scholarly journals A standardized genome architecture for bacterial synthetic biology (SEGA)

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Carolyn N. Bayer ◽  
Maja Rennig ◽  
Anja K. Ehrmann ◽  
Morten H. H. Nørholm
Keyword(s):  
Synthetic Biology ◽  
Genome Engineering ◽  
Bacterial Genome ◽  
Genome Architecture ◽  
Bacterial Cells ◽  
Level Control ◽  
Recombinant Gene Expression ◽  
Application Range ◽  
Recombinant Plasmids ◽  
Strain Collection

AbstractChromosomal recombinant gene expression offers a number of advantages over plasmid-based synthetic biology. However, the methods applied for bacterial genome engineering are still challenging and far from being standardized. Here, in an attempt to realize the simplest recombinant genome technology imaginable and facilitate the transition from recombinant plasmids to genomes, we create a simplistic methodology and a comprehensive strain collection called the Standardized Genome Architecture (SEGA). In its simplest form, SEGA enables genome engineering by combining only two reagents: a DNA fragment that can be ordered from a commercial vendor and a stock solution of bacterial cells followed by incubation on agar plates. Recombinant genomes are identified by visual inspection using green-white colony screening akin to classical blue-white screening for recombinant plasmids. The modular nature of SEGA allows precise multi-level control of transcriptional, translational, and post-translational regulation. The SEGA architecture simultaneously supports increased standardization of genetic designs and a broad application range by utilizing well-characterized parts optimized for robust performance in the context of the bacterial genome. Ultimately, its adaption and expansion by the scientific community should improve predictability and comparability of experimental outcomes across different laboratories.

scholarly journals Bacterial genome engineering and synthetic biology: combating pathogens

2016 ◽  
Vol 16 (1) ◽  
Author(s):  
Malathy Krishnamurthy ◽  
Richard T. Moore ◽  
Sathish Rajamani ◽  
Rekha G. Panchal
Keyword(s):  
Synthetic Biology ◽  
Genome Engineering ◽  
Bacterial Genome

scholarly journals The Hidden Charm of Life

Life ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 5 ◽  
Author(s):  
Manuel Porcar
Keyword(s):  
Synthetic Biology ◽  
Genetic Engineering ◽  
Complex Systems ◽  
Bacterial Cells ◽  
Living Organisms

Synthetic biology is an engineering view on biotechnology, which has revolutionized genetic engineering. The field has seen a constant development of metaphors that tend to highlight the similarities of cells with machines. I argue here that living organisms, particularly bacterial cells, are not machine-like, engineerable entities, but, instead, factory-like complex systems shaped by evolution. A change of the comparative paradigm in synthetic biology from machines to factories, from hardware to software, and from informatics to economy is discussed.

Enabling plant synthetic biology through genome engineering

2015 ◽  
Vol 33 (2) ◽  
pp. 120-131 ◽  
Author(s):  
Nicholas J. Baltes ◽  
Daniel F. Voytas
Keyword(s):  
Synthetic Biology ◽  
Genome Engineering ◽  
Plant Synthetic Biology

Rhizobial plasmids — replication, structure and biological role

2012 ◽  
Vol 7 (4) ◽  
pp. 571-586 ◽  
Author(s):  
Andrzej Mazur ◽  
Piotr Koper
Keyword(s):  
Genome Organization ◽  
Soil Bacteria ◽  
Evolutionary History ◽  
Bacterial Genome ◽  
Biological Role ◽  
Genome Architecture ◽  
Genomic Knowledge ◽  
History Of ◽  
Cryptic Plasmids ◽  
Complex Evolutionary History

AbstractSoil bacteria, collectively named rhizobia, can establish mutualistic relationships with legume plants. Rhizobia often have multipartite genome architecture with a chromosome and several extrachromosomal replicons making these bacteria a perfect candidate for plasmid biology studies. Rhizobial plasmids are maintained in the cells using a tightly controlled and uniquely organized replication system. Completion of several rhizobial genome-sequencing projects has changed the view that their genomes are simply composed of the chromosome and cryptic plasmids. The genetic content of plasmids and the presence of some important (or even essential) genes contribute to the capability of environmental adaptation and competitiveness with other bacteria. On the other hand, their mosaic structure results in the plasticity of the genome and demonstrates a complex evolutionary history of plasmids. In this review, a genomic perspective was employed for discussion of several aspects regarding rhizobial plasmids comprising structure, replication, genetic content, and biological role. A special emphasis was placed on current post-genomic knowledge concerning plasmids, which has enriched the view of the entire bacterial genome organization by the discovery of plasmids with a potential chromosome-like role.

In the fast lane: Large-scale bacterial genome engineering

2012 ◽  
Vol 160 (1-2) ◽  
pp. 72-79 ◽  
Author(s):  
Tamás Fehér ◽  
Valerie Burland ◽  
György Pósfai
Keyword(s):  
Large Scale ◽  
Genome Engineering ◽  
Bacterial Genome ◽  
Fast Lane

scholarly journals Development of oriC-Based Plasmids for Mesoplasma florum

2017 ◽  
Vol 83 (7) ◽  
Author(s):  
Dominick Matteau ◽  
Marie-Eve Pepin ◽  
Vincent Baby ◽  
Samuel Gauthier ◽  
Mélissa Arango Giraldo ◽  
...  
Keyword(s):  
Systems Biology ◽  
Synthetic Biology ◽  
Genetic Engineering ◽  
Genome Engineering ◽  
Antibiotic Resistance Genes ◽  
Viable Cell ◽  
Pathogenic Potential ◽  
Strong Basis ◽  
Content Type ◽  
Basic Biology

ABSTRACT The near-minimal bacterium Mesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. However, the lack of genetic engineering tools for this microorganism has limited our capacity to understand its basic biology and modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first generation of artificial plasmids able to replicate in this bacterium. Selected regions of the predicted M. florum chromosomal origin of replication (oriC) were used to create different plasmid versions that were tested for their transformation frequency and stability. Using polyethylene glycol-mediated transformation, we observed that plasmids harboring both rpmH-dnaA and dnaA-dnaN intergenic regions, interspaced or not with a copy of the dnaA gene, resulted in a frequency of ∼4.1 × 10−6 transformants per viable cell and were stably maintained throughout multiple generations. In contrast, plasmids containing only one M. florum oriC intergenic region or the heterologous oriC region of Mycoplasma capricolum, Mycoplasma mycoides, or Spiroplasma citri failed to produce any detectable transformants. We also developed alternative transformation procedures based on electroporation and conjugation from Escherichia coli, reaching frequencies up to 7.87 × 10−6 and 8.44 × 10−7 transformants per viable cell, respectively. Finally, we demonstrated the functionality of antibiotic resistance genes active against tetracycline, puromycin, and spectinomycin/streptomycin in M. florum. Taken together, these valuable genetic tools will facilitate efforts toward building an M. florum-based near-minimal cellular chassis for synthetic biology. IMPORTANCE Mesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. M. florum is closely related to the mycoides cluster of mycoplasmas, which has become a model for whole-genome cloning, genome transplantation, and genome minimization. However, M. florum shows higher growth rates than other Mollicutes, has no known pathogenic potential, and possesses a significantly smaller genome that positions this species among some of the simplest free-living organisms. So far, the lack of genetic engineering tools has limited our capacity to understand the basic biology of M. florum in order to modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first artificial plasmids and transformation methods for this bacterium. This represents a strong basis for ongoing genome engineering efforts using this near-minimal microorganism.

scholarly journals Characterizing a thermostable Cas9 for bacterial genome editing and silencing

2017 ◽  
Author(s):  
Ioannis Mougiakos ◽  
Prarthana Mohanraju ◽  
Elleke F. Bosma ◽  
Valentijn Vrouwe ◽  
Max Finger Bou ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Gene Deletion ◽  
Elevated Temperatures ◽  
Bacterial Genome ◽  
Thermophilic Bacterium ◽  
Geobacillus Thermodenitrificans ◽  
Temperature Range ◽  
Fundamental Research

AbstractCRISPR-Cas9 based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here, we identify and characterize ThermoCas9: an RNA-guided DNA-endonuclease from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that ThermoCas9 is active in vitro between 20°C and 70°C, a temperature range much broader than that of the currently used Cas9 orthologues. Additionally, we demonstrate that ThermoCas9 activity at elevated temperatures is strongly associated with the structure of the employed sgRNA. Subsequently, we develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55°C in Bacillus smithii and for gene deletion at 37°C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish the first Cas9-based bacterial genome editing and silencing tool with a broad temperature range.

scholarly journals Coexistence of Wolbachia with Buchnera aphidicola and a Secondary Symbiont in the Aphid Cinara cedri

2004 ◽  
Vol 186 (19) ◽  
pp. 6626-6633 ◽  
Author(s):  
Laura Gómez-Valero ◽  
Mario Soriano-Navarro ◽  
Vicente Pérez-Brocal ◽  
Abdelaziz Heddi ◽  
Andrés Moya ◽  
...  
Keyword(s):  
Bacterial Genome ◽  
Pcr Amplification ◽  
Bacterial Cells ◽  
Buchnera Aphidicola ◽  
Rdna Genes ◽  
16S Ribosomal Dna ◽  
Secondary Symbiont ◽  
Rdna Sequences ◽  
16S Rdna Sequences

ABSTRACT Intracellular symbiosis is very common in the insect world. For the aphid Cinara cedri, we have identified by electron microscopy three symbiotic bacteria that can be characterized by their different sizes, morphologies, and electrodensities. PCR amplification and sequencing of the 16S ribosomal DNA (rDNA) genes showed that, in addition to harboring Buchnera aphidicola, the primary endosymbiont of aphids, C. cedri harbors a secondary symbiont (S symbiont) that was previously found to be associated with aphids (PASS, or R type) and an α-proteobacterium that belongs to the Wolbachia genus. Using in situ hybridization with specific bacterial probes designed for symbiont 16S rDNA sequences, we have shown that Wolbachia was represented by only a few minute bacteria surrounding the S symbionts. Moreover, the observed B. aphidicola and the S symbionts had similar sizes and were housed in separate specific bacterial cells, the bacteriocytes. Interestingly, in contrast to the case for all aphids examined thus far, the S symbionts were shown to occupy a similarly sized or even larger bacteriocyte space than B. aphidicola. These findings, along with the facts that C. cedri harbors the B. aphidicola strain with the smallest bacterial genome and that the S symbionts infect all Cinara spp. analyzed so far, suggest the possibility of bacterial replacement in these species.

scholarly journals Attenuation of a Pathogenic Mycoplasma Strain by Modification of the obg Gene by Using Synthetic Biology Approaches

mSphere ◽  
2019 ◽  
Vol 4 (3) ◽  
Author(s):  
Carole Lartigue ◽  
Yanina Valverde Timana ◽  
Fabien Labroussaa ◽  
Elise Schieck ◽  
Anne Liljander ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Genome Engineering ◽  
Recipient Cell ◽  
Single Point ◽  
Chemical Mutagenesis ◽  
Economic Losses ◽  
Temperature Sensitive ◽  
Content Type ◽  
Mycoplasma Mycoides ◽  
Virulent Phenotype

ABSTRACT Mycoplasma species are responsible for several economically significant livestock diseases for which there is a need for new and improved vaccines. Most of the existing mycoplasma vaccines are attenuated strains that have been empirically obtained by serial passages or by chemical mutagenesis. The recent development of synthetic biology approaches has opened the way for the engineering of live mycoplasma vaccines. Using these tools, the essential GTPase-encoding gene obg was modified directly on the Mycoplasma mycoides subsp. capri genome cloned in yeast, reproducing mutations suspected to induce a temperature-sensitive (TS+) phenotype. After transplantation of modified genomes into a recipient cell, the phenotype of the resulting M. mycoides subsp. capri mutants was characterized. Single-point obg mutations did not result in a strong TS+ phenotype in M. mycoides subsp. capri, but a clone presenting three obg mutations was shown to grow with difficulty at temperatures of ≥40°C. This particular mutant was then tested in a caprine septicemia model of M. mycoides subsp. capri infection. Five out of eight goats infected with the parental strain had to be euthanized, in contrast to one out of eight goats infected with the obg mutant, demonstrating an attenuation of virulence in the mutant. Moreover, the strain isolated from the euthanized animal in the group infected with the obg mutant was shown to carry a reversion in the obg gene associated with the loss of the TS+ phenotype. This study demonstrates the feasibility of building attenuated strains of mycoplasma that could contribute to the design of novel vaccines with improved safety. IMPORTANCE Animal diseases due to mycoplasmas are a major cause of morbidity and mortality associated with economic losses for farmers all over the world. Currently used mycoplasma vaccines exhibit several drawbacks, including low efficacy, short time of protection, adverse reactions, and difficulty in differentiating infected from vaccinated animals. Therefore, there is a need for improved vaccines to control animal mycoplasmoses. Here, we used genome engineering tools derived from synthetic biology approaches to produce targeted mutations in the essential GTPase-encoding obg gene of Mycoplasma mycoides subsp. capri. Some of the resulting mutants exhibited a marked temperature-sensitive phenotype. The virulence of one of the obg mutants was evaluated in a caprine septicemia model and found to be strongly reduced. Although the obg mutant reverted to a virulent phenotype in one infected animal, we believe that these results contribute to a strategy that should help in building new vaccines against animal mycoplasmoses.

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