Synthetic Circuits for Robust Orthogonal Production

Deliverables

Theory for robust circuit design with optimal specific fluxes
Transcription and translation map of the rewired E. coli and S. cerevisiae for optimal production

Transcription and translation map of the rewired E coli and S cerevisiae for optimal production

Tunable expression tools for MSCs in E. coli

Tunable expression tools for MSCs in E coli

Markers for membrane integrity
A method to determine the major fraction of membrane lipids and annotation protocol for modifications
Chromosomally located fluorescently-tagged expression strains
Map of the position of translating ribosomes in S. cerevisiae and in E. coli when expressing different production circuits
Whole-cell model of translation

Wholecell model of translation

Data set of single-cell of stress-responsive transcription factors
Coarse-grained mathematical model of glycolysis and proton motive force

Coarsegrained mathematical model of glycolysis and proton motive force

Maps of internal pH, ionic strength, crowding and ATP levels in E. coli/yeast

Maps of internal pH ionic strength crowding and ATP levels in E coliyeast

Spatiotemporal maps of live yeast cells
Project website
Map of charged tRNAome in S. cerevisiae and in E. coli when expressing different production circuits
Public dissemination of project results

Public dissemination of project resultsThe deliverable is planed to accompany the project throughout its duration

Parameterized model of E. coli central metabolism

Parameterized model of E coli central metabolism

Parameterised kinetic model of E. coli glycolysis/gluconeogenesis and pentose phosphate pathway
Data set of single-cell measurements of global physiological variables
Sensor to counteract macromolecular crowding by employing a semi-orthogonal HOG signalling

Sensor to counteract macromolecular crowding by employing a semiorthogonal HOG signalling

Genome-scale models of metabolism and expression in E. coli and S. cerevisiae

Genomescale models of metabolism and expression in E coli and S cerevisiae

Proteome map of the metabolism reorientation upon circuit expression stress
Coarse-grained whole-cell model of gene expression, tRNA levels, and translation

Coarsegrained wholecell model of gene expression tRNA levels and translation

Panel of biosensors directed to specific locations in the cell (organelles in S. cerevisiae; cytosol or membrane of E. coli)
Mid-term special session at an international conference CF1

Mid-term special session at an international conference CF1 Co-responsible partner BSY

Overall Recruitment

Overall Recruitment of ESR All partners reponsible

Researcher Declarations on Conformity

Researcher Declarations on Conformity All partners co-responsible

Transferable skills courses

Transferable skills courses months: 7, 12, 18, 21

Scientific courses

Scientific courses Months: 7, 10, 12, 21, 24

Award of doctoral degrees

Award of doctoral degrees54 month for PhD programmes of 4 years VUA ETHZ RUG

Sensor to monitor metabolic effects on energy homeostasis
Satellite Meeting at an International conference CF2

Satellite Meeting at an International conference CF2Coresponsible partner CHAL

Single-molecule environmental-sensing optical microscope and image analysis tools

Singlemolecule environmentalsensing optical microscope and image analysis tools

Supervisory Board constitution

Constitution of the supervisory board

Systems for monitoring and quantifying crowding stress

Publications

Investigating molecular crowding during cell division and hyperosmotic stress in budding yeast with FRET

Author(s): Sarah Lecinski, Jack W Shepherd, Lewis Frame, Imogen Hayton, Chris MacDonald, Mark C Leake
Published in: Current Topics in Membranes, Issue 88, 2021, Page(s) 75-118, ISSN 1063-5823
Publisher: Current Topics in Membranes
DOI: 10.1101/2021.07.29.454334

Fluorescence-based sensing of the bioenergetic and physicochemical status of the cell.

Author(s): Mantovanelli L, Gaastra BF, Poolman B.
Published in: Current Topics in Membranes, 2021, Page(s) 1-54
Publisher: ScienceDirect
DOI: 10.1016/bs.ctm.2021.10.002

Codon Resolution Analysis of Ribosome Profiling Data

Author(s): Alexander Bartholomäus, Zoya Ignatova
Published in: Ribosome Profiling - Methods and Protocols, Issue 2252, 2021, Page(s) 251-268, ISBN 978-1-0716-1149-4
Publisher: Springer US
DOI: 10.1007/978-1-0716-1150-0_12

Characterizing Genetic Parts and Devices Using RNA Sequencing

Author(s): Deepti Vipin, Zoya Ignatova, Thomas E. Gorochowski
Published in: Synthetic Gene Circuits - Methods and Protocols, Issue 2229, 2021, Page(s) 175-187, ISBN 978-1-0716-1031-2
Publisher: Springer US
DOI: 10.1007/978-1-0716-1032-9_8

Molecular crowding in single eukaryotic cells: Using cell environment biosensing and single-molecule optical microscopy to probe dependence on extracellular ionic strength, local glucose conditions, and sensor copy number

Author(s): Jack W Shepherd, Sarah Lecinski, Jasmine Wragg, Sviatlana Shashkova, Chris MacDonald, Mark C Leake
Published in: Methods, Issue 193, 2021, Page(s) 54-61, ISSN 1046-2023
Publisher: Academic Press
DOI: 10.1016/j.ymeth.2020.10.015

Expanding the Scope of Orthogonal Translation with Pyrrolysyl-tRNA Synthetases Dedicated to Aromatic Amino Acids

Author(s): Hsueh-Wei Tseng, Tobias Baumann, Huan Sun, Yane-Shih Wang, Zoya Ignatova, Nediljko Budisa
Published in: Molecules, Issue 25/19, 2020, Page(s) 4418, ISSN 1420-3049
Publisher: Multidisciplinary Digital Publishing Institute (MDPI)
DOI: 10.3390/molecules25194418

Improving the robustness of engineered bacteria to nutrient stress using programmed proteolysis

Author(s): Szydlo K, Ignatova Z, Godochowski TE
Published in: ACS Synth Biol, Issue 11, 2021, Page(s) 1049-1059, ISSN 2161-5063
Publisher: American Chemical Society
DOI: 10.1021/acssynbio.1c00490

Protein cost allocation explains metabolic strategies in Escherichia coli

Author(s): Grigaitis, P., Olivier, B. G., Fiedler, T., Teusink, B., Kummer, U., Veith, N.
Published in: J Biotechnol, Issue 327, 2021, Page(s) 54-63, ISSN 0168-1656
Publisher: Elsevier BV
DOI: 10.1016/j.jbiotec.2020.11.003

Estimation of peptide elongation times from ribosome profiling spectra

Author(s): Michael Y Pavlov, Gustaf Ullman, Zoya Ignatova, Måns Ehrenberg
Published in: Nucleic Acids Research, Issue 49/9, 2021, Page(s) 5124-5142, ISSN 0305-1048
Publisher: Oxford University Press
DOI: 10.1093/nar/gkab260

Constraints on error rate revealed by computational study of G•U tautomerization in translation

Author(s): Andriy Kazantsev, Zoya Ignatova
Published in: Nucleic Acids Reserach, 2021, Page(s) 11823–11833, ISSN 1362-4962
Publisher: Oxfrod University Press
DOI: 10.1093/nar/gkab947

CRISPR/Cas12a Multiplex Genome Editing of <em>Saccharomyces cerevisiae</em> and the Creation of Yeast Pixel Art

Author(s): Klaudia Ciurkot, Brenda Vonk, Thomas E. Gorochowski, Johannes A. Roubos, René Verwaal
Published in: Journal of Visualized Experiments, Issue 147, 2019, ISSN 1940-087X
Publisher: MYJoVE Corporation
DOI: 10.3791/59350

Absolute quantification of translational regulation and burden using combined sequencing approaches

Author(s): Thomas E Gorochowski, Irina Chelysheva, Mette Eriksen, Priyanka Nair, Steen Pedersen, Zoya Ignatova
Published in: Molecular Systems Biology, Issue 15/5, 2019, ISSN 1744-4292
Publisher: Nature Publishing Group
DOI: 10.15252/msb.20188719

Harnessing the metabolic potential of Streptococcus thermophilus for new biotechnological applications

Author(s): Sofia Markakiou, Paula Gaspar, Eric Johansen, Ahmad A Zeidan, Ana Rute Neves
Published in: Current Opinion in Biotechnology, Issue 61, 2020, Page(s) 142-152, ISSN 0958-1669
Publisher: Elsevier BV
DOI: 10.1016/j.copbio.2019.12.019

Influence of Fluorescent Protein Maturation on FRET Measurements in Living Cells

Author(s): Boqun Liu, Sara N. Mavrova, Jonas van den Berg, Sebastian K. Kristensen, Luca Mantovanelli, Liesbeth M. Veenhoff, Bert Poolman, Arnold J. Boersma
Published in: ACS Sensors, Issue 3/9, 2018, Page(s) 1735-1742, ISSN 2379-3694
Publisher: ACS Publications
DOI: 10.1021/acssensors.8b00473

Discovery and Characterization of a New Cold-Active Protease From an Extremophilic Bacterium via Comparative Genome Analysis and in vitro Expression

Author(s): Amedea Perfumo, Georg Johannes Freiherr von Sass, Eva-Lena Nordmann, Nediljko Budisa, Dirk Wagner
Published in: Frontiers in Microbiology, Issue 11, 2020, ISSN 1664-302X
Publisher: Frontiers Media
DOI: 10.3389/fmicb.2020.00881

Synthesis of New Aza‐ and Thia‐Crown Ether Based Amino Acids

Author(s): Tobias Schneider, Nico Brüssow, Alev Yuvanc, Nediljko Budisa
Published in: ChemistrySelect, Issue 5/9, 2020, Page(s) 2854-2857, ISSN 2365-6549
Publisher: Wiley-VCH
DOI: 10.1002/slct.202000122

Efficient multiplexed gene regulation in Saccharomyces cerevisiae using dCas12a

Author(s): Klaudia Ciurkot, Thomas E Gorochowski, Johannes A Roubos, René Verwaal
Published in: Nucleic Acids Research, Issue 49/13, 2021, Page(s) 7775-7790, ISSN 0305-1048
Publisher: Oxford University Press
DOI: 10.1093/nar/gkab529

Use and limitations of genome-scale metabolic models in food microbiologyAuthor links open overlay panel

Author(s): Somerville, V., Grigaitis, P., Battjes, J., Moro, F., Teusink, B.
Published in: Current Open Food Sci, Issue 43, 2021, Page(s) 225-231, ISSN 2214-7993
Publisher: Elsevier BV
DOI: 10.1016/j.cofs.2021.12.010

he Effect of Lithium on the Budding Yeast Saccharomyces cerevisiae upon Stress Adaptation

Author(s): Patrick Reith, Svenja Braam , Niek Welkenhuysen, Sarah Lecinski, Jack Shepherd, Chris MacDonald, Mark C Leake, Stefan Hohmann, Sviatlana Shashkova, Marija Cvijovic
Published in: Microorganisms, 2022, ISSN 2076-2607
Publisher: MDPI
DOI: 10.3390/microorganisms10030590

Combating Antimicrobial Resistance With New-To-Nature Lanthipeptides Created by Genetic Code Expansion

Author(s): Hamid Reza Karbalaei-Heidari, Nediljko Budisa
Published in: Frontiers in Microbiology, Issue 11, 2020, ISSN 1664-302X
Publisher: Frontiers Media
DOI: 10.3389/fmicb.2020.590522

Membrane thickness, lipid phase and sterol type are determining factors in the permeability of membranes to small solutes.

Author(s): Frallicciardi J, Melcr J, Siginou E, Marrink SJ & Poolman B
Published in: bioRxiv, 2022
Publisher: bioRxiv
DOI: 10.1101/2021.07.16.452599

Evolving a mitigation of the stress response pathway to change the basic chemistry of life

Author(s): Isabella Tolle Stefan Oehm Michael Georg Hoesl Christin Treiber-Kleinke Lauri Peil Abdul-Rahman Adamu Bukari Torsten Semmler Juri Rappsilber Zoya Ignatova Aleeza Gerstein Nediljko Budisa
Published in: bioRxiv, 2021
Publisher: bioRxiv
DOI: 10.1101/2021.09.23.461486

Whole-cell modeling in yeast predicts compartment-specific proteome constraints that drive metabolic strategies

Author(s): Ibrahim E. Elsemman, Angelica Rodriguez Prado, Pranas Grigaitis, Manuel Garcia Albornoz, Victoria Harman, Stephen W. Holman, Johan van Heerden, Frank J. Bruggeman, Mark M.M. Bisschops, Nikolaus Sonnenschein, Simon Hubbard, Rob Beynon, Pascale Daran-Lapujade, Jens Nielsen, Bas Teusink
Published in: bioRxiv, 2021
Publisher: bioRxiv
DOI: 10.1101/2021.06.11.448029

PEPSDI: Scalable and flexible inference framework for stochastic dynamic single-cell models

Author(s): Sebastian Persson, Niek Welkenhuysen, View ORCID ProfileSviatlana Shashkova, Samuel Wiqvist, Patrick Reith, Gregor W. Schmidt, Umberto Picchini, View ORCID ProfileMarija Cvijovic
Published in: bioRxiv, 2021
Publisher: Gold Spring Harbor Laboratiry
DOI: 10.1101/2021.07.01.450748

Deliverables

Publications

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