Life | Special Issue : Synthetic Genetic Elements, Devices, and Systems

Editorial

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3 pages, 175 KiB

Open AccessEditorial

Synthetic Genetic Elements, Devices, and Systems

by Yusuke Kato and Chunbo Lou

Life 2022, 12(7), 945; https://doi.org/10.3390/life12070945 - 23 Jun 2022

Viewed by 1195

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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16 pages, 2907 KiB

Open AccessArticle

Detection of pks Island mRNAs Using Toehold Sensors in Escherichia coli

by Taeyang Heo, Hansol Kang, Seungdo Choi and Jongmin Kim

Life 2021, 11(11), 1280; https://doi.org/10.3390/life11111280 - 22 Nov 2021

Cited by 4 | Viewed by 2728

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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21 pages, 3096 KiB

Open AccessArticle

Cloning, Expression and Characterization of UDP-Glucose Dehydrogenases

by Márcia R. Couto, Joana L. Rodrigues and Lígia R. Rodrigues

Life 2021, 11(11), 1201; https://doi.org/10.3390/life11111201 - 7 Nov 2021

Cited by 5 | Viewed by 3144

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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16 pages, 19195 KiB

Open AccessArticle

Context-Dependent Stability and Robustness of Genetic Toggle Switches with Leaky Promoters

by Andras Gyorgy

Life 2021, 11(11), 1150; https://doi.org/10.3390/life11111150 - 28 Oct 2021

Cited by 2 | Viewed by 2475

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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13 pages, 1643 KiB

Open AccessArticle

An Unnatural Amino Acid-Regulated Growth Controller Based on Informational Disturbance

by Yusuke Kato

Life 2021, 11(9), 920; https://doi.org/10.3390/life11090920 - 5 Sep 2021

Cited by 3 | Viewed by 1691

Abstract

We designed a novel growth controller regulated by feeding of an unnatural amino acid, N^ε-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, N^ε-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 (tRNA^pyl). 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 tRNA^pyl gene. Taking advantage of the low selectivity of PylRS for tRNA^pyl anticodons, we experimentally determined the most effective anticodon sequence among all 64 nucleotide sequences in the anticodon region of tRNA^pyl 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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8 pages, 1010 KiB

Open AccessCommunication

Enhancement of Transgene Expression by Mild Hypothermia Is Promoter Dependent in HEK293 Cells

by Min Ho Jang, Honggi Min and Jae Seong Lee

Life 2021, 11(9), 901; https://doi.org/10.3390/life11090901 - 30 Aug 2021

Cited by 4 | Viewed by 2710

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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20 pages, 1557 KiB

Open AccessReview

Engineering of Synthetic Transcriptional Switches in Yeast

by Masahiro Tominaga, Akihiko Kondo and Jun Ishii

Life 2022, 12(4), 557; https://doi.org/10.3390/life12040557 - 8 Apr 2022

Cited by 5 | Viewed by 4162

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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19 pages, 5697 KiB

Open AccessReview

Protein-Based Systems for Translational Regulation of Synthetic mRNAs in Mammalian Cells

by Hideyuki Nakanishi

Life 2021, 11(11), 1192; https://doi.org/10.3390/life11111192 - 5 Nov 2021

Cited by 7 | Viewed by 2739

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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10 pages, 1142 KiB

Open AccessReview

Synthetic Protein Circuits and Devices Based on Reversible Protein-Protein Interactions: An Overview

by Stefano Rosa, Chiara Bertaso, Paolo Pesaresi, Simona Masiero and Andrea Tagliani

Life 2021, 11(11), 1171; https://doi.org/10.3390/life11111171 - 3 Nov 2021

Cited by 4 | Viewed by 3237

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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15 pages, 1393 KiB

Open AccessReview

Aptamers, Riboswitches, and Ribozymes in S. cerevisiae Synthetic Biology

by Huanhuan Ge and Mario Andrea Marchisio

Life 2021, 11(3), 248; https://doi.org/10.3390/life11030248 - 17 Mar 2021

Cited by 14 | Viewed by 4909

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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