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Global identification and characterization of essential genome features by random transposon mutagenesis

English title Global identification and characterization of essential genome features by random transposon mutagenesis
Applicant Christen Beat
Number 166476
Funding scheme Project funding (Div. I-III)
Research institution Institut für Molekulare Systembiologie ETH Zürich
Institution of higher education ETH Zurich - ETHZ
Main discipline Experimental Microbiology
Start/End 01.04.2016 - 31.03.2019
Approved amount 332'461.00
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All Disciplines (2)

Discipline
Experimental Microbiology
Genetics

Keywords (6)

essential genome analysis; Sinorhizobium meliloti; transposon mutagenesis; synthetic genomics; experimental systems biology; caulobacter crescentus

Lay Summary (German)

Lead
Der genetische Bauplan einer Zelle ist in ihrer DNA Sequenz abgelegt. Essentielle DNA Abschnitte beinhalten die genetischen Instruktionen für alle wichtigen zellulären Prozesse wie Wachstum, Vermehrung und das Aufrechterhalten des Stoffwechsels. Das Forschungsprojekt verwendet experimentelle Hochdurchsatzverfahren, um neuartige Einblicke in die fundamentalen genetischen Bausteine von bakteriellen Lebewesen zu gewinnen.
Lay summary

Inhalt und Ziel des Forschungsprojekts

Im ersten Projektteil wird die biologische Funktion von essentiellen Genom-Elementen erforscht indem ein Teil der überlebenswichtigen DNA Sequenzen eines freilebenden Bakteriums umgeschrieben und auf ihre zelluläre Funktion hin überprüft wird. In einem zweiten Ansatz entschlüsseln wir die genetischen Bausteine, die für das spezialisierte Zusammenleben eines Bakteriums in den Wurzelzellen einer Nutzpflanze  verantwortlich sind. Zur Durchführung beider Forschungsansätze werden neuartige Hochdurchsatzverfahren der Experimentellen System Biologie angewendet. Unsere Untersuchung wird dazu beitragen, neue essentielle DNA Bausteine, welche für bakterielle Lebewesen von fundamentaler Bedeutung sind, aufzuspüren und deren Funktion zu verstehen.

 

Wissenschaftlicher und gesellschaftlicher Kontext des Forschungsprojekts

In den letzten 10 Jahren konnte anhand der neuen Möglichkeiten der Hochdurchsatz-Sequenzierung die genaue DNA Basenabfolge von tausenden von bakteriellen Genomsequenzen bestimmt werden. Die Entschlüsselung von biologischen Funktionen und deren Zuordnung zu bestimmten DNA Sequenzabschnitten ist zentral für das Verständnis wie eine Zelle auf der molekularen Ebene aufgebaut ist. Der gewählte Forschungsansatz ermöglicht das Aufspüren aller essentiellen DNA Sequenzen einer bakteriellen Zelle und leistet dadurch einen wertvollen Beitrag zur Grundlagenforschung. Aufbauend auf diesen Erkenntnissen können in Zukunft neuartige biologische Systeme aus definierten DNA Sequenzen aufgebaut werden, um biologische Moleküle mit Anwendungen in der Biotechnologie, Medizin und der Landwirtschaft nachhaltig zu produzieren.

Direct link to Lay Summary Last update: 01.04.2016

Responsible applicant and co-applicants

Name Institute

Employees

Publications

Publication
Chemical synthesis rewriting of a bacterial genome to achieve design flexibility and biological functionality
Venetz Jonathan E., Del Medico Luca, Wölfle Alexander, Schächle Philipp, Bucher Yves, Appert Donat, Tschan Flavia, Flores-Tinoco Carlos E., van Kooten Mariëlle, Guennoun Rym, Deutsch Samuel, Christen Matthias, Christen Beat (2019), Chemical synthesis rewriting of a bacterial genome to achieve design flexibility and biological functionality, in Proceedings of the National Academy of Sciences, 116(16), 8070-8079.
Identification of Small‐Molecule Modulators of Diguanylate Cyclase by FRET‐Based High‐Throughput Screening
Christen Matthias, Kamischke Cassandra, Kulasekara Hemantha D., Olivas Kathleen C., Kulasekara Bridget R., Christen Beat, Kline Toni, Miller Samuel I. (2018), Identification of Small‐Molecule Modulators of Diguanylate Cyclase by FRET‐Based High‐Throughput Screening, in ChemBioChem, 20(3), 394-407.
Transposon Sequencing of Brucella abortus Uncovers Essential Genes for Growth In Vitro and Inside Macrophages
Sternon Jean-François, Godessart Pierre, Gonçalves de Freitas Rosa, Van der Henst Mathilde, Poncin Katy, Francis Nayla, Willemart Kevin, Christen Matthias, Christen Beat, Letesson Jean-Jacques, De Bolle Xavier (2018), Transposon Sequencing of Brucella abortus Uncovers Essential Genes for Growth In Vitro and Inside Macrophages, in Infection and Immunity, 86(8), 1-20.
Transposon Sequencing Uncovers an Essential Regulatory Function of Phosphoribulokinase for Methylotrophy
Ochsner Andrea M., Christen Matthias, Hemmerle Lucas, Peyraud Rémi, Christen Beat, Vorholt Julia A. (2017), Transposon Sequencing Uncovers an Essential Regulatory Function of Phosphoribulokinase for Methylotrophy, in Current Biology, 27(17), 2579-2588.e6.
Gene Transfer Agent Promotes Evolvability within the Fittest Subpopulation of a Bacterial Pathogen
Québatte Maxime, Christen Matthias, Harms Alexander, Körner Jonas, Christen Beat, Dehio Christoph (2017), Gene Transfer Agent Promotes Evolvability within the Fittest Subpopulation of a Bacterial Pathogen, in Cell Systems, 4(6), 611-621.e6.
Genome Partitioner: A web tool for multi-level partitioning of large-scale DNA constructs for synthetic biology applications
Christen Matthias, Del Medico Luca, Christen Heinz, Christen Beat (2017), Genome Partitioner: A web tool for multi-level partitioning of large-scale DNA constructs for synthetic biology applications, in PLOS ONE, 12(5), e0177234-e0177234.

Datasets

Synthetic Caulobacter sp. 'ethensis' strain CETH2.0 chromosome, complete genome

Author Venetz, Jonathan; Christen, Matthias; Christen, Beat
Publication date 16.04.2019
Persistent Identifier (PID) CP035535
Repository NCBI, GenBank
Abstract
Understanding how to program biological functions into artificial DNA sequences remains a key challenge in synthetic genomics. Here, we report the chemical synthesis and testing of Caulobacter ethensis-2.0 ( C. eth-2.0 ), a rewritten bacterial genome composed of the most fundamental functions of a bacterial cell. We rebuilt the essential genome of Caulobacter crescentus through the process of chemical synthesis rewriting and studied the genetic information content at the level of its essential genes. Within the 785,701-bp genome, we used sequence rewriting to reduce the number of encoded genetic features from 6,290 to 799. Overall, we introduced 133,313 base substitutions, resulting in the rewriting of 123,562 codons. We tested the biological functionality of the genome design in C. crescentus by transposon mutagenesis. Our analysis revealed that 432 essential genes of C. eth-2.0 , corresponding to 81.5% of the design, are equal in functionality to natural genes. These findings suggest that neither changing mRNA structure nor changing the codon context have significant influence on biological functionality of synthetic genomes. Discovery of 98 genes that lost their function identified essential genes with incorrect annotation, including a limited set of 27 genes where we uncovered noncoding control features embedded within protein-coding sequences. In sum, our results highlight the promise of chemical synthesis rewriting to decode fundamental genome functions and its utility toward the design of improved organisms for industrial purposes and health benefits.

Data_S01

Author Venetz, Jonathan E.; Del Medico, Luca; Wölfle, Alexander; Schächle, Philipp; Bucher, Yves; Appert, Donat; Tschan, Flavia; Flores-Tinoco, Carlos E.; van Kooten, Mariëlle; Guennoun, Rym; Deutsch, Samuel; Christen, Matthias; Christen, Beat
Publication date 16.04.2019
Persistent Identifier (PID) pnas.1818259116.sd01
Repository PNAS
Abstract
Understanding how to program biological functions into artificial DNA sequences remains a key challenge in synthetic genomics. Here, we report the chemical synthesis and testing of Caulobacter ethensis-2.0 ( C. eth-2.0 ), a rewritten bacterial genome composed of the most fundamental functions of a bacterial cell. We rebuilt the essential genome of Caulobacter crescentus through the process of chemical synthesis rewriting and studied the genetic information content at the level of its essential genes. Within the 785,701-bp genome, we used sequence rewriting to reduce the number of encoded genetic features from 6,290 to 799. Overall, we introduced 133,313 base substitutions, resulting in the rewriting of 123,562 codons. We tested the biological functionality of the genome design in C. crescentus by transposon mutagenesis. Our analysis revealed that 432 essential genes of C. eth-2.0 , corresponding to 81.5% of the design, are equal in functionality to natural genes. These findings suggest that neither changing mRNA structure nor changing the codon context have significant influence on biological functionality of synthetic genomes. Discovery of 98 genes that lost their function identified essential genes with incorrect annotation, including a limited set of 27 genes where we uncovered noncoding control features embedded within protein-coding sequences. In sum, our results highlight the promise of chemical synthesis rewriting to decode fundamental genome functions and its utility toward the design of improved organisms for industrial purposes and health benefits.

Data_S02

Author Venetz, Jonathan E.; Del Medico, Luca; Wölfle, Alexander; Schächle, Philipp; Bucher, Yves; Appert, Donat; Tschan, Flavia; Flores-Tinoco, Carlos E.; van Kooten, Mariëlle; Guennoun, Rym; Deutsch, Samuel; Christen, Matthias; Christen, Beat
Publication date 16.04.2019
Persistent Identifier (PID) pnas.1818259116.sd02
Repository PNAS
Abstract
Understanding how to program biological functions into artificial DNA sequences remains a key challenge in synthetic genomics. Here, we report the chemical synthesis and testing of Caulobacter ethensis-2.0 ( C. eth-2.0 ), a rewritten bacterial genome composed of the most fundamental functions of a bacterial cell. We rebuilt the essential genome of Caulobacter crescentus through the process of chemical synthesis rewriting and studied the genetic information content at the level of its essential genes. Within the 785,701-bp genome, we used sequence rewriting to reduce the number of encoded genetic features from 6,290 to 799. Overall, we introduced 133,313 base substitutions, resulting in the rewriting of 123,562 codons. We tested the biological functionality of the genome design in C. crescentus by transposon mutagenesis. Our analysis revealed that 432 essential genes of C. eth-2.0 , corresponding to 81.5% of the design, are equal in functionality to natural genes. These findings suggest that neither changing mRNA structure nor changing the codon context have significant influence on biological functionality of synthetic genomes. Discovery of 98 genes that lost their function identified essential genes with incorrect annotation, including a limited set of 27 genes where we uncovered noncoding control features embedded within protein-coding sequences. In sum, our results highlight the promise of chemical synthesis rewriting to decode fundamental genome functions and its utility toward the design of improved organisms for industrial purposes and health benefits.

Data_S03

Author Venetz, Jonathan E.; Del Medico, Luca; Wölfle, Alexander; Schächle, Philipp; Bucher, Yves; Appert, Donat; Tschan, Flavia; Flores-Tinoco, Carlos E.; van Kooten, Mariëlle; Guennoun, Rym; Deutsch, Samuel; Christen, Matthias; Christen, Beat
Publication date 16.04.2019
Persistent Identifier (PID) pnas.1818259116.sd03
Repository PNAS
Abstract
Understanding how to program biological functions into artificial DNA sequences remains a key challenge in synthetic genomics. Here, we report the chemical synthesis and testing of Caulobacter ethensis-2.0 ( C. eth-2.0 ), a rewritten bacterial genome composed of the most fundamental functions of a bacterial cell. We rebuilt the essential genome of Caulobacter crescentus through the process of chemical synthesis rewriting and studied the genetic information content at the level of its essential genes. Within the 785,701-bp genome, we used sequence rewriting to reduce the number of encoded genetic features from 6,290 to 799. Overall, we introduced 133,313 base substitutions, resulting in the rewriting of 123,562 codons. We tested the biological functionality of the genome design in C. crescentus by transposon mutagenesis. Our analysis revealed that 432 essential genes of C. eth-2.0 , corresponding to 81.5% of the design, are equal in functionality to natural genes. These findings suggest that neither changing mRNA structure nor changing the codon context have significant influence on biological functionality of synthetic genomes. Discovery of 98 genes that lost their function identified essential genes with incorrect annotation, including a limited set of 27 genes where we uncovered noncoding control features embedded within protein-coding sequences. In sum, our results highlight the promise of chemical synthesis rewriting to decode fundamental genome functions and its utility toward the design of improved organisms for industrial purposes and health benefits.

Transposon Sequencing Uncovers an Essential Regulatory Function of Phosphoribulokinase for Methylotrophy

Author Ochsner, Andrea M.; Christen, Matthias; Hemmerle, Lucas; Peyraud, Rémi; Christen, Beat; Vorholt, Julia A.
Publication date 01.09.2017
Persistent Identifier (PID) PXD006834
Repository proteomecentral
Abstract
Proteomics data set

Collaboration

Group / person Country
Types of collaboration
Prof. Julia Vorholt, PhD, Institute of Microbiology, ETHZ Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Rudolf Aebersold, PhD, Institute of Molecular Systems Biology, ETHZ Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof. Wolf-Dietrich Hardt, PhD, Institute of Microbiology, ETHZ Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Sam Deutsch, PhD, Synthetic Biology Unit, Joint Genome Institute, US Department of Energy United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof. Sharon Long, PhD, Stanford University United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Christoph Dehio, PhD, Biozentrum, University of Basel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Xavier De Bolle, University of Namure Belgium (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Christa Pennachio, PhD, Genome Sequencing Unit, Joint Genome Institute, US Department of Energy United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof. Hans-Martin, Fischer, PhD, Institute of Microbiology, ETHZ Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Department Seminar D-BSSE Individual talk Chemical synthesis rewriting of genomes 05.11.2018 Basel, Switzerland Christen Beat;
D-Biol Symposium Poster Systems-biology of nitrogen-fixing microbe-plant symbiosis 11.06.2018 Davos, Switzerland Flores Tinoco Carlos Eduardo;
D-Biol Symposium Talk given at a conference Design and testing of a synthetic genome 11.06.2018 Davos, Switzerland Christen Beat;
MIM Seminar, UZH Individual talk Multiplexed transposon sequencing strategies to illuminate complex traits in bacteria 11.05.2017 Zürich, Switzerland Christen Beat;
Microbiology Seminar, University of Geneva Individual talk Transposon sequencing strategies to illuminate complex traits in bacteria 14.11.2016 Geneva, Switzerland Christen Beat;
SGM Meeting 2017 Poster Insights into the temperature dependent plasticity of bacterial core networks 30.08.2016 Basel, Switzerland Del Medico Luca;
NBO Seminar, ZHAW Wädenswil Individual talk Systems and synthetic biology approaches to accelerate bio-systems engineering 26.07.2016 Waedenswil, Switzerland Christen Beat;
6th UK Algea Meeting Talk given at a conference Bridging systems biology and computational DNA design for bottum up bio-engineering 07.07.2016 Sheffield, Great Britain and Northern Ireland Christen Beat;
BioTech Conference 2017 Talk given at a conference Systems and synthetic biology approaches to accelerate bio- systems engineering 13.06.2016 Prag, Czech Republic Christen Beat;
D-Biol Symposium Poster The essential genome of the N2-fixing plant-symbiont S. meliloti 13.06.2016 Davos, Switzerland Flores Tinoco Carlos Eduardo;
D-Biol Symposium Poster Temperature dependent plasticity of bacterial core networks 13.06.2016 Davos, Switzerland Del Medico Luca;
SGM Meeting 2016 Talk given at a conference The essential genome of the plant endosymbiont Sinorhizobium meliloti cultured in rich and minimal media 13.06.2016 Bern, Switzerland Flores Tinoco Carlos Eduardo;


Self-organised

Title Date Place
Synthetic Biology Session, SGM Meeting 2017 30.08.2017 Basel, Switzerland
Systems Microbiology Session, SSM Meeting 2016 11.06.2016 Bern, Switzerland

Knowledge transfer events

Active participation

Title Type of contribution Date Place Persons involved
SynBio Symposium, LTC Talk 12.12.2018 Regensburg, Germany Christen Beat;
Synthetic Genomics, Seminar DSM, Talk 14.11.2018 Birsfelden, Switzerland Christen Beat;
Bio-systems engineering, Seminar, Lonza Talk 17.02.2017 Visp, Switzerland Christen Beat;
BioTech Conference 2017 Talk 13.06.2016 Prag, Czech Republic Christen Beat;


Communication with the public

Communication Title Media Place Year
Media relations: radio, television Bakterien-Erbgut aus dem Computer SRF German-speaking Switzerland 2019
Media relations: print media, online media Bauanleitung für eine synthetische Lebensform Scinexx das Wissensmagazin International 2019
Media relations: print media, online media Caulobacter ethensis 2.0, il primo batterio progettato al computer Wired.it International 2019
Media relations: print media, online media Creado el primer genoma 100% digital National Geographics Espania International 2019
Media relations: print media, online media Durchbruch an der ETH Zürich: Forscher erzeugen komplett künstliches Bakteriengenom Aargauer Zeitung German-speaking Switzerland 2019
Media relations: print media, online media El primer genoma de un ser vivo diseñado al 100% por un ordenador El Mundo International 2019
Media relations: print media, online media Erbgut am PC erschaffen VDI nachrichten International 2019
Media relations: print media, online media Erstes komplett am Computer erzeugtes Bakterien-Genom Computerworld German-speaking Switzerland 2019
Media relations: radio, television Erstes künstliches Bakterium gebaut deutschlandfunknova International 2019
Media relations: print media, online media First bacterial genome created entirely with a computer ScienceDaily International 2019
Media relations: print media, online media First-ever computer-generated genome could revolutionise biotechnology European Scientist International 2019
Media relations: print media, online media Forscher erzeugen komplett künstliches Bakteriengenom NZZ Western Switzerland German-speaking Switzerland 2019
Media relations: radio, television Komplett künstliches Erbgut eines Bakteriums erzeugt ORF International 2019
Media relations: print media, online media Kuenstliches Bakteriengenom hergestellt Der Standard International 2019
Media relations: print media, online media Leben aus dem Labor RIFF reporter International 2019
New media (web, blogs, podcasts, news feeds etc.) Rewriting genomes to understand life Synbiobeta International 2019
Media relations: print media, online media Scientists present first computer-generated artificial genome ZME science International 2019
Media relations: print media, online media Synthetic biology leap does not have to lead to monstrous outcomes the irish times International 2019
Media relations: print media, online media Synthetic Genome of a New Bacterial Species Chemistry views, Wiley-VCH International 2019
Media relations: print media, online media Un nuovo batterio esiste in natura. Il suo Dna creato dal computer la repubblica International 2019

Use-inspired outputs


Start-ups

Name Year

Associated projects

Number Title Start Funding scheme
184664 Chemical synthesis rewriting of a bacterial genome 01.04.2019 Project funding (Div. I-III)
126243 Transposomics: A System-Level Analysis of Whole Genomes by Global High-Resolution Transposon Mutagenesis. 01.11.2009 Fellowships for advanced researchers

Abstract

Systems biology aims to define, understand and predict the properties, functions and behaviors of living systems and their subsystems. From the systems biology perspective, a living cell represents a complex, adaptive, self-organized network formed by a multitude of interdependent molecular component. The ensemble of all essential DNA stretches within a chromosome defines the essential genome of an organism. It encodes all relevant components of the cellular core network and contains the complete set of genetic instructions needed to sustain life. In recent years, comparative genomics approaches as well as large-scale genome perturbation methods have been used to gain insights into universally conserved sequences and essential genes. These studies revealed a large variability in essential genome composition between organisms and in response to changes in environmental conditions. Building on these novel and surprising findings, we aim in this proposal to study the genome compositions of essential DNA features in the two closely related alpha-proteobacterial species Caulobacter and Sinorhizobium. Using transposon mutagenesis coupled to high-throughput sequencing, we aim in project A to illuminate the repertoire of essential DNA sequences to sustain free-living growth of Caulobacter. In project B we aim to assess the essential genome components that mediate microbe-plant interactions of Sinorhizobium with its plant host. Accordingly in project A, we will i) rebuild essential genome features from Caulobacter by de novo DNA synthesis and ii) probe their function on the level of DNA, RNA and protein expression by three orthogonal systems biology approaches (TnSeq, RNA-Seq and SWATH-MS). In project B we aim at i) comprehensive identification of essential genome features of Sinorhizobium that are required for endosymbiosis with host plants. Further, we will ii) phenotypically characterize symbiosis genes using single and competitive plant infection assays and iii) elucidate their role by global approaches and in planta reporter assays. For both subprojects, we propose hypothesis driven follow up experiments to address the function of selected essential genome features using global orthogonal approaches and experimental phenotypic mutant characterization.
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