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Gynnå, Arvid H.
Publications (4 of 4) Show all publications
Yang, S., Kim, S., Kim, D.-K., An, H. J., Son, J. B., Gynnå, A. H. & Lee, N. K. (2019). Transcription and translation contribute to gene locus relocation to the nucleoid periphery in E. coli. Nature Communications, 10, Article ID 5131.
Open this publication in new window or tab >>Transcription and translation contribute to gene locus relocation to the nucleoid periphery in E. coli
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2019 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 10, article id 5131Article in journal (Refereed) Published
Abstract [en]

Transcription by RNA polymerase (RNAP) is coupled with translation in bacteria. Here, we observe the dynamics of transcription and subcellular localization of a specific gene locus (encoding a non-membrane protein) in living E. coli cells at subdiffraction-limit resolution. The movement of the gene locus to the nucleoid periphery correlates with transcription, driven by either E. coli RNAP or T7 RNAP, and the effect is potentiated by translation.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2019
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-398017 (URN)10.1038/s41467-019-13152-y (DOI)000495858500012 ()31719538 (PubMedID)
Available from: 2019-12-04 Created: 2019-12-04 Last updated: 2019-12-04Bibliographically approved
Liljeruhm, J., Funk, S. K., Tietscher, S., Edlund, A. D., Jamal, S., Yuen, P., . . . Forster, A. C. (2018). Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology. Journal of Biological Engineering, 12, Article ID 8.
Open this publication in new window or tab >>Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology
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2018 (English)In: Journal of Biological Engineering, ISSN 1754-1611, E-ISSN 1754-1611, Vol. 12, article id 8Article in journal (Refereed) Published
Abstract [en]

Background: Coral reefs are colored by eukaryotic chromoproteins (CPs) that are homologous to green fluorescent protein. CPs differ from fluorescent proteins (FPs) by intensely absorbing visible light to give strong colors in ambient light. This endows CPs with certain advantages over FPs, such as instrument-free detection uncomplicated by ultra-violet light damage or background fluorescence, efficient Forster resonance energy transfer (FRET) quenching, and photoacoustic imaging. Thus, CPs have found utility as genetic markers and in teaching, and are attractive for potential cell biosensor applications in the field. Most near-term applications of CPs require expression in a different domain of life: bacteria. However, it is unclear which of the eukaryotic CP genes might be suitable and how best to assay them.

Results: Here, taking advantage of codon optimization programs in 12 cases, we engineered 14 CP sequences (meffRed, eforRed, asPink, spisPink, scOrange, fwYellow, amilGFP, amajLime, cjBlue, mefiBlue, aeBlue, amilCP, tsPurple and gfasPurple) into a palette of Escherichia coil BioBrick plasmids. BioBricks comply with synthetic biology's most widely used, simplified, cloning standard. Differences in color intensities, maturation times and fitness costs of expression were compared under the same conditions, and visible readout of gene expression was quantitated. A surprisingly large variation in cellular fitness costs was found, resulting in loss of color in some overnight liquid cultures of certain high-copy-plasmid-borne CPs, and cautioning the use of multiple CPs as markers in competition assays. We solved these two problems by integrating pairs of these genes into the chromosome and by engineering versions of the same CP with very different colors.

Conclusion: Availability of 14 engineered CP genes compared in E coil, together with chromosomal mutants suitable for competition assays, should simplify and expand CP study and applications. There was no single plasmid-borne CP that combined all of the most desirable features of intense color, fast maturation and low fitness cost, so this study should help direct future engineering efforts.

Place, publisher, year, edition, pages
BIOMED CENTRAL LTD, 2018
Keywords
Chromoprotein, Fluorescent protein, Coral, Escherichia coli, Genetic marker, Reporter gene, Integration, Fitness cost, BioBrick, iGEM
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-356454 (URN)10.1186/s13036-018-0100-0 (DOI)000432246200001 ()29760772 (PubMedID)
Funder
VINNOVASwedish Research Council, 349-2006-267Swedish Research Council, 2011-5787Swedish Research Council, 2016-1Swedish Research Council, 2017-04148Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Available from: 2018-07-30 Created: 2018-07-30 Last updated: 2019-01-25Bibliographically approved
Balzarotti, F., Eilers, Y., Gwosch, K. C., Gynnå, A. H., Westphal, V., Stefani, F. D., . . . Hell, S. W. (2017). Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science, 355(6325), 606-612
Open this publication in new window or tab >>Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes
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2017 (English)In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 355, no 6325, p. 606-612Article in journal (Refereed) Published
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.

National Category
Biophysics
Identifiers
urn:nbn:se:uu:diva-317586 (URN)10.1126/science.aak9913 (DOI)000393636700043 ()28008086 (PubMedID)
Funder
EU, European Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2017-03-22 Created: 2017-03-22 Last updated: 2017-11-29Bibliographically 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
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