptibility testing, from loading of sample to diagnostic readout, is less than 30 min, which allows the development of a point-of-care test that can guide correct treatment of urinary tract infection.

  • 3.
    Balzarotti, Francisco
    et al.
    Max Planck Inst Biophys Chem, Dept NanoBiophoton, Gottingen, Germany..
    Eilers, Yvan
    Max Planck Inst Biophys Chem, Dept NanoBiophoton, Gottingen, Germany..
    Gwosch, Klaus C.
    Max Planck Inst Biophys Chem, Dept NanoBiophoton, Gottingen, Germany..
    Gynnå, Arvid H.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Westphal, Volker
    Max Planck Inst Biophys Chem, Dept NanoBiophoton, Gottingen, Germany..
    Stefani, Fernando D.
    Consejo Nacl Invest Cient & Tecn, Ctr Invest Bionanociencias CIBION, Buenos Aires, DF, Argentina.;Univ Buenos Aires, Fac Ciencias Exactas & Nat, Dept Fis, Buenos Aires, DF, Argentina..
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hell, Stefan W.
    Max Planck Inst Biophys Chem, Dept NanoBiophoton, Gottingen, Germany.;Max Planck Inst Med Res, Dept Opt Nanoscopy, Heidelberg, Germany.;German Canc Res Ctr, Opt Nanoscopy Div, Heidelberg, Germany..
    Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes2017In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 355, no 6325, p. 606-612Article in journal (Refereed)
    Abstract [en]

    We introduce MINFLUX, a concept for localizing photon emitters in space. By probing the emitter with a local intensity minimum of excitation light, MINFLUX minimizes the fluorescence photons needed for high localization precision. In our experiments, 22 times fewer fluorescence photons are required as compared to popular centroid localization. In superresolutionmicroscopy, MINFLUXattained similar to 1-nanometer precision, resolving molecules only 6 nanometers apart. MINFLUX tracking of single fluorescent proteins increased the temporal resolution and the number of localizations per trace by a factor of 100, as demonstrated with diffusing 30S ribosomal subunits in living Escherichia coli. As conceptual limits have not been reached, we expect this localization modality to break new ground for observing the dynamics, distribution, and structure of macromolecules in living cells and beyond.

  • 4.
    Berg, Otto G.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Mahmutovic, Anel
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Marklund, Emil
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    The helical structure of DNA facilitates binding2016In: Journal of Physics A: Mathematical and Theoretical, ISSN 1751-8113, E-ISSN 1751-8121, Vol. 9, no 36, article id 364002Article in journal (Other academic)
    Abstract [en]

    The helical structure of DNA imposes constraints on the rate of diffusion-limited protein binding. Here we solve the reaction-diffusion equations for DNA-like geometries and extend with simulations when necessary. We find that the helical structure can make binding to the DNA more than twice as fast compared to a case where DNA would be reactive only along one side. We also find that this rate advantage remains when the contributions from steric constraints and rotational diffusion of the DNA-binding protein are included. Furthermore, we find that the association rate is insensitive to changes in the steric constraints on the DNA in the helix geometry, while it is much more dependent on the steric constraints on the DNA-binding protein. We conclude that the helical structure of DNA facilitates the nonspecific binding of transcription factors and structural DNA-binding proteins in general.

  • 5. Dittmar, Kimberly A
    et al.
    Sörensen, Michael A
    Elf, Johan
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Ehrenberg, Måns
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Pan, Tao
    Selective charging of tRNA isoacceptors induced by amino-acid starvation.2005In: EMBO Rep, ISSN 1469-221X, Vol. 6, no 2, p. 151-7Article in journal (Other scientific)
  • 6.
    Doudna, Jennifer
    et al.
    Univ Calif Berkeley, Berkeley, CA 94720 USA..
    Bar-Ziv, Roy
    Weizmann Inst Sci, IL-76100 Rehovot, Israel..
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Noireaux, Vincent
    Univ Minnesota, Minneapolis, MN 55455 USA..
    Berro, Julien
    Yale Univ, New Haven, CT 06520 USA..
    Saiz, Leonor
    Univ Calif Davis, Davis, CA 95616 USA..
    Vavylonis, Dimitrios
    Lehigh Univ, Bethlehem, PA 18015 USA..
    Faulon, Jean-Loup
    INRA, Jouy En Josas, France..
    Fordyce, Polly
    Stanford Univ, Stanford, CA 94305 USA..
    How Will Kinetics and Thermodynamics Inform Our Future Efforts to Understand and Build Biological Systems?2017In: CELL SYSTEMS, ISSN 2405-4712, Vol. 4, no 2, p. 144-146Article in journal (Refereed)
  • 7.
    Ehrenberg, M.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. MOLECULAR BIOLOGY.
    Elf, J
    Aurell, E
    Sandberg, R
    Tegnér, J
    Systems biology is taking off.2003In: Genome Research, Vol. 13, p. 2475-2484Article in journal (Refereed)
  • 8.
    Elf, J.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. MOLECULAR BIOLOGY.
    An efficient algorithm for mesoscopic reaction-diffusion simulation2003In: Proceedings of the SPIE conference on Fluctuations and Noise in Biological, Biophysical, and Biomedical SystemsArticle in journal (Refereed)
  • 9.
    Elf, J.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. MOLECULAR BIOLOGY.
    Fast evaluation of fluctuations in biochemical networks with the linear noise approximation.2003In: Genome Research, Vol. 13, p. 2475-2484Article in journal (Refereed)
  • 10.
    Elf, J
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. MOLEKYLÄRBIOLOGI.
    Near critical phenomena in metabolite pools2003In: Journal, Vol. 84, p. 154-170Article in journal (Refereed)
  • 11.
    Elf, J
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolutionary Biology.
    Berg, OG
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Ehrenberg, M
    Comparison of repressor and transcriptional attenuator systems for control of amino acid biosynthetic operons2001In: JOURNAL OF MOLECULAR BIOLOGY, ISSN 0022-2836, Vol. 313, no 5, p. 941-954Article in journal (Refereed)
    Abstract [en]

    In bacteria, expression from amino acid biosynthetic operons is transcriptionally controlled by two main mechanisms with principally different modes of action. When the supply of an amino acid is in excess over demand, its concentration will be high and w

  • 12.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Direct Measurements of Transcription Factor Binding and Dissociation at Individual Chromosomal Operators2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 106, no 2, p. 444A-444AArticle in journal (Other academic)
    Abstract [en]

    I will discuss some of our recent progress in studying transcription factor kinetics at the level of individual molecules in E. coli. I will in particular describe an assay for measuring the rate of dissociation for a LacI repressor from an individual chromosomal operator site. When combined with the corresponding association rate measurement, the assay allows us to test the commonly used assumption that TF kinetics can be considered to be at equilibrium and that the gene expression is proportional to the time the operator is free .

  • 13.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hypothesis: Homologous Recombination Depends on Parallel Search2016In: CELL SYSTEMS, ISSN 2405-4712, Vol. 3, no 4, p. 325-327Article in journal (Refereed)
    Abstract [en]

    It is not known how a cell manages to find a specific DNA sequence sufficiently fast to repair a broken chromosome through homologous recombination. I propose a solution based on freely diffusing molecules that are programmed with sequences corresponding to those flanking the break site. In such a process, the target search would be parallelized.

  • 14.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Staying Clear of the Dragons2016In: CELL SYSTEMS, ISSN 2405-4712, Vol. 2, no 4, p. 219-220Article in journal (Refereed)
  • 15.
    Elf, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Barkefors, Irmeli
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Single-Molecule Kinetics in Living Cells2019In: Annual Review of Biochemistry, ISSN 0066-4154, E-ISSN 1545-4509, Vol. 88, p. 635-659Article, review/survey (Refereed)
    Abstract [en]

    In the past decades, advances in microscopy have made it possible to study the dynamics of individual biomolecules in vitro and resolve intramolecular kinetics that would otherwise be hidden in ensemble averages. More recently, single-molecule methods have been used to image, localize, and track individually labeled macromolecules in the cytoplasm of living cells, allowing investigations of intermolecular kinetics under physiologically relevant conditions. In this review, we illuminate the particular advantages of single-molecule techniques when studying kinetics in living cells and discuss solutions to specific challenges associated with these methods.

  • 16.
    Elf, Johan
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Ehrenberg, Måns
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Near-critical behavior of aminoacyl-tRNA pools in E. coli at rate-limiting supply of amino acids.2005In: Biophys J, ISSN 0006-3495, Vol. 88, no 1, p. 132-46Article in journal (Other scientific)
  • 17.
    Elf, Johan
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Ehrenberg, Måns
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Spontaneous separation of bi-stable biochemical systems into spatial domains of opposite phases2004In: Systems Biology, ISSN 1741-2471, Vol. 1, no 2, p. 230-236Article in journal (Refereed)
  • 18.
    Elf, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    What makes ribosome-mediated transcriptional attenuation sensitive to amino2005In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 1, no 1, p. e2-Article in journal (Refereed)
  • 19.
    Elf, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Li, Gene-Wei
    Xie, X Sunney
    Probing transcription factor dynamics at the single-molecule level in a living cell.2007In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 316, no 5828, p. 1191-1194Article in journal (Other academic)
    Abstract [en]

    Transcription factors regulate gene expression through their binding to DNA. In a living Escherichia coli cell, we directly observed specific binding of a lac repressor, labeled with a fluorescent protein, to a chromosomal lac operator. Using single-molecule detection techniques, we measured the kinetics of binding and dissociation of the repressor in response to metabolic signals. Furthermore, we characterized the nonspecific binding to DNA, one-dimensional (1D) diffusion along DNA segments, and 3D translocation among segments through cytoplasm at the single-molecule level. In searching for the operator, a lac repressor spends ~90% of time nonspecifically bound to and diffusing along DNA with a residence time of <5 milliseconds. The methods and findings can be generalized to other nucleic acid binding proteins.

  • 20.
    Elf, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Lötstedt, Per
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Scientific Computing. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Numerical Analysis.
    Sjöberg, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Scientific Computing. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Numerical Analysis.
    Problems of high dimension in molecular biology2003In: Proc. 19th GAMM Seminar Leipzig on High-dimensional problems: Numerical treatment and applications, Leipzig, Germany: Max Planck Institute for Mathematics in the Sciences , 2003, p. 21-30Conference paper (Refereed)
  • 21.
    Elf, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Nilsson, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Tenson, Tanel
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Bistable bacterial growth rate in response to antibiotics with low membrane permeability2006In: Physical Review Letters, ISSN 0031-9007, E-ISSN 1079-7114, Vol. 97, no 25, p. 258104-Article in journal (Refereed)
    Abstract [en]

    We demonstrate that growth rate bistability for bacterial cells growing exponentially at a fixed external antibiotic concentration can emerge when the cell wall permeability for the drug is low and the growth rate sensitivity to the intracellular drug concentration is high. Under such conditions, an initially high growth rate can remain high, due to dilution of the intracellular drug concentration by rapid cell volume increase, while an initially low growth rate can remain low, due to slow cell volume increase and insignificant drug dilution. Our findings have implications for the testing of novel antibiotics on growing bacterial strains.

  • 22.
    Elf, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Paulsson, Johan
    Berg, Otto
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Mesoscopic kinetics and its applications in protein synthesis2005In: Systems Biology: Definitions and Perspectives / [ed] Lila Alberghina and H.V. Westerhoff, Heidelberg: Springer-Verlag GmbH , 2005, p. 95-118Chapter in book (Other academic)
    Abstract [en]

    Molecular biology emerged through unification of genetics and nucleic acid chemistry that took place with the discovery of the double helix (Watson and Crick 1953). Accordingly, molecular biology could be defined as the sum of all techniques used to perform genetic experiments by manipulating DNA. One consequence of the development of these techniques is large-scale sequencing of genomes from an ever increasing number of organisms. However, it became clear from this development that genetic information per se is not enough to grasp the most interesting functional and evolutionary aspects of cells and multi-cellular organisms. In fact, understanding how genotype leads to phenotype depends on concepts and techniques from areas that so far have been largely alien to molecular biological research, like physics, mathematics, and engineering. From the bits and pieces from these and other scientific fields new tools must be generated to make possible an understanding of the dynamic, adapting, and developing living systems that somehow take shape from the instructions given by their genomes. The growing total of these tools and their integration in experimental and theoretical approaches to understand complex biological processes in ways previously out of reach could be a way to define systems biology, in analogy with the above definition of molecular biology.

  • 23.
    Elf, Johan
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Daniel.
    Tenson, Tanel
    Ehrenberg, Måns
    Selective loss of charging of tRNA isoacceptors during amino acid limitation.2003In: Science, Vol. 300, p. 1718-1722Article in journal (Refereed)
  • 24.
    English, Brian P.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Hauryliuk, Vasili
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Sanamrad, Arash
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Tankov, Stoyan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Dekker, Nynke H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Single-molecule investigations of the stringent response machinery in living bacterial cells2011In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, no 31, p. E365-E373Article in journal (Refereed)
    Abstract [en]

    The RelA-mediated stringent response is at the heart of bacterial adaptation to starvation and stress, playing a major role in the bacterial cell cycle and virulence. RelA integrates several environmental cues and synthesizes the alarmone ppGpp, which globally reprograms transcription, translation, and replication. We have developed and implemented novel single-molecule tracking methodology to characterize the intracellular catalytic cycle of RelA. Our single-molecule experiments show that RelA is on the ribosome under nonstarved conditions and that the individual enzyme molecule stays off the ribosome for an extended period of time after activation. This suggests that the catalytically active part of the RelA cycle is performed off, rather than on, the ribosome, and that rebinding to the ribosome is not necessary to trigger each ppGpp synthesis event. Furthermore, we find fast activation of RelA in response to heat stress followed by RelA rapidly being reset to its inactive state, which makes the system sensitive to new environmental cues and hints at an underlying excitable response mechanism.

  • 25.
    Fange, David
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Berg, Otto G
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Sjöberg, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Stochastic reaction-diffusion kinetics in the microscopic limit2010In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 107, no 46, p. 19820-19825Article in journal (Refereed)
    Abstract [en]

    Quantitative analysis of biochemical networks often requires consideration of both spatial and stochastic aspects of chemical processes. Despite significant progress in the field, it is still computationally prohibitive to simulate systems involving many reactants or complex geometries using a microscopic framework that includes the finest length and time scales of diffusion-limited molecular interactions. For this reason, spatially or temporally discretized simulations schemes are commonly used when modeling intracellular reaction networks. The challenge in defining such coarse-grained models is to calculate the correct probabilities of reaction given the microscopic parameters and the uncertainty in the molecular positions introduced by the spatial or temporal discretization. In this paper we have solved this problem for the spatially discretized Reaction-Diffusion Master Equation; this enables a seamless and physically consistent transition from the microscopic to the macroscopic frameworks of reaction-diffusion kinetics. We exemplify the use of the methods by showing that a phosphorylation-dephosphorylation motif, commonly observed in eukaryotic signaling pathways, is predicted to display fluctuations that depend on the geometry of the system.

     

     

  • 26.
    Fange, David
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Noise-induced Min phenotypes in E. coli2006In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 2, no 6, p. 637-648Article in journal (Refereed)
    Abstract [en]

    The spatiotemporal oscillations of the Escherichia coli proteins MinD and MinE direct cell division to the region between the chromosomes. Several quantitative models of the Min system have been suggested before, but no one of them accounts for the behavior of all documented mutant phenotypes. We analyzed the stochastic reaction-diffusion kinetics of the Min proteins for several E. coli mutants and compared the results to the corresponding deterministic mean-field description. We found that wild-type (wt) and filamentous (ftsZ(-)) cells are well characterized by the mean-field model, but that a stochastic model is necessary to account for several of the characteristics of the spherical (rodA(-)) and phospathedylethanolamide-deficient (PE-) phenotypes. For spherical cells, the mean-field model is bistable, and the system can get trapped in a non-oscillatory state. However, when the intrinsic noise is considered, only the experimentally observed oscillatory behavior remains. The stochastic model also reproduces the change in oscillation directions observed in the spherical phenotype and the occasional gliding of the MinD region along the inner membrane. For the PE- mutant, the stochastic model explains the appearance of randomly localized and dense MinD clusters as a nucleation phenomenon, in which the stochastic kinetics at low copy number causes local discharges of the high MinD(ATP) to MinD(ADP) potential. We find that a simple five-reaction model of the Min system can explain all documented Min phenotypes, if stochastic kinetics and three-dimensional diffusion are accounted for. Our results emphasize that local copy number fluctuation may result in phenotypic differences although the total number of molecules of the relevant species is high.

  • 27.
    Fange, David
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Fange, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    MesoRD 1.0: Stochastic reaction-diffusion simulations in the microscopic limit2012In: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811Article in journal (Refereed)
  • 28.
    Fange, David
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mahmutovic, Anel
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    MesoRD 1.0: Stochastic reaction-diffusion simulations in the microscopic limit2012In: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 28, no 23, p. 3155-3157Article in journal (Refereed)
    Abstract [en]

    MesoRD is a tool for simulating stochastic reaction-diffusion systems as modeled by the reaction diffusion master equation. The simulated systems are defined in the Systems Biology Markup Language with additions to define compartment geometries. MesoRD 1.0 supports scale-dependent reaction rate constants and reactions between reactants in neighbouring subvolumes. These new features make it possible to construct physically consistent models of diffusion-controlled reactions also at fine spatial discretization.

  • 29.
    Fender, Aurelie
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hampel, Kornelia
    Zimmermann, Bastian
    Wagner, Gerhart E. H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    RNAs actively cycle on the Sm-like protein Hfq2010In: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 24, no 23, p. 2621-2626Article in journal (Refereed)
    Abstract [en]

    Hfq, a protein required for small RNA (sRNA)-mediated regulation in bacteria, binds RNA with low-nanomolar K-d values and long half-lives of complexes (>100 min). This cannot be reconciled with the 1-2-min response time of regulation in vivo. We show that RNAs displace each other on Hfq on a short time scale by RNA concentration-driven (active) cycling. Already at submicromolar concentrations of competitor RNA, half-lives of RNA-Hfq complexes are approximate to 1 min. We propose that competitor RNA associates transiently with RNA-Hfq complexes, RNAs exchange binding sites, and one of the RNAs eventually dissociates. This solves the "strong binding-high turn-over" paradox and permits efficient use of the Hfq pool.

  • 30.
    Ghosh, Anirban
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Baltekin, Özden
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Wäneskog, Marcus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Elkhalifa, Dina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Larsson, Disa
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Contact-dependent growth inhibition induces high levels of antibiotic-tolerant persister cells in clonal bacterial populations2018In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 37, no 9, article id UNSP e98026Article in journal (Refereed)
    Abstract [en]

    Bacterial populations can use bet-hedging strategies to cope with rapidly changing environments. One example is non-growing cells in clonal bacterial populations that are able to persist antibiotic treatment. Previous studies suggest that persisters arise in bacterial populations either stochastically through variation in levels of global signalling molecules between individual cells, or in response to various stresses. Here, we show that toxins used in contact-dependent growth inhibition (CDI) create persisters upon direct contact with cells lacking sufficient levels of CdiI immunity protein, which would otherwise bind to and neutralize toxin activity. CDI-mediated persisters form through a feedforward cycle where the toxic activity of the CdiA toxin increases cellular (p)ppGpp levels, which results in Lon-mediated degradation of the immunity protein and more free toxin. Thus, CDI systems mediate a population density-dependent bet-hedging strategy, where the fraction of non-growing cells is increased only when there are many cells of the same genotype. This may be one of the mechanisms of how CDI systems increase the fitness of their hosts.

  • 31.
    Grönlund, Andreas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Mathematics, Analysis and Applied Mathematics.
    Lötstedt, Per
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Scientific Computing. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Numerical Analysis.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Costs and constraints from time-delayed feedback in small gene regulatory motifs2010In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 107, p. 8171-8176Article in journal (Refereed)
  • 32.
    Grönlund, Andreas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Mathematics, Analysis and Applied Mathematics.
    Lötstedt, Per
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Scientific Computing. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Numerical Analysis.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Delay-induced anomalous fluctuations in intracellular regulation2011In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 2, p. 419:1-7Article in journal (Refereed)
  • 33.
    Grönlund, Andreas
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lötstedt, Per
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Scientific Computing. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Numerical Analysis.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Transcription factor binding kinetics constrain noise suppression via negative feedback2013In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 4, p. 1864:1-5Article in journal (Refereed)
  • 34.
    Hammar, Petter
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Leroy, Prune
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mahmutovic, Anel
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Marklund, Erik G
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Berg, Otto G
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    The lac repressor displays facilitated diffusion in living cells2012In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 336, no 6088, p. 1595-1598Article in journal (Refereed)
    Abstract [en]

    Transcription factors (TFs) are proteins that regulate the expression of genes by binding sequence-specific sites on the chromosome. It has been proposed that to find these sites fast and accurately, TFs combine one-dimensional (1D) sliding on DNA with 3D diffusion in the cytoplasm. This facilitated diffusion mechanism has been demonstrated in vitro, but it has not been shown experimentally to be exploited in living cells. We have developed a single-molecule assay that allows us to investigate the sliding process in living bacteria. Here we show that the lac repressor slides 45 ± 10 base pairs on chromosomal DNA and that sliding can be obstructed by other DNA-bound proteins near the operator. Furthermore, the repressor frequently (>90%) slides over its natural lacO(1) operator several times before binding. This suggests a trade-off between rapid search on nonspecific sequences and fast binding at the specific sequence.

  • 35.
    Hammar, Petter
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Walldén, Mats
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Fange, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Baltekin, Özden
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Ullman, Gustaf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Leroy, Prune
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Transcription factor dissociation measurements using single molecule chase in living cellsManuscript (preprint) (Other academic)
  • 36.
    Hammar, Petter
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Walldén, Mats
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Fange, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Baltekin, Özden
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Ullman, Gustaf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Leroy, Prune
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Direct measurement of transcription factor dissociation excludes a simple operator occupancy model for gene regulation2014In: Nature Genetics, ISSN 1061-4036, E-ISSN 1546-1718, Vol. 46, no 4, p. 405-+Article in journal (Refereed)
    Abstract [en]

    Transcription factors mediate gene regulation by site-specific binding to chromosomal operators. It is commonly assumed that the level of repression is determined solely by the equilibrium binding of a repressor to its operator. However, this assumption has not been possible to test in living cells. Here we have developed a single-molecule chase assay to measure how long an individual transcription factor molecule remains bound at a specific chromosomal operator site. We find that the lac repressor dimer stays bound on average 5 min at the native lac operator in Escherichia coli and that a stronger operator results in a slower dissociation rate but a similar association rate. Our findings do not support the simple equilibrium model. The discrepancy with this model can, for example, be accounted for by considering that transcription initiation drives the system out of equilibrium. Such effects need to be considered when predicting gene activity from transcription factor binding strengths.

  • 37.
    Hattne, Johan
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Fange, David
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Elf, Johan
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Stochastic reaction-diffusion simulation with MesoRD.2005In: Bioinformatics, ISSN 1367-4803, Vol. 21, no 12, p. 2923-4Article in journal (Refereed)
  • 38.
    Ishaq, Omer
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Elf, Johan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Wählby, Carolina
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Uppsala University, Science for Life Laboratory, SciLifeLab.
    An Evaluation of the Faster STORM Method for Super-resolution Microscopy2014In: Proceedings of the 22nd International Conference on Pattern Recognition, 2014, p. 4435-4440Conference paper (Refereed)
    Abstract [en]

    Development of new stochastic super-resolution methods together with fluorescence microscopy imaging enables visualization of biological processes at increasing spatial and temporal resolution. Quantitative evaluation of such imaging experiments call for computational analysis methods that localize the signals with high precision and recall. Furthermore, it is desirable that the methods are fast and possible to parallelize so that the ever increasing amounts of collected data can be handled in an efficient way. We here in address signal detection in super-resolution microscopy by approaches based on compressed sensing. We describe how a previously published approach can be parallelized, reducing processing time at least four times. We also evaluate the effect of a greedy optimization approach on signal recovery at high noise and molecule density. Furthermore, our evaluation reveals how previously published compressed sensing algorithms have a performance that degrades to that of a random signal detector at high molecule density. Finally, we show the approximation of the imaging system's point spread function affects recall and precision of signal detection, illustrating the importance of parameter optimization. We evaluate the methods on synthetic data with varying signal to noise ratio and increasing molecular density, and visualize performance on realsuper-resolution microscopy data from a time-lapse sequence of livingcells.

  • 39.
    Jones, Daniel
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Elf, Johan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Bursting onto the scene?: Exploring stochastic mRNA production in bacteria2018In: Current Opinion in Microbiology, ISSN 1369-5274, E-ISSN 1879-0364, Vol. 45, p. 124-130Article, review/survey (Refereed)
    Abstract [en]

    Recent large-scale measurements of gene expression variability (or noise) in E. coli have led to the unexpected conclusion that the variability is in large part dictated by and increasing with the mean level of expression. Here we review the evidence for this apparent universal trend in variability, as well as for the related idea that transcription is fundamentally bursty. We examine recently proposed mechanisms for burstiness and universality and argue that they do not explain important features of observed data. Finally, we discuss potential limitations and pitfalls in the interpretation of experimental measurements of cell-to-cell variability.

  • 40.
    Jones, Daniel
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Leroy, Prune
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Unoson, Cecilia
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Fange, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Curic, Vladimir
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lawson, Michael J.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Elf, Johan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala Univ, Dept Cell & Mol Biol, Sci Life Lab, Uppsala, Sweden..
    Kinetics of dCas9 target search in Escherichia coli2017In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 357, no 6358, p. 1420-1423Article in journal (Refereed)
    Abstract [en]

    How fast can a cell locate a specific chromosomal DNA sequence specified by a single-stranded oligonucleotide? To address this question, we investigate the intracellular search processes of the Cas9 protein, which can be programmed by a guide RNA to bind essentially any DNA sequence. This targeting flexibility requires Cas9 to unwind the DNA double helix to test for correct base pairing to the guide RNA. Here we study the search mechanisms of the catalytically inactive Cas9 (dCas9) in living Escherichia coli by combining single-molecule fluorescence microscopy and bulk restriction-protection assays. We find that it takes a single fluorescently labeled dCas9 6 hours to find the correct target sequence, which implies that each potential target is bound for less than 30 milliseconds. Once bound, dCas9 remains associated until replication. To achieve fast targeting, both Cas9 and its guide RNA have to be present at high concentrations.

  • 41.
    Jones, Daniel
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Unoson, Cecilia
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Leroy, Prune
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Curic, Vladimir
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Kinetics of dCas9 Target Search in Escherichia Coli2017In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 112, no 3, p. 314A-314AArticle in journal (Other academic)
  • 42.
    Kecheril Sadanandan, Sajith
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction.
    Baltekin, Özden
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Magnusson, Klas E. G.
    Boucharin, Alexis
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Ranefall, Petter
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction.
    Jaldén, Joakim
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Wählby, Carolina
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction.
    Segmentation and track-analysis in time-lapse imaging of bacteria2016In: IEEE Journal on Selected Topics in Signal Processing, ISSN 1932-4553, E-ISSN 1941-0484, Vol. 10, no 1, p. 174-184Article in journal (Refereed)
    Abstract [en]

    In this paper, we have developed tools to analyze prokaryotic cells growing in monolayers in a microfluidic device. Individual bacterial cells are identified using a novel curvature based approach and tracked over time for several generations. The resulting tracks are thereafter assessed and filtered based on track quality for subsequent analysis of bacterial growth rates. The proposed method performs comparable to the state-of-the-art methods for segmenting phase contrast and fluorescent images, and we show a 10-fold increase in analysis speed.

  • 43.
    Kipper, Kalle
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Eremina, Nadja
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Marklund, Emil
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Tubasum, Sumera
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mao, Guanzhong
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lehmann, Laura Christina
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Elf, Johan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Deindl, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Structure-guided approach to site-specific fluorophore labeling of the lac repressor LacI2018In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 6, article id e0198416Article in journal (Refereed)
    Abstract [en]

    The lactose operon repressor protein LacI has long served as a paradigm of the bacterial transcription factors. However, the mechanisms whereby LacI rapidly locates its cognate binding site on the bacterial chromosome are still elusive. Single-molecule fluorescence imaging approaches are well suited for the study of these mechanisms but rely on a functionally compatible fluorescence labeling of LacI. Particularly attractive for protein fluorescence labeling are synthetic fluorophores due to their small size and favorable photophysical characteristics. Synthetic fluorophores are often conjugated to natively occurring cysteine residues using maleimide chemistry. For a site-specific and functionally compatible labeling with maleimide fluorophores, the target protein often needs to be redesigned to remove unwanted native cysteines and to introduce cysteines at locations better suited for fluorophore attachment. Biochemical screens can then be employed to probe for the functional activity of the redesigned protein both before and after dye labeling. Here, we report a mutagenesis- based redesign of LacI to enable a functionally compatible labeling with maleimide fluorophores. To provide an easily accessible labeling site in LacI, we introduced a single cysteine residue at position 28 in the DNA-binding headpiece of LacI and replaced two native cysteines with alanines where derivatization with bulky substituents is known to compromise the protein's activity. We find that the redesigned LacI retains a robust activity in vitro and in vivo, provided that the third native cysteine at position 281 is retained in LacI. In a total internal reflection microscopy assay, we observed individual Cy3-labeled LacI molecules bound to immobilized DNA harboring the cognate O-1 operator sequence, indicating that the dye-labeled LacI is functionally active. We have thus been able to generate a functional fluorescently labeled LacI that can be used to unravel mechanistic details of LacI target search at the single molecule level.

  • 44.
    Kipper, Kalle
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lundius, Ebba G.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Ćurić, Vladimir
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Nikic, Ivana
    European Mol Biol Lab, Cell Biol & Biophys Unit, Struct & Computat Biol Unit, D-69117 Heidelberg, Germany..
    Wiessler, Manfred
    Deutsch Krebsforschungszentrum, Biol Chem, D-69120 Heidelberg, Germany..
    Lemke, Edward A.
    European Mol Biol Lab, Cell Biol & Biophys Unit, Struct & Computat Biol Unit, D-69117 Heidelberg, Germany..
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Application of Noncanonical Amino Acids for Protein Labeling in a Genomically Recoded Escherichia coli2017In: ACS Photonics, E-ISSN 2330-4022, Vol. 6, no 2, p. 233-255Article in journal (Refereed)
    Abstract [en]

    Small synthetic fluorophores are in many ways superior to fluorescent proteins as labels for imaging. A major challenge is to use them for a protein-specific labeling in living cells. Here, we report on our use of noncanonical amino acids that are genetically encoded via the pyrrolysyl-tRNA/pyrrolysyl-RNA synthetase pair at artificially introduced TAG codons in a recoded E. coli strain. The strain is lacking endogenous TAG codons and the TAG-specific release factor RF1. The amino acids contain bioorthogonal groups that can be clicked to externally supplied dyes, thus enabling protein-specific labeling in live cells. We find that the noncanonical amino acid incorporation into the target protein is robust for diverse amino acids and that the usefulness of the recoded E. coli strain mainly derives from the absence of release factor RF1. However, the membrane permeable dyes display high nonspecific binding in intracellular environment and the electroporation of hydrophilic nonmembrane permeable dyes severely impairs growth of the recoded strain. In contrast, proteins exposed on the outer membrane of E. coli can be labeled with hydrophilic dyes with a high specificity as demonstrated by labeling of the osmoporin OmpC. Here, labeling can be made sufficiently specific to enable single molecule studies as exemplified by OmpC single particle tracking.

  • 45.
    Koskiniemi, Sanna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Arming the Neighborhood2016In: Developmental Cell, ISSN 1534-5807, E-ISSN 1878-1551, Vol. 39, no 1, p. 5-6Article in journal (Other academic)
    Abstract [en]

    Bacteria use type 6 secretion systems in antagonistic behavior to compete for resources with other bacteria. In a recent issue of Cell, Vettiger and Basler (2016) show that bacteria can also use these systems to arm neighboring cells and force them to pass on a signal in the bacterial population.

  • 46. Kuzmenko, Anton
    et al.
    Tankov, Stoyan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    English, Brian P.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Tarassov, Ivan
    Tenson, Tanel
    Kamenski, Piotr
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Hauryliuk, Vasili
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Single molecule tracking fluorescence microscopy in mitochondria reveals highly dynamic but confined movement of Tom402011In: Scientific reports, ISSN 2045-2322, Vol. 1, p. 195-Article in journal (Refereed)
    Abstract [en]

    Tom40 is an integral protein of the mitochondrial outer membrane, which as the central component of the Translocase of the Outer Membrane (TOM) complex forms a channel for protein import. We characterize the diffusion properties of individual Tom40 molecules fused to the photoconvertable fluorescent protein Dendra2 with millisecond temporal resolution. By imaging individual Tom40 molecules in intact isolated yeast mitochondria using photoactivated localization microscopy with sub-diffraction limited spatial precision, we demonstrate that Tom40 movement in the outer mitochondrial membrane is highly dynamic but confined in nature, suggesting anchoring of the TOM complex as a whole.

  • 47.
    Lawson, Michael J.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Camsund, Daniel
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Larsson, Jimmy
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Baltekin, Özden
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Fange, David
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Elf, Johan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    In situ genotyping of a pooled strain library after characterizing complex phenotypes2017In: Molecular Systems Biology, ISSN 1744-4292, E-ISSN 1744-4292, Vol. 13, no 10, article id 947Article in journal (Refereed)
    Abstract [en]

    In this work, we present a proof-of-principle experiment that extends advanced live cell microscopy to the scale of pool-generated strain libraries. We achieve this by identifying the genotypes for individual cells in situ after a detailed characterization of the phenotype. The principle is demonstrated by single-molecule fluorescence time-lapse imaging of Escherichia coli strains harboring barcoded plasmids that express a sgRNA which suppresses different genes in the E.coli genome through dCas9 interference. In general, the method solves the problem of characterizing complex dynamic phenotypes for diverse genetic libraries of cell strains. For example, it allows screens of how changes in regulatory or coding sequences impact the temporal expression, location, or function of a gene product, or how the altered expression of a set of genes impacts the intracellular dynamics of a labeled reporter.

  • 48. Li, Gene-Wei
    et al.
    Berg, Otto G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioinformatics.
    Effects of macromolecular crowding and DNA looping on gene regulation kinetics.2009In: Nature Physics, ISSN 1745-2473, E-ISSN 1745-2481, Vol. 5, no 4, p. 294-297Article in journal (Refereed)
    Abstract [en]

    DNA-binding proteins control how genomes function. The theory of facilitated diffusion(1) explains how DNA-binding proteins can find targets apparently faster than the diffusion limit by using reduced dimensionality(2,3)-combining three-dimensional (3D) diffusion through cytoplasm with 1D sliding along DNA (refs 3-15). However, it does not include a description of macromolecular crowding on DNA as observed in living cells. Here, we show that such a physical constraint to sliding greatly reduces the search speed, in agreement with single-molecule measurements. Interestingly, the generalized theory also reveals significant insights into the design principles of biology. First, it places a hard constraint on the total number of DNA-binding proteins per cell. Remarkably, the number measured for Escherichia coli fits within the optimal range. Secondly, it defines a new role for DNA looping, a ubiquitous topological motif in genomes. DNA looping can speed up the search process by bypassing proteins that block the sliding track close to the target.

  • 49. Li, Gene-Wei
    et al.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Single molecule approaches to transcription factor kinetics in living cells2009In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 583, no 24, p. 3979-3983Article in journal (Refereed)
    Abstract [en]

    Quantitative modeling of intracellular processes often requires information about intracellular rate constants as well as the concentrations of low abundance species in individual cells. Single molecule imaging techniques offer not only new ways for obtaining such information but also the possibilities to test model-based hypotheses that have previously been out of reach for experiments. In this review we highlight some advantages of single molecule techniques and exemplify by their capability to help understanding how transcription factors find their chromosomal binding sites in bacterial cells.

  • 50.
    Liao, Qinghua
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Lüking, Malin
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Krueger, Dennis M.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab. German Ctr Neurodegenerat Dis, Bioinformat Unit, Dept Epigenet & Syst Med Neurodegenerat Dis, Siebold Str 3A, D-37075 Gottingen, Germany.
    Deindl, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kasson, Peter M.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kamerlin, Shina Caroline Lynn
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Long Time-Scale Atomistic Simulations of the Structure and Dynamics of Transcription Factor-DNA Recognition2019In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 123, no 17, p. 3576-3590Article in journal (Refereed)
    Abstract [en]

    Recent years have witnessed an explosion of interest in computational studies of DNA binding proteins, including both coarse grained and atomistic simulations of transcription factor-DNA recognition, to understand how these transcription factors recognize their binding sites on the DNA with such exquisite specificity. The present study performs microsecond time scale all-atom simulations of the dimeric form of the lactose repressor (Lad), both in the absence of any DNA and in the presence of both specific and nonspecific complexes, considering three different DNA sequences. We examine, specifically, the conformational differences between specific and nonspecific protein DNA interactions, as well as the behavior of the helix-turn-helix motif of Lad when interacting with the DNA. Our simulations suggest that stable Lad binding occurs primarily to bent A-form DNA, with a loss of Lad conformational entropy and optimization of correlated conformational equilibria across the protein. In addition, binding to the specific operator sequence involves a slightly larger number of stabilizing DNA protein hydrogen bonds (in comparison to nonspecific complexes), which may account for the experimentally observed specificity for this operator. In doing so, our simulations provide a detailed atomistic description of potential structural drivers for LacI selectivity.

12 1 - 50 of 81
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