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5 results on '"Synthetic Biology and Bioengineering"'

1. Targeted-antibacterial-plasmids (TAPs) combining conjugation and CRISPR/Cas systems achieve strain-specific antibacterial activity

Author

Sarah Bigot, Christian Lesterlin, Sophie Lematre, Cécile Hilpert, Audrey Reuter, Guillaume Launay, Annick Dedieu-Berne, Erwan Gueguen, Microbiologie moléculaire et biochimie structurale / Molecular Microbiology and Structural Biochemistry (MMSB), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Microbiologie, adaptation et pathogénie (MAP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon, Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), and Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
MESH: CRISPR-Cas Systems, AcademicSubjects/SCI00010, Computational biology, MESH: Carbapenem-Resistant Enterobacteriaceae, medicine.disease_cause, Genome, 03 medical and health sciences, MESH: Enterobacteriaceae, MESH: Software, Plasmid, Enterobacteriaceae, Species Specificity, MESH: Plasmids, MESH: RNA, Genetics, medicine, Escherichia coli, CRISPR, MESH: Species Specificity, Gene, 030304 developmental biology, 0303 health sciences, biology, 030306 microbiology, MESH: Escherichia coli, Bacterial conjugation, Pathogenic bacteria, MESH: Conjugation, Genetic, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Carbapenem-Resistant Enterobacteriaceae, Conjugation, Genetic, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, RNA, CRISPR-Cas Systems, Antibacterial activity, Synthetic Biology and Bioengineering, Bacteria, Software, Plasmids
Abstract

The global emergence of drug-resistant bacteria leads to the loss of efficacy of our antibiotics arsenal and severely limits the success of currently available treatments. Here, we developed an innovative strategy based on Targeted-Antibacterial-Plasmids (TAPs) that use bacterial conjugation to deliver CRISPR/Cas systems exerting a strain-specific antibacterial activity. TAPs are highly versatile as they can be directed against any specific genomic or plasmid DNA using the custom algorithm (CSTB) that identifies appropriate targeting spacer sequences. We demonstrate the ability of TAPs to induce strain-selective killing by introducing lethal double strand breaks (DSBs) into the targeted genomes. TAPs directed against a plasmid-born carbapenem resistance gene efficiently resensitise the strain to the drug. This work represents an essential step towards the development of an alternative to antibiotic treatments, which could be used for in situ microbiota modification to eradicate targeted resistant and/or pathogenic bacteria without affecting other non-targeted bacterial species.

Published
2021

2. In vivo stabilization of endogenous chloroplast RNAs by customized artificial pentatricopeptide repeat proteins

Author

Alice Barkan, Kamel Hammani, Nikolay Manavski, Sébastien Mathieu, Louis-Valentin Meteignier, Margarita Rojas, Andreas Brachmann, Institut de biologie moléculaire des plantes (IBMP), Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA), ANR-18-CE20-0013,SynPPR,Exploiter des protéines synthétiques de liaison à l'ARN à motifs PPR pour le contrôle ciblé de l'expression génétique dans les organismes vivants(2018), and Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0106 biological sciences, Untranslated region, Chloroplasts, AcademicSubjects/SCI00010, Ribulose-Bisphosphate Carboxylase, Arabidopsis, Gene Expression, RNA-binding protein, Computational biology, Plasma protein binding, Biology, Protein Engineering, 01 natural sciences, 03 medical and health sciences, Protein-fragment complementation assay, Gene expression, Genetics, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Messenger RNA, RNA, Chloroplast, Chemistry, Arabidopsis Proteins, RNA, RNA-Binding Proteins, biology.organism_classification, Plants, Genetically Modified, Recombinant Proteins, Amino acid, RNA-Binding Motifs, Pentatricopeptide repeat, Synthetic Biology and Bioengineering, 5' Untranslated Regions, 010606 plant biology & botany, Protein Binding
Abstract

Pentatricopeptide repeat (PPR) proteins are helical repeat-proteins that bind RNA in a modular fashion with a sequence-specificity that can be manipulated by the use of an amino acid code. As such, PPR repeats are promising scaffolds for the design of RNA binding proteins for synthetic biology applications. However, the in vivo functional capabilities of artificial PPR proteins built from consensus PPR motifs are just starting to be explored. Here, we report in vivo functions of an artificial PPR protein, dPPRrbcL, made of consensus PPR motifs that were designed to bind a sequence near the 5’ end of rbcL transcripts in Arabidopsis chloroplasts. We used a functional complementation assay to demonstrate that this protein bound its intended RNA target with specificity in vivo and that it substituted for a natural PPR protein by stabilizing processed rbcL mRNA. We targeted a second protein of analogous design to the petL 5’ UTR, where it substituted for the native stabilizing PPR protein PGR3, albeit inefficiently. These results showed that artificial PPRs can be engineered to functionally mimic the class of native PPR proteins that serve as physical barriers against exoribonucleases.

Published
2020

3. C3P3-G1: first generation of a eukaryotic artificial cytoplasmic expression system

Author

Jaïs, Philippe, Decroly, Etienne, Jacquet, Eric, Le Boulch, Marine, Jaïs, Aurélien, Jean-Jean, Olivier, Eaton, Heather, Ponien, Prishila, Verdier, Frédérique, Canard, Bruno, Gonçalves, Sergio, Chiron, Stéphane, Le Gall, Maude, Mayeux, Patrick, Shmulevitz, Maya, LE GALL, Maude, Eukarÿs SAS [Evry, France] (Génopole Campus), Architecture et fonction des macromolécules biologiques (AFMB), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie des Substances Naturelles (ICSN), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Adaptation Biologique et Vieillissement = Biological Adaptation and Ageing (B2A), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Medical Microbiology and Immunology [Alberta, Canada], University of Alberta, Institut Cochin (IC UM3 (UMR 8104 / U1016)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre de recherche sur l'Inflammation (CRI (UMR_S_1149 / ERL_8252 / U1149)), Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Eukar¨ys SAS. Funding for open access charge: Eukar¨ys SAS. Conflict of interest statement. P.J. has equity ownership in Eukar¨ys SAS. M.L.B., S.G. and S.C. employed by Eukar¨ys SAS., Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7), Vieillissement Cellulaire Intégré et Inflammation (VCII), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Paris Seine (IBPS)

Subjects
Cytoplasm, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Polyadenylation, Transcription, Genetic, [SDV]Life Sciences [q-bio], CHO Cells, Biology, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Plasmid, Cricetulus, RNA polymerase, Protein biosynthesis, Genetics, Animals, Humans, Polymerase, 030304 developmental biology, Cell Nucleus, 0303 health sciences, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, Chinese hamster ovary cell, [SDV.MHEP.HEG]Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology, DNA-Directed RNA Polymerases, [SDV.MHEP.HEG] Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology, Cell biology, [SDV.BIO] Life Sciences [q-bio]/Biotechnology, Eukaryotic Cells, chemistry, biology.protein, Synthetic Biology and Bioengineering, Poly A, 030217 neurology & neurosurgery, DNA, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract

International audience; Most eukaryotic expression systems make use of host-cell nuclear transcriptional and post-transcriptional machineries. Here, we present the first generation of the chimeric cytoplasmic capping-prone phage polymerase (C3P3-G1) expression system developed by biological engineering, which generates capped and polyadenylated transcripts in host-cell cytoplasm by means of two components. First, an artificial single-unit chimeric enzyme made by fusing an mRNA capping enzyme and a DNA-dependent RNA polymerase. Second, specific DNA templates designed to operate with the C3P3-G1 enzyme , which encode for the transcripts and their artificial polyadenylation. This system, which can potentially be adapted to any in cellulo or in vivo eu-karyotic expression applications, was optimized for transient expression in mammalian cells. C3P3-G1 shows promising results for protein production in Chinese Hamster Ovary (CHO-K1) cells. This work also provides avenues for enhancing the performances for next generation C3P3 systems.

Published
2019

4. Enhancing multiplex genome editing by natural transformation (MuGENT) via inactivation of ssDNA exonucleases

Author

Christopher M. Waters, Elisa Galli, François Xavier Barre, Ankur B. Dalia, Soo Hun Yoon, Triana N. Dalia, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Evolution et maintenance des chromosomes circulaires (EMC2), Département Biologie des Génomes (DBG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Exonuclease, DNA, Bacterial, Exonucleases, [SDV]Life Sciences [q-bio], Mutant, DNA, Single-Stranded, Computational biology, Genome, DNA Mismatch Repair, 03 medical and health sciences, chemistry.chemical_compound, Genome editing, Bacterial Proteins, Genetics, Nucleoid, Homologous Recombination, Vibrio cholerae, 030304 developmental biology, Whole genome sequencing, Gene Editing, 0303 health sciences, biology, Acinetobacter, 030306 microbiology, Oligonucleotide, Escherichia coli Proteins, 030104 developmental biology, chemistry, RNA splicing, Mutation, biology.protein, Transformation, Bacterial, Phosphorus-Oxygen Lyases, Homologous recombination, Synthetic Biology and Bioengineering, Multiplex Polymerase Chain Reaction, DNA, Genome, Bacterial
Abstract

Recently, we described a method for multiplex genome editing by natural transformation (MuGENT). Mutant constructs for MuGENT require large arms of homology (>2000 bp) surrounding each genome edit, which necessitates laboriousin vitroDNA splicing. InVibriocholerae, we uncover that this requirement is due to cytoplasmic ssDNA exonucleases, which inhibit natural transformation. In ssDNA exonuclease mutants, one arm of homology can be reduced to as little as 40 bp while still promoting integration of genome edits at rates of ~50% without selectionin cis. Consequently, editing constructs are generated in a single PCR reaction where one homology arm is oligonucleotide encoded. To further enhance editing efficiencies, we also developed a strain for transient inactivation of the mismatch repair system. As a proof-of-concept, we used these advances to rapidly mutate 10 high-affinity binding sites for the nucleoid occlusion protein SlmA and generated a duodecuple mutant of 12 diguanylate cyclases inV. cholerae. Whole genome sequencing revealed little to no off-target mutations in these strains. Finally, we show that ssDNA exonucleases inhibit natural transformation inAcinetobacter baylyi. Thus, rational removal of ssDNA exonucleases may be broadly applicable for enhancing the efficacy and ease of MuGENT in diverse naturally transformable species.

Published
2017

5. Impact of donor–recipient phylogenetic distance on bacterial genome transplantation

Author

Dominick Matteau, Sébastien Rodrigue, Anne Lebaudy, Géraldine Gourgues, Carole Lartigue, Fabien Labroussaa, Vincent Baby, Sanjay Vashee, Pascal Sirand-Pugnet, Biologie du fruit et pathologie (BFP), Université Bordeaux Segalen - Bordeaux 2-Institut National de la Recherche Agronomique (INRA)-Université Sciences et Technologies - Bordeaux 1, Département de biologie [Sherbrooke] (UdeS), Faculté des sciences [Sherbrooke] (UdeS), Université de Sherbrooke (UdeS)-Université de Sherbrooke (UdeS), and J. Craig Venter Institute [La Jolla, USA] (JCVI)

Subjects
DNA Replication, DNA, Bacterial, Genetic Markers, 0301 basic medicine, Genotype, 030106 microbiology, Spiroplasma, Saccharomyces cerevisiae, Bacterial genome size, Genome, Mycoplasma capricolum, Genome engineering, 03 medical and health sciences, Transformation, Genetic, transplantation de génome, Genetics, mollicute, Cloning, Molecular, Phylogeny, ComputingMilieux_MISCELLANEOUS, Phylogenetic tree, biology, génome, Microbiology and Parasitology, Mycoplasma mycoides, Reproducibility of Results, biology.organism_classification, Microbiologie et Parasitologie, Transplantation, Mutagenesis, Insertional, Phenotype, 030104 developmental biology, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, biologie synthétique, spiroplasme, bactérie pathogène, Synthetic Biology and Bioengineering, Genome, Bacterial, Plasmids
Abstract

Genome transplantation (GT) allows the installation of purified chromosomes into recipient cells, causing the resulting organisms to adopt the genotype and the phenotype conferred by the donor cells. This key process remains a bottleneck in synthetic biology, especially for genome engineering strategies of intractable and economically important microbial species. So far, this process has only been reported using two closely related bacteria, Mycoplasma mycoides subsp. capri (Mmc) and Mycoplasma capricolum subsp. capricolum (Mcap), and the main factors driving the compatibility between a donor genome and a recipient cell are poorly understood. Here, we investigated the impact of the evolutionary distance between donor and recipient species on the efficiency of GT. Using Mcap as the recipient cell, we successfully transplanted the genome of six bacteria belonging to the Spiroplasma phylogenetic group but including species of two distinct genera. Our results demonstrate that GT efficiency is inversely correlated with the phylogenetic distance between donor and recipient bacteria but also suggest that other species-specific barriers to GT exist. This work constitutes an important step toward understanding the cellular factors governing the GT process in order to better define and eventually extend the existing genome compatibility limit.

Published
2016

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