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Fange, David
Publications (10 of 12) Show all publications
Lawson, M. J., Camsund, D., Larsson, J., Baltekin, Ö., Fange, D. & Elf, J. (2017). In situ genotyping of a pooled strain library after characterizing complex phenotypes. Molecular Systems Biology, 13(10), Article ID 947.
Open this publication in new window or tab >>In situ genotyping of a pooled strain library after characterizing complex phenotypes
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2017 (English)In: Molecular Systems Biology, ISSN 1744-4292, E-ISSN 1744-4292, Vol. 13, no 10, article id 947Article in journal (Refereed) Published
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.

Keywords
DuMPLING, live cell, microfluidic, single cell, strain libraries
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-342924 (URN)10.15252/msb.20177951 (DOI)000416160000004 ()29042431 (PubMedID)
Funder
Knut and Alice Wallenberg FoundationSwedish Research CouncilEU, European Research Council
Available from: 2018-02-26 Created: 2018-02-26 Last updated: 2018-02-26Bibliographically approved
Jones, D., Leroy, P., Unoson, C., Fange, D., Curic, V., Lawson, M. J. & Elf, J. (2017). Kinetics of dCas9 target search in Escherichia coli. Science, 357(6358), 1420-1423
Open this publication in new window or tab >>Kinetics of dCas9 target search in Escherichia coli
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2017 (English)In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 357, no 6358, p. 1420-1423Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
AMER ASSOC ADVANCEMENT SCIENCE, 2017
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-337092 (URN)10.1126/science.aah7084 (DOI)000411880800052 ()28963258 (PubMedID)
Funder
EU, European Research CouncilSwedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2018-01-25 Created: 2018-01-25 Last updated: 2018-01-25Bibliographically approved
Hammar, P., Walldén, M., Fange, D., Persson, F., Baltekin, Ö., Ullman, G., . . . Elf, J. (2014). Direct measurement of transcription factor dissociation excludes a simple operator occupancy model for gene regulation [Letter to the editor]. Nature Genetics, 46(4), 405-+
Open this publication in new window or tab >>Direct measurement of transcription factor dissociation excludes a simple operator occupancy model for gene regulation
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2014 (English)In: Nature Genetics, ISSN 1061-4036, E-ISSN 1546-1718, Vol. 46, no 4, p. 405-+Article in journal, Letter (Refereed) Published
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.

National Category
Cell Biology Bioinformatics and Systems Biology
Identifiers
urn:nbn:se:uu:diva-225087 (URN)10.1038/ng.2905 (DOI)000334510100020 ()
Note

Hammar and Walldén contributed equally to this work.

Available from: 2014-06-13 Created: 2014-05-27 Last updated: 2017-12-05Bibliographically approved
Sanamrad, A., Persson, F., Lundius, E. G., Fange, D., Gynnå, A. H. & Elf, J. (2014). Single-particle tracking reveals that free ribosomal subunits are not excluded from the Escherichia coli nucleoid. Proceedings of the National Academy of Sciences of the United States of America, 111(31), 11413-11418
Open this publication in new window or tab >>Single-particle tracking reveals that free ribosomal subunits are not excluded from the Escherichia coli nucleoid
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2014 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 111, no 31, p. 11413-11418Article in journal (Refereed) Published
Abstract [en]

Biochemical and genetic data show that ribosomes closely follow RNA polymerases that are transcribing protein-coding genes in bacteria. At the same time, electron and fluorescence microscopy have revealed that ribosomes are excluded from the Escherichia coli nucleoid, which seems to be inconsistent with fast translation initiation on nascent mRNA transcripts. The apparent paradox can be reconciled if translation of nascent mRNAs can start throughout the nucleoid before they relocate to the periphery. However, this mechanism requires that free ribosomal subunits are not excluded from the nucleoid. Here, we use single-particle tracking in living E. coli cells to determine the fractions of free ribosomal subunits, classify individual subunits as free or mRNA-bound, and quantify the degree of exclusion of bound and free subunits separately. We show that free subunits are not excluded from the nucleoid. This finding strongly suggests that translation of nascent mRNAs can start throughout the nucleoid, which reconciles the spatial separation of DNA and ribosomes with cotranscriptional translation. We also show that, after translation inhibition, free subunit precursors are partially excluded from the compacted nucleoid. This finding indicates that it is active translation that normally allows ribosomal subunits to assemble on nascent mRNAs throughout the nucleoid and that the effects of translation inhibitors are enhanced by the limited access of ribosomal subunits to nascent mRNAs in the compacted nucleoid.

Keywords
nucleoid exclusion, transcription-translation coupling, antibiotics, single-molecule tracking, single-molecule imaging
National Category
Bioinformatics and Systems Biology
Identifiers
urn:nbn:se:uu:diva-229101 (URN)10.1073/pnas.1411558111 (DOI)000339807200043 ()25056965 (PubMedID)
Available from: 2014-07-30 Created: 2014-07-30 Last updated: 2017-12-05Bibliographically approved
Fange, D., Mellenius, H., Dennis, P. P. & Ehrenberg, M. (2014). Thermodynamic Modeling of Variations in the Rate of RNA Chain Elongation of E-coli rrn Operons. Biophysical Journal, 106(1), 55-64
Open this publication in new window or tab >>Thermodynamic Modeling of Variations in the Rate of RNA Chain Elongation of E-coli rrn Operons
2014 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 106, no 1, p. 55-64Article in journal (Refereed) Published
Abstract [en]

Previous electron-microscopic imaging has shown high RNA polymerase occupation densities in the 16S and 23S encoding regions and low occupation densities in the noncoding leader, spacer, and trailer regions of the rRNA (rrn) operons in E. coli. This indicates slower transcript elongation within the coding regions and faster elongation within the noncoding regions of the operon. Inactivation of four of the seven rrn operons increases the transcript initiation frequency at the promoters of the three intact operons and reduces the time for RNA polymerase to traverse the operon. We have used the DNA sequence-dependent standard free energy variation of the transcription complex to model the experimentally observed changes in the elongation rate along the rrnB operon. We also model the stimulation of the average transcription rate over the whole operon by increasing rate of transcript initiation. Monte Carlo simulations, taking into account initiation of transcription, translocation, and backward and forward tracking of RNA polymerase, partially reproduce the observed transcript elongation rate variations along the rrn operon and fully account for the increased average rate in response to increased frequency of transcript initiation.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-217653 (URN)10.1016/j.bpj.2013.11.4487 (DOI)000329407700010 ()
Available from: 2014-02-12 Created: 2014-02-04 Last updated: 2017-12-06Bibliographically approved
Marklund, E. G., Mahmutovic, A., Berg, O. G., Hammar, P., van der Spoel, D., Fange, D. & Elf, J. (2013). Transcription-factor binding and sliding on DNA studied using micro- and macroscopic models. Proceedings of the National Academy of Sciences of the United States of America, 110(49), 19796-19801
Open this publication in new window or tab >>Transcription-factor binding and sliding on DNA studied using micro- and macroscopic models
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2013 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, no 49, p. 19796-19801Article in journal (Refereed) Published
Abstract [en]

Transcription factors search for specific operator sequences by alternating rounds of 3D diffusion with rounds of 1D diffusion (sliding) along the DNA. The details of such sliding have largely been beyond direct experimental observation. For this purpose we devised an analytical formulation of umbrella sampling along a helical coordinate, and from extensive and fully atomistic simulations we quantified the free-energy landscapes that underlie the sliding dynamics and dissociation kinetics for the LacI dimer. The resulting potential of mean force distributions show a fine structure with an amplitude of 1 k(B)T for sliding and 12 kBT for dissociation. Based on the free-energy calculations the repressor slides in close contact with DNA for 8 bp on average before making a microscopic dissociation. By combining the microscopic molecular-dynamics calculations with Brownian simulation including rotational diffusion from the microscopically dissociated state we estimate a macroscopic residence time of 48 ms at the same DNA segment and an in vitro sliding distance of 240 bp. The sliding distance is in agreement with previous in vitro sliding-length estimates. The in vitro prediction for the macroscopic residence time also compares favorably to what we measure by single-molecule imaging of nonspecifically bound fluorescently labeled LacI in living cells. The investigation adds to our understanding of transcription-factor search kinetics and connects the macro-/mesoscopic rate constants to the microscopic dynamics.

Keywords
facilitated diffusion, lac operon, lac repressors, gene regulation
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-213898 (URN)10.1073/pnas.1307905110 (DOI)000327744900041 ()
External cooperation:
Available from: 2014-01-06 Created: 2014-01-05 Last updated: 2017-12-06Bibliographically approved
Mahmutovic, A., Fange, D., Berg, O. G. & Elf, J. (2012). Lost in presumption: stochastic reactions in spatial models. Nature Methods, 9(12), 1163-1166
Open this publication in new window or tab >>Lost in presumption: stochastic reactions in spatial models
2012 (English)In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 9, no 12, p. 1163-1166Article in journal (Refereed) Published
Abstract [en]

Physical modeling is increasingly important for generating insights into intracellular processes. We describe situations in which combined spatial and stochastic aspects of chemical reactions are needed to capture the relevant dynamics of biochemical systems.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-191793 (URN)10.1038/nmeth.2253 (DOI)000312093500016 ()
Available from: 2013-01-14 Created: 2013-01-14 Last updated: 2017-12-06
Fange, D., Mahmutovic, A. & Elf, J. (2012). MesoRD 1.0: Stochastic reaction-diffusion simulations in the microscopic limit. Bioinformatics, 28(23), 3155-3157
Open this publication in new window or tab >>MesoRD 1.0: Stochastic reaction-diffusion simulations in the microscopic limit
2012 (English)In: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 28, no 23, p. 3155-3157Article in journal (Refereed) Published
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.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-192453 (URN)10.1093/bioinformatics/bts584 (DOI)000311902700025 ()
Available from: 2013-01-23 Created: 2013-01-21 Last updated: 2017-12-06Bibliographically approved
Fange, D. (2010). Modelling Approaches to Molecular Systems Biology. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Modelling Approaches to Molecular Systems Biology
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Systembiologisk modellering på molekylär nivå
Abstract [en]

Implementation and analysis of mathematical models can serve as a powerful tool in understanding how intracellular processes in bacteria affect the bacterial phenotype. In this thesis I have implemented and analysed models of a number of different parts of the bacterium E. coli in order to understand these types of connections. I have also developed new tools for analysis of stochastic reaction-diffusion models.

Resistance mutations in the E. coli ribosomes make the bacteria less susceptible to treatment with the antibiotic drug erythromycin compared to bacteria carrying wildtype ribosomes. The effect is dependent on efficient drug efflux pumps. In the absence of pumps for erythromycin, there is no difference in growth between wildtype and drug target resistant bacteria. I present a model explaining this unexpected phenotype, and also give the conditions for its occurrence.

Stochastic fluctuations in gene expression in bacteria, such as E. coli, result in stochastic fluctuations in biosynthesis pathways. I have characterised the effect of stochastic fluctuations in the parallel biosynthesis pathways of amino acids. I show how the average protein synthesis rate decreases with an increasing number of fluctuating amino acid production pathways. I further show how the cell can remedy this problem by using sensitive feedback control of transcription, and by optimising its expression levels of amino acid biosynthetic enzymes.

The pole-to-pole oscillations of the Min-proteins in E. coli are required for accurate mid-cell division. The phenotype of the Min-oscillations is altered in three different mutants: filamentous cells, round cells and cells with changed membrane lipid composition. I have shown that the wildtype and mutant phenotypes can be explained using a stochastic reaction-diffusion model.

In E. coli, the transcription elongation rate on the ribosmal RNA operon increases with increasing transcription initiation rate. In addition, the polymerase density varies along the ribosomal RNA operons. I present a DNA sequence dependent model that explains the transcription elongation rate speed-up, and also the density variation along the ribosomal operons. Both phenomena are explained by the RNA polymerase backtracking on the DNA.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. p. 57
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 785
Keywords
stochastic reaction-diffuion kinetics, antibiotic drugs, efflux pumps, amino acid biosynthesis, Min-system, rRNA operon, transcription
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-132864 (URN)978-91-554-7941-1 (ISBN)
Public defence
2010-12-16, B21, BMC, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Note
Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 715Available from: 2010-11-24 Created: 2010-10-27 Last updated: 2011-03-21Bibliographically approved
Fange, D. & Elf, J. (2006). Noise-induced Min phenotypes in E. coli. PloS Computational Biology, 2(6), 637-648
Open this publication in new window or tab >>Noise-induced Min phenotypes in E. coli
2006 (English)In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 2, no 6, p. 637-648Article in journal (Refereed) Published
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.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-18951 (URN)10.1371/journal.pcbi.0020080 (DOI)000239494000016 ()16846247 (PubMedID)
Available from: 2006-11-24 Created: 2006-11-24 Last updated: 2017-12-08Bibliographically approved
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