Special Issue "Synthetic Genetic Elements, Devices, and Systems"

A special issue of Life (ISSN 2075-1729). This special issue belongs to the section "Synthetic Biology and Systems Biology".

Deadline for manuscript submissions: closed (15 March 2022) | Viewed by 9733

Special Issue Editors

Dr. Yusuke Kato
E-Mail Website
Guest Editor
Division of Biotechnology, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization (NARO), Oowashi 1-2, Tsukuba, Ibaraki 305-8634, Japan
Interests: biological containment; synthetic biology; development of novel genetic parts; unnatural amino acids; heterologous protein production
Prof. Dr. Chunbo Lou
E-Mail Website
Guest Editor
CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, 1068 Xueyuan Avenue, University Town, Nanshan, Shenzhen 518055, China
Interests: genetic circuit; modular design; promoter design; insulator

Special Issue Information

Dear Colleagues,

Since the beginning of life on Earth, over the course of 3–4 billion years, nature has created vast numbers of genetic elements. These freshly created elements faced natural selection. Winners survived and losers disappeared. Nature selected only the genetically stable elements that contributed to the maintenance of life. From a contrary perspective, natural selection restricted the diversity of genetic elements. In the last 20 years, i.e., 2 × 10−8 billion years, synthetic biologists have tried to create novel genetic elements that “nature has not invented or cannot invent”. The objective of this research is to go beyond the restriction of natural selection and obtain novel genetic elements that are “useful for human use”. From the limited modification of characteristics of natural elements to originally designed elements, various synthetic genetic elements have been reported. “Genetic devices”, such as logic gates and memory elements, and higher order “genetic systems”, such as metabolite factories and biological containment systems, can be constructed using these synthetic genetic elements in combination with other genetic elements. Through this approach, the incorporation of synthetic genetic elements is dramatically expanding biological functions.

This Special Issue "Synthetic Genetic Elements, Devices, and Systems" will explore the current state-of-the-art in this growing field. We hereby invite articles (full articles, short communications, and reviews) covering a broad range of topics.

Topics for this Special Issue include, but are not limited to:

a) Synthetic genetic elements in prokaryotes and eukaryotes (promoters, transcription factors, RBS, degradation tags, transcriptional terminators, sensors, indicators, ribozymes and riboswitches, enzymes such as recombinases and proteases as regulatory elements, etc.).

b) Synthetic genetic circuits as devices and systems (either involving or not involving synthetic genetic elements, but with the purpose of eliciting designed behavior).

c) Methods to develop synthetic genetic elements, devices, and systems.

d) Applications for laboratory and industrial use.

e) History and future perspectives.

Dr. Yusuke Kato
Prof. Dr. Chunbo Lou
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Life is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 1800 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • synthetic biology
  • synthetic genetic elements/parts
  • biobricks
  • artificial gene synthesis
  • biocomputing
  • directed evolution
  • metabolic engineering/cell factory
  • optogenetics
  • genetic code expansion
  • xenobiology

Published Papers (12 papers)

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Editorial

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Editorial
Synthetic Genetic Elements, Devices, and Systems
Life 2022, 12(7), 945; https://doi.org/10.3390/life12070945 - 23 Jun 2022
Viewed by 132
Abstract
Since the beginning of life on Earth, over the course of 3 to 4 billion years, nature has created vast quantities of genetic elements [...] Full article
(This article belongs to the Special Issue Synthetic Genetic Elements, Devices, and Systems)

Research

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Article
Multiplexed Promoter Engineering for Improving Thaxtomin A Production in Heterologous Streptomyces Hosts
Life 2022, 12(5), 689; https://doi.org/10.3390/life12050689 - 06 May 2022
Cited by 2 | Viewed by 490
Abstract
Thaxtomin A is a potent bioherbicide in both organic and conventional agriculture; however, its low yield hinders its wide application. Here, we report the direct cloning and heterologous expression of the thaxtomin A gene cluster in three well-characterized Streptomyces hosts. Then, we present [...] Read more.
Thaxtomin A is a potent bioherbicide in both organic and conventional agriculture; however, its low yield hinders its wide application. Here, we report the direct cloning and heterologous expression of the thaxtomin A gene cluster in three well-characterized Streptomyces hosts. Then, we present an efficient, markerless and multiplex large gene cluster editing method based on in vitro CRISPR/Cas9 digestion and yeast homologous recombination. With this method, we successfully engineered the thaxtomin A cluster by simultaneously replacing the native promoters of the txtED operon, txtABH operon and txtC gene with strong constitutive promoters, and the yield of thaxtomin A improved to 289.5 µg/mL in heterologous Streptomyces coelicolor M1154. To further optimize the biosynthetic pathway, we used constraint-based combinatorial design to build 27 refactored gene clusters by varying the promoter strength of every operon, and the highest titer of thaxtomin A production reached 504.6 μg/mL. Taken altogether, this work puts forward a multiplexed promoter engineering strategy to engineer secondary metabolism gene clusters for efficiently improving fermentation titers. Full article
(This article belongs to the Special Issue Synthetic Genetic Elements, Devices, and Systems)
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Article
Detection of pks Island mRNAs Using Toehold Sensors in Escherichia coli
Life 2021, 11(11), 1280; https://doi.org/10.3390/life11111280 - 22 Nov 2021
Cited by 1 | Viewed by 672
Abstract
Synthetic biologists have applied biomolecular engineering approaches toward the goal of novel biological devices and have shown progress in diverse areas of medicine and biotechnology. Especially promising is the application of synthetic biological devices towards a novel class of molecular diagnostics. As an [...] Read more.
Synthetic biologists have applied biomolecular engineering approaches toward the goal of novel biological devices and have shown progress in diverse areas of medicine and biotechnology. Especially promising is the application of synthetic biological devices towards a novel class of molecular diagnostics. As an example, a de-novo-designed riboregulator called toehold switch, with its programmability and compatibility with field-deployable devices showed promising in vitro applications for viral RNA detection such as Zika and Corona viruses. However, the in vivo application of high-performance RNA sensors remains challenging due to the secondary structure of long mRNA species. Here, we introduced ‘Helper RNAs’ that can enhance the functionality of toehold switch sensors by mitigating the effect of secondary structures around a target site. By employing the helper RNAs, previously reported mCherry mRNA sensor showed improved fold-changes in vivo. To further generalize the Helper RNA approaches, we employed automatic design pipeline for toehold sensors that target the essential genes within the pks island, an important target of biomedical research in connection with colorectal cancer. The toehold switch sensors showed fold-changes upon the expression of full-length mRNAs that apparently depended sensitively on the identity of the gene as well as the predicted local structure within the target region of the mRNA. Still, the helper RNAs could improve the performance of toehold switch sensors in many instances, with up to 10-fold improvement over no helper cases. These results suggest that the helper RNA approaches can further assist the design of functional RNA devices in vivo with the aid of the streamlined automatic design software developed here. Further, our solutions for screening and stabilizing single-stranded region of mRNA may find use in other in vivo mRNA-sensing applications such as cas13 crRNA design, transcriptome engineering, and trans-cleaving ribozymes. Full article
(This article belongs to the Special Issue Synthetic Genetic Elements, Devices, and Systems)
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Article
Cloning, Expression and Characterization of UDP-Glucose Dehydrogenases
Life 2021, 11(11), 1201; https://doi.org/10.3390/life11111201 - 07 Nov 2021
Cited by 2 | Viewed by 813
Abstract
Uridine diphosphate-glucose dehydrogenase (UGD) is an enzyme that produces uridine diphosphate-glucuronic acid (UDP-GlcA), which is an intermediate in glycosaminoglycans (GAGs) production pathways. GAGs are generally extracted from animal tissues. Efforts to produce GAGs in a safer way have been conducted by constructing artificial [...] Read more.
Uridine diphosphate-glucose dehydrogenase (UGD) is an enzyme that produces uridine diphosphate-glucuronic acid (UDP-GlcA), which is an intermediate in glycosaminoglycans (GAGs) production pathways. GAGs are generally extracted from animal tissues. Efforts to produce GAGs in a safer way have been conducted by constructing artificial biosynthetic pathways in heterologous microbial hosts. This work characterizes novel enzymes with potential for UDP-GlcA biotechnological production. The UGD enzymes from Zymomonas mobilis (ZmUGD) and from Lactobacillus johnsonii (LbjUGD) were expressed in Escherichia coli. These two enzymes and an additional eukaryotic one from Capra hircus (ChUGD) were also expressed in Saccharomyces cerevisiae strains. The three enzymes herein studied represent different UGD phylogenetic groups. The UGD activity was evaluated through UDP-GlcA quantification in vivo and after in vitro reactions. Engineered E. coli strains expressing ZmUGD and LbjUGD were able to produce in vivo 28.4 µM and 14.9 µM UDP-GlcA, respectively. Using S. cerevisiae as the expression host, the highest in vivo UDP-GlcA production was obtained for the strain CEN.PK2-1C expressing ZmUGD (17.9 µM) or ChUGD (14.6 µM). Regarding the in vitro assays, under the optimal conditions, E. coli cell extract containing LbjUGD was able to produce about 1800 µM, while ZmUGD produced 407 µM UDP-GlcA, after 1 h of reaction. Using engineered yeasts, the in vitro production of UDP-GlcA reached a maximum of 533 µM using S. cerevisiae CEN.PK2-1C_pSP-GM_LbjUGD cell extract. The UGD enzymes were active in both prokaryotic and eukaryotic hosts, therefore the genes and expression chassis herein used can be valuable alternatives for further industrial applications. Full article
(This article belongs to the Special Issue Synthetic Genetic Elements, Devices, and Systems)
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Article
Context-Dependent Stability and Robustness of Genetic Toggle Switches with Leaky Promoters
Life 2021, 11(11), 1150; https://doi.org/10.3390/life11111150 - 28 Oct 2021
Cited by 2 | Viewed by 683
Abstract
Multistable switches are ubiquitous building blocks in both systems and synthetic biology. Given their central role, it is thus imperative to understand how their fundamental properties depend not only on the tunable biophysical properties of the switches themselves, but also on their genetic [...] Read more.
Multistable switches are ubiquitous building blocks in both systems and synthetic biology. Given their central role, it is thus imperative to understand how their fundamental properties depend not only on the tunable biophysical properties of the switches themselves, but also on their genetic context. To this end, we reveal in this article how these factors shape the essential characteristics of toggle switches implemented using leaky promoters such as their stability and robustness to noise, both at single-cell and population levels. In particular, our results expose the roles that competition for scarce transcriptional and translational resources, promoter leakiness, and cell-to-cell heterogeneity collectively play. For instance, the interplay between protein expression from leaky promoters and the associated cost of relying on shared cellular resources can give rise to tristable dynamics even in the absence of positive feedback. Similarly, we demonstrate that while promoter leakiness always acts against multistability, resource competition can be leveraged to counteract this undesirable phenomenon. Underpinned by a mechanistic model, our results thus enable the context-aware rational design of multistable genetic switches that are directly translatable to experimental considerations, and can be further leveraged during the synthesis of large-scale genetic systems using computer-aided biodesign automation platforms. Full article
(This article belongs to the Special Issue Synthetic Genetic Elements, Devices, and Systems)
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Article
An Unnatural Amino Acid-Regulated Growth Controller Based on Informational Disturbance
Life 2021, 11(9), 920; https://doi.org/10.3390/life11090920 - 05 Sep 2021
Cited by 2 | Viewed by 709
Abstract
We designed a novel growth controller regulated by feeding of an unnatural amino acid, Nε-benzyloxycarbonyl-l-lysine (ZK), using a specific incorporation system at a sense codon. This system is constructed by a pair of modified pyrrolisyl-tRNA synthetase (PylRS) and its [...] Read more.
We designed a novel growth controller regulated by feeding of an unnatural amino acid, Nε-benzyloxycarbonyl-l-lysine (ZK), using a specific incorporation system at a sense codon. This system is constructed by a pair of modified pyrrolisyl-tRNA synthetase (PylRS) and its cognate tRNA (tRNApyl). Although ZK is non-toxic for normal organisms, the growth of Escherichia coli carrying the ZK incorporation system was inhibited in a ZK concentration-dependent manner without causing rapid bacterial death, presumably due to generation of non-functional or toxic proteins. The extent of growth inhibition strongly depended on the anticodon sequence of the tRNApyl gene. Taking advantage of the low selectivity of PylRS for tRNApyl anticodons, we experimentally determined the most effective anticodon sequence among all 64 nucleotide sequences in the anticodon region of tRNApyl gene. The results suggest that the ZK-regulated growth controller is a simple, target-specific, environmental noise-resistant and titratable system. This technique may be applicable to a wide variety of organisms because the growth inhibitory effects are caused by “informational disturbance”, in which the highly conserved system for transmission of information from DNA to proteins is perturbed. Full article
(This article belongs to the Special Issue Synthetic Genetic Elements, Devices, and Systems)
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Communication
Enhancement of Transgene Expression by Mild Hypothermia Is Promoter Dependent in HEK293 Cells
Life 2021, 11(9), 901; https://doi.org/10.3390/life11090901 - 30 Aug 2021
Cited by 1 | Viewed by 763
Abstract
Mild hypothermia has been widely used to enhance transgene expression and improve the cellular productivity of mammalian cells. This study investigated mild hypothermia-responsive exogenous promoters in human embryonic kidney 293 (HEK293) cells using site-specific integration of various promoter sequences, including CMV, EF1α, SV40, [...] Read more.
Mild hypothermia has been widely used to enhance transgene expression and improve the cellular productivity of mammalian cells. This study investigated mild hypothermia-responsive exogenous promoters in human embryonic kidney 293 (HEK293) cells using site-specific integration of various promoter sequences, including CMV, EF1α, SV40, and TK promoters, into the well-known genomic safe harbor site, AAVS1. EGFP expression driven by the CMV promoter increased up to 1.5-fold at 32 °C versus 37 °C under stable expression, while others showed no hypothermic response. Integration of short CMV variants revealed that the CMV-enhancer region is responsible for the positive hypothermic response. CMV-enhancer-specific transcription factors (TFs) were then predicted through in silico analysis and RNA-sequencing analysis, resulting in the selection of one TF, NKX3-1. At 37 °C, overexpression of NKX3-1 in recombinant HEK293 cells expressing EGFP through the CMV promoter (CMV-EGFP) increased EGFP expression up to 1.6-fold, compared with that in CMV-EGFP, the expression level of which was comparable to that of CMV-EGFP at 32 °C. Taken together, this work demonstrates promoter-dependent hypothermia responses in HEK293 cells and emphasizes interactions between endogenous TFs and promoter sequences. Full article
(This article belongs to the Special Issue Synthetic Genetic Elements, Devices, and Systems)
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Review

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Review
Engineering of Synthetic Transcriptional Switches in Yeast
Life 2022, 12(4), 557; https://doi.org/10.3390/life12040557 - 08 Apr 2022
Cited by 1 | Viewed by 615
Abstract
Transcriptional switches can be utilized for many purposes in synthetic biology, including the assembly of complex genetic circuits to achieve sophisticated cellular systems and the construction of biosensors for real-time monitoring of intracellular metabolite concentrations. Although to date such switches have mainly been [...] Read more.
Transcriptional switches can be utilized for many purposes in synthetic biology, including the assembly of complex genetic circuits to achieve sophisticated cellular systems and the construction of biosensors for real-time monitoring of intracellular metabolite concentrations. Although to date such switches have mainly been developed in prokaryotes, those for eukaryotes are increasingly being reported as both rational and random engineering technologies mature. In this review, we describe yeast transcriptional switches with different modes of action and how to alter their properties. We also discuss directed evolution technologies for the rapid and robust construction of yeast transcriptional switches. Full article
(This article belongs to the Special Issue Synthetic Genetic Elements, Devices, and Systems)
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Review
CRISPR-Based Genetic Switches and Other Complex Circuits: Research and Application
Life 2021, 11(11), 1255; https://doi.org/10.3390/life11111255 - 17 Nov 2021
Cited by 2 | Viewed by 935
Abstract
CRISPR-based enzymes have offered a unique capability to the design of genetic switches, with advantages in designability, modularity and orthogonality. CRISPR-based genetic switches operate on multiple levels of life, including transcription and translation. In both prokaryotic and eukaryotic cells, deactivated CRISPR endonuclease and [...] Read more.
CRISPR-based enzymes have offered a unique capability to the design of genetic switches, with advantages in designability, modularity and orthogonality. CRISPR-based genetic switches operate on multiple levels of life, including transcription and translation. In both prokaryotic and eukaryotic cells, deactivated CRISPR endonuclease and endoribonuclease have served in genetic switches for activating or repressing gene expression, at both transcriptional and translational levels. With these genetic switches, more complex circuits have been assembled to achieve sophisticated functions including inducible switches, non-linear response and logical biocomputation. As more CRISPR enzymes continue to be excavated, CRISPR-based genetic switches will be used in a much wider range of applications. Full article
(This article belongs to the Special Issue Synthetic Genetic Elements, Devices, and Systems)
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Review
Protein-Based Systems for Translational Regulation of Synthetic mRNAs in Mammalian Cells
Life 2021, 11(11), 1192; https://doi.org/10.3390/life11111192 - 05 Nov 2021
Viewed by 548
Abstract
Synthetic mRNAs, which are produced by in vitro transcription, have been recently attracting attention because they can express any transgenes without the risk of insertional mutagenesis. Although current synthetic mRNA medicine is not designed for spatiotemporal or cell-selective regulation, many preclinical studies have [...] Read more.
Synthetic mRNAs, which are produced by in vitro transcription, have been recently attracting attention because they can express any transgenes without the risk of insertional mutagenesis. Although current synthetic mRNA medicine is not designed for spatiotemporal or cell-selective regulation, many preclinical studies have developed the systems for the translational regulation of synthetic mRNAs. Such translational regulation systems will cope with high efficacy and low adverse effects by producing the appropriate amount of therapeutic proteins, depending on the context. Protein-based regulation is one of the most promising approaches for the translational regulation of synthetic mRNAs. As synthetic mRNAs can encode not only output proteins but also regulator proteins, all components of protein-based regulation systems can be delivered as synthetic mRNAs. In addition, in the protein-based regulation systems, the output protein can be utilized as the input for the subsequent regulation to construct multi-layered gene circuits, which enable complex and sophisticated regulation. In this review, I introduce what types of proteins have been used for translational regulation, how to combine them, and how to design effective gene circuits. Full article
(This article belongs to the Special Issue Synthetic Genetic Elements, Devices, and Systems)
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Review
Synthetic Protein Circuits and Devices Based on Reversible Protein-Protein Interactions: An Overview
Life 2021, 11(11), 1171; https://doi.org/10.3390/life11111171 - 03 Nov 2021
Cited by 1 | Viewed by 917
Abstract
Protein-protein interactions (PPIs) contribute to regulate many aspects of cell physiology and metabolism. Protein domains involved in PPIs are important building blocks for engineering genetic circuits through synthetic biology. These domains can be obtained from known proteins and rationally engineered to produce orthogonal [...] Read more.
Protein-protein interactions (PPIs) contribute to regulate many aspects of cell physiology and metabolism. Protein domains involved in PPIs are important building blocks for engineering genetic circuits through synthetic biology. These domains can be obtained from known proteins and rationally engineered to produce orthogonal scaffolds, or computationally designed de novo thanks to recent advances in structural biology and molecular dynamics prediction. Such circuits based on PPIs (or protein circuits) appear of particular interest, as they can directly affect transcriptional outputs, as well as induce behavioral/adaptational changes in cell metabolism, without the need for further protein synthesis. This last example was highlighted in recent works to enable the production of fast-responding circuits which can be exploited for biosensing and diagnostics. Notably, PPIs can also be engineered to develop new drugs able to bind specific intra- and extra-cellular targets. In this review, we summarize recent findings in the field of protein circuit design, with particular focus on the use of peptides as scaffolds to engineer these circuits. Full article
(This article belongs to the Special Issue Synthetic Genetic Elements, Devices, and Systems)
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Review
Aptamers, Riboswitches, and Ribozymes in S. cerevisiae Synthetic Biology
Life 2021, 11(3), 248; https://doi.org/10.3390/life11030248 - 17 Mar 2021
Cited by 6 | Viewed by 1353
Abstract
Among noncoding RNA sequences, riboswitches and ribozymes have attracted the attention of the synthetic biology community as circuit components for translation regulation. When fused to aptamer sequences, ribozymes and riboswitches are enabled to interact with chemicals. Therefore, protein synthesis can be controlled at [...] Read more.
Among noncoding RNA sequences, riboswitches and ribozymes have attracted the attention of the synthetic biology community as circuit components for translation regulation. When fused to aptamer sequences, ribozymes and riboswitches are enabled to interact with chemicals. Therefore, protein synthesis can be controlled at the mRNA level without the need for transcription factors. Potentially, the use of chemical-responsive ribozymes/riboswitches would drastically simplify the design of genetic circuits. In this review, we describe synthetic RNA structures that have been used so far in the yeast Saccharomyces cerevisiae. We present their interaction mode with different chemicals (e.g., theophylline and antibiotics) or proteins (such as the RNase III) and their recent employment into clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 9 (CRISPR-Cas) systems. Particular attention is paid, throughout the whole paper, to their usage and performance into synthetic gene circuits. Full article
(This article belongs to the Special Issue Synthetic Genetic Elements, Devices, and Systems)
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