scholarly journals Properties of alternative microbial hosts used in synthetic biology: towards the design of a modular chassis

2016 ◽  
Vol 60 (4) ◽  
pp. 303-313 ◽  
Author(s):  
Juhyun Kim ◽  
Manuel Salvador ◽  
Elizabeth Saunders ◽  
Jaime González ◽  
Claudio Avignone-Rossa ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Genetic Information ◽  
Essential Element ◽  
Model Organisms ◽  
Biotechnological Applications ◽  
Cell Models ◽  
Genetic Circuits ◽  
Metabolic Diversity ◽  
Translational Machinery ◽  
Metabolic Fluxes

The chassis is the cellular host used as a recipient of engineered biological systems in synthetic biology. They are required to propagate the genetic information and to express the genes encoded in it. Despite being an essential element for the appropriate function of genetic circuits, the chassis is rarely considered in their design phase. Consequently, the circuits are transferred to model organisms commonly used in the laboratory, such as Escherichia coli, that may be suboptimal for a required function. In this review, we discuss some of the properties desirable in a versatile chassis and summarize some examples of alternative hosts for synthetic biology amenable for engineering. These properties include a suitable life style, a robust cell wall, good knowledge of its regulatory network as well as of the interplay of the host components with the exogenous circuits, and the possibility of developing whole-cell models and tuneable metabolic fluxes that could allow a better distribution of cellular resources (metabolites, ATP, nucleotides, amino acids, transcriptional and translational machinery). We highlight Pseudomonas putida, widely used in many different biotechnological applications as a prominent organism for synthetic biology due to its metabolic diversity, robustness and ease of manipulation.

scholarly journals CRIMoClo plasmids for modular assembly and orthogonal chromosomal integration of synthetic circuits in Escherichia coli

2019 ◽  
Vol 13 (1) ◽  
Author(s):  
Stefano Vecchione ◽  
Georg Fritz
Keyword(s):  
Escherichia Coli ◽  
Synthetic Biology ◽  
Integrated Circuits ◽  
High Efficiency ◽  
Genetic Circuit ◽  
Dna Assembly ◽  
Chromosomal Integration ◽  
Golden Gate ◽  
Genetic Circuits ◽  
E Coli

Abstract Background Synthetic biology heavily depends on rapid and simple techniques for DNA engineering, such as Ligase Cycling Reaction (LCR), Gibson assembly and Golden Gate assembly, all of which allow for fast, multi-fragment DNA assembly. A major enhancement of Golden Gate assembly is represented by the Modular Cloning (MoClo) system that allows for simple library propagation and combinatorial construction of genetic circuits from reusable parts. Yet, one limitation of the MoClo system is that all circuits are assembled in low- and medium copy plasmids, while a rapid route to chromosomal integration is lacking. To overcome this bottleneck, here we took advantage of the conditional-replication, integration, and modular (CRIM) plasmids, which can be integrated in single copies into the chromosome of Escherichia coli and related bacteria by site-specific recombination at different phage attachment (att) sites. Results By combining the modularity of the MoClo system with the CRIM plasmids features we created a set of 32 novel CRIMoClo plasmids and benchmarked their suitability for synthetic biology applications. Using CRIMoClo plasmids we assembled and integrated a given genetic circuit into four selected phage attachment sites. Analyzing the behavior of these circuits we found essentially identical expression levels, indicating orthogonality of the loci. Using CRIMoClo plasmids and four different reporter systems, we illustrated a framework that allows for a fast and reliable sequential integration at the four selected att sites. Taking advantage of four resistance cassettes the procedure did not require recombination events between each round of integration. Finally, we assembled and genomically integrated synthetic ECF σ factor/anti-σ switches with high efficiency, showing that the growth defects observed for circuits encoded on medium-copy plasmids were alleviated. Conclusions The CRIMoClo system enables the generation of genetic circuits from reusable, MoClo-compatible parts and their integration into 4 orthogonal att sites into the genome of E. coli. Utilizing four different resistance modules the CRIMoClo system allows for easy, fast, and reliable multiple integrations. Moreover, utilizing CRIMoClo plasmids and MoClo reusable parts, we efficiently integrated and alleviated the toxicity of plasmid-borne circuits. Finally, since CRIMoClo framework allows for high flexibility, it is possible to utilize plasmid-borne and chromosomally integrated circuits simultaneously. This increases our ability to permute multiple genetic modules and allows for an easier design of complex synthetic metabolic pathways in E. coli.

scholarly journals Nucleic Acids Chemistry and Engineering: Special Issue on Nucleic Acid Conjugates for Biotechnological Applications

2021 ◽  
Vol 11 (8) ◽  
pp. 3594
Author(s):  
Tamaki Endoh ◽  
Eriks Rozners ◽  
Takashi Ohtsuki
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Genetic Information ◽  
Biotechnological Applications ◽  
Biological Functions ◽  
Primary Sequence ◽  
Special Issue

Nucleic acids not only store genetic information in their primary sequence but also exhibit biological functions through the formation of their unique structures [...]

scholarly journals On the Diversification of the Translation Apparatus across Eukaryotes

2012 ◽  
Vol 2012 ◽  
pp. 1-14 ◽  
Author(s):  
Greco Hernández ◽  
Christopher G. Proud ◽  
Thomas Preiss ◽  
Armen Parsyan
Keyword(s):  
Model Organisms ◽  
Ecological Niches ◽  
Translational Machinery ◽  
Functional Differences ◽  
Profound Knowledge ◽  
Genome Wide ◽  
Translation Apparatus ◽  
Fundamental Process ◽  
Organismal Complexity ◽  
Living Organisms

Diversity is one of the most remarkable features of living organisms. Current assessments of eukaryote biodiversity reaches 1.5 million species, but the true figure could be several times that number. Diversity is ingrained in all stages and echelons of life, namely, the occupancy of ecological niches, behavioral patterns, body plans and organismal complexity, as well as metabolic needs and genetics. In this review, we will discuss that diversity also exists in a key biochemical process, translation, across eukaryotes. Translation is a fundamental process for all forms of life, and the basic components and mechanisms of translation in eukaryotes have been largely established upon the study of traditional, so-called model organisms. By using modern genome-wide, high-throughput technologies, recent studies of many nonmodel eukaryotes have unveiled a surprising diversity in the configuration of the translation apparatus across eukaryotes, showing that this apparatus is far from being evolutionarily static. For some of the components of this machinery, functional differences between different species have also been found. The recent research reviewed in this article highlights the molecular and functional diversification the translational machinery has undergone during eukaryotic evolution. A better understanding of all aspects of organismal diversity is key to a more profound knowledge of life.

scholarly journals Societal impact of synthetic biology: responsible research and innovation (RRI)

2016 ◽  
Vol 60 (4) ◽  
pp. 371-379 ◽  
Author(s):  
Daniel Gregorowius ◽  
Anna Deplazes-Zemp
Keyword(s):  
Synthetic Biology ◽  
Technology Assessment ◽  
Ethical Issues ◽  
Responsible Research And Innovation ◽  
Societal Impact ◽  
Biotechnological Applications ◽  
Research And Innovation ◽  
Challenges And Opportunities ◽  
Living Organisms ◽  
Responsible Research

Synthetic biology is an emerging field at the interface between biology and engineering, which has generated many expectations for beneficial biomedical and biotechnological applications. At the same time, however, it has also raised concerns about risks or the aim of producing new forms of living organisms. Researchers from different disciplines as well as policymakers and the general public have expressed the need for a form of technology assessment that not only deals with technical aspects, but also includes societal and ethical issues. A recent and very influential model of technology assessment that tries to implement these aims is known as RRI (Responsible Research and Innovation). In this paper, we introduce this model and its historical precursor strategies. Based on the societal and ethical issues which are presented in the current literature, we discuss challenges and opportunities of applying the RRI model for the assessment of synthetic biology.

scholarly journals Synthetic Biology Parts for the Storage of Increased Genetic Information in Cells

2017 ◽  
Vol 6 (10) ◽  
pp. 1834-1840 ◽  
Author(s):  
Sydney E. Morris ◽  
Aaron W. Feldman ◽  
Floyd E. Romesberg
Keyword(s):  
Synthetic Biology ◽  
Genetic Information ◽  
In Cells

CRISPR-Based Tools in Immunity

2019 ◽  
Vol 37 (1) ◽  
pp. 571-597 ◽  
Author(s):  
Dimitre R. Simeonov ◽  
Alexander Marson
Keyword(s):  
Genome Engineering ◽  
Disease Risk ◽  
Ease Of Use ◽  
Model Organisms ◽  
Mechanistic Studies ◽  
Genetic Circuits ◽  
New Era ◽  
Wide Range ◽  
Health And Disease ◽  
Cancer Immunotherapies

CRISPR technology has opened a new era of genome interrogation and genome engineering. Discovered in bacteria, where it protects against bacteriophage by cleaving foreign nucleic acid sequences, the CRISPR system has been repurposed as an adaptable tool for genome editing and multiple other applications. CRISPR's ease of use, precision, and versatility have led to its widespread adoption, accelerating biomedical research and discovery in human cells and model organisms. Here we review CRISPR-based tools and discuss how they are being applied to decode the genetic circuits that control immune function in health and disease. Genetic variation in immune cells can affect autoimmune disease risk, infectious disease pathogenesis, and cancer immunotherapies. CRISPR provides unprecedented opportunities for functional mechanistic studies of coding and noncoding genome sequence function in immunity. Finally, we discuss the potential of CRISPR technology to engineer synthetic cellular immunotherapies for a wide range of human diseases.

scholarly journals Engineering polymerases for applications in synthetic biology

2020 ◽  
Vol 53 ◽  
Author(s):  
Ali Nikoomanzar ◽  
Nicholas Chim ◽  
Eric J. Yik ◽  
John C. Chaput
Keyword(s):  
Cell Division ◽  
Synthetic Biology ◽  
Substrate Specificity ◽  
Physicochemical Properties ◽  
Genetic Information ◽  
Biomedical Applications ◽  
Dna Polymerases ◽  
Enzyme Engineering ◽  
Practical Applications ◽  
Naturally Occurring

Abstract DNA polymerases play a central role in biology by transferring genetic information from one generation to the next during cell division. Harnessing the power of these enzymes in the laboratory has fueled an increase in biomedical applications that involve the synthesis, amplification, and sequencing of DNA. However, the high substrate specificity exhibited by most naturally occurring DNA polymerases often precludes their use in practical applications that require modified substrates. Moving beyond natural genetic polymers requires sophisticated enzyme-engineering technologies that can be used to direct the evolution of engineered polymerases that function with tailor-made activities. Such efforts are expected to uniquely drive emerging applications in synthetic biology by enabling the synthesis, replication, and evolution of synthetic genetic polymers with new physicochemical properties.

scholarly journals Small but Smart: On the Diverse Role of Small Proteins in the Regulation of Cyanobacterial Metabolism

Life ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 322
Author(s):  
Fabian Brandenburg ◽  
Stephan Klähn
Keyword(s):  
Metabolic Pathways ◽  
Chemical Compounds ◽  
Industrial Processes ◽  
Biotechnological Applications ◽  
Major Focus ◽  
The Past ◽  
Metabolic Fluxes ◽  
Reactor Technology ◽  
Small Proteins

Over the past few decades, bioengineered cyanobacteria have become a major focus of research for the production of energy carriers and high value chemical compounds. Besides improvements in cultivation routines and reactor technology, the integral understanding of the regulation of metabolic fluxes is the key to designing production strains that are able to compete with established industrial processes. In cyanobacteria, many enzymes and metabolic pathways are regulated differently compared to other bacteria. For instance, while glutamine synthetase in proteobacteria is mainly regulated by covalent enzyme modifications, the same enzyme in cyanobacteria is controlled by the interaction with unique small proteins. Other prominent examples, such as the small protein CP12 which controls the Calvin–Benson cycle, indicate that the regulation of enzymes and/or pathways via the attachment of small proteins might be a widespread mechanism in cyanobacteria. Accordingly, this review highlights the diverse role of small proteins in the control of cyanobacterial metabolism, focusing on well-studied examples as well as those most recently described. Moreover, it will discuss their potential to implement metabolic engineering strategies in order to make cyanobacteria more definable for biotechnological applications.

scholarly journals SynMyco transposon: engineering transposon vectors for efficient transformation of minimal genomes

DNA Research ◽  
2019 ◽  
Vol 26 (4) ◽  
pp. 327-339 ◽  
Author(s):  
Ariadna Montero-Blay ◽  
Samuel Miravet-Verde ◽  
Maria Lluch-Senar ◽  
Carlos Piñero-Lambea ◽  
Luis Serrano
Keyword(s):  
Antibiotic Resistance ◽  
Synthetic Biology ◽  
Genome Editing ◽  
Mycoplasma Pneumoniae ◽  
Mycoplasma Gallisepticum ◽  
Model Organisms ◽  
Mycoplasma Species ◽  
Resistance Marker ◽  
Antibiotic Resistance Marker ◽  
Reference Tool

Abstract Mycoplasmas are important model organisms for Systems and Synthetic Biology, and are pathogenic to a wide variety of species. Despite their relevance, many of the tools established for genome editing in other microorganisms are not available for Mycoplasmas. The Tn4001 transposon is the reference tool to work with these bacteria, but the transformation efficiencies (TEs) reported for the different species vary substantially. Here, we explore the mechanisms underlying these differences in four Mycoplasma species, Mycoplasma agalactiae, Mycoplasma feriruminatoris, Mycoplasma gallisepticum and Mycoplasma pneumoniae, selected for being representative members of each cluster of the Mycoplasma genus. We found that regulatory regions (RRs) driving the expression of the transposase and the antibiotic resistance marker have a major impact on the TEs. We then designed a synthetic RR termed SynMyco RR to control the expression of the key transposon vector elements. Using this synthetic RR, we were able to increase the TE for M. gallisepticum, M. feriruminatoris and M. agalactiae by 30-, 980- and 1036-fold, respectively. Finally, to illustrate the potential of this new transposon, we performed the first essentiality study in M. agalactiae, basing our study on more than 199,000 genome insertions.

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