scholarly journals Robust gene expression control in human cells with a novel universal TetR aptamer splicing module

2019 ◽  
Vol 47 (20) ◽  
pp. e132-e132 ◽  
Author(s):  
Adam A Mol ◽  
Florian Groher ◽  
Britta Schreiber ◽  
Ciaran Rühmkorff ◽  
Beatrix Suess
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
Dynamic Range ◽  
Intron Retention ◽  
Human Cells ◽  
Fine Tuning ◽  
Genomic Context ◽  
Mammalian Cell Lines ◽  
Gene Expression Control ◽  
Wide Range

Abstract Fine-tuning of gene expression is desirable for a wide range of applications in synthetic biology. In this context, RNA regulatory devices provide a powerful and highly functional tool. We developed a versatile, robust and reversible device to control gene expression by splicing regulation in human cells using an aptamer that is recognized by the Tet repressor TetR. Upon insertion in proximity to the 5′ splice site, intron retention can be controlled via the binding of TetR to the aptamer. Although we were able to demonstrate regulation for different introns, the genomic context had a major impact on regulation. In consequence, we advanced the aptamer to develop a splice device. Our novel device contains the aptamer integrated into a context of exonic and intronic sequences that create and maintain an environment allowing a reliable and robust splicing event. The exon-born, additional amino acids will then be cleaved off by a self-cleaving peptide. This design allows portability of the splicing device, which we confirmed by demonstrating its functionality in different gene contexts. Intriguingly, our splicing device shows a high dynamic range and low basal activity, i.e. desirable features that often prove a major challenge when implementing synthetic biology in mammalian cell lines.

scholarly journals Dual UTR-A novel 5′ untranslated region design for synthetic biology applications

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Simone Balzer Le ◽  
Ingerid Onsager ◽  
Jon Andreas Lorentzen ◽  
Rahmi Lale
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
De Novo ◽  
Expression Profiles ◽  
Regulation Of Gene Expression ◽  
Design Concept ◽  
Fine Tuning ◽  
The Novel ◽  
Wide Range ◽  
Application Potential

Abstract Bacterial 5′ untranslated regions of mRNA (UTR) involve in a complex regulation of gene expression; however, the exact sequence features contributing to gene regulation are not yet fully understood. In this study, we report the design of a novel 5′ UTR, dual UTR, utilizing the transcriptional and translational characteristics of 5′ UTRs in a single expression cassette. The dual UTR consists of two 5′ UTRs, each separately leading to either increase in transcription or translation of the reporter, that are separated by a spacer region, enabling de novo translation initiation. We rationally create dual UTRs with a wide range of expression profiles and demonstrate the functionality of the novel design concept in Escherichia coli and Pseudomonas putida using different promoter systems and coding sequences. Overall, we demonstrate the application potential of dual UTR design concept in various synthetic biology applications ranging from fine-tuning of gene expression to maximization of protein production.

scholarly journals Dual UTR-A novel 5′ untranslated region design for synthetic biology applications

2019 ◽  
Author(s):  
Simone Balzer Le ◽  
Ingerid Onsager ◽  
Jon Andreas Lorentzen ◽  
Rahmi Lale
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
De Novo ◽  
Expression Profiles ◽  
Regulation Of Gene Expression ◽  
Design Concept ◽  
Fine Tuning ◽  
The Novel ◽  
Wide Range ◽  
Application Potential

ABSTRACTBacterial 5′ untranslated regions of mRNA (UTR) involve in a complex regulation of gene expression; however, the exact sequence features contributing to gene regulation are not yet fully understood. In this study, we report the design of a novel 5′ UTR, dual UTR, utilising the transcriptional and translational characteristics of 5′ UTRs in a single expression cassette. The dual UTR consists of two 5′ UTRs, each separately leading to either increase in transcription or translation of the reporter, that are separated by a spacer region, enabling de novo translation initiation. We rationally create dual UTRs with a wide range of expression profiles and demonstrate the functionality of the novel design concept in Escherichia coli and in Pseudomonas putida using different promoter systems and coding sequences. Overall, we demonstrate the application potential of dual UTR design concept in various synthetic biology applications ranging from fine-tuning of gene expression to maximisation of protein production.

scholarly journals Refactoring of a synthetic raspberry ketone pathway with EcoFlex

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Simon J. Moore ◽  
Yonek B. Hleba ◽  
Sarah Bischoff ◽  
David Bell ◽  
Karen M. Polizzi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
Combinatorial Libraries ◽  
Value Added ◽  
Microtiter Plate ◽  
Fine Tuning ◽  
Golden Gate ◽  
E Coli ◽  
Fine Chemical ◽  
Raspberry Ketone

Abstract Background  A key focus of synthetic biology is to develop microbial or cell-free based biobased routes to value-added chemicals such as fragrances. Originally, we developed the EcoFlex system, a Golden Gate toolkit, to study genes/pathways flexibly using Escherichia coli heterologous expression. In this current work, we sought to use EcoFlex to optimise a synthetic raspberry ketone biosynthetic pathway. Raspberry ketone is a high-value (~ £20,000 kg−1) fine chemical farmed from raspberry (Rubeus rubrum) fruit. Results  By applying a synthetic biology led design-build-test-learn cycle approach, we refactor the raspberry ketone pathway from a low level of productivity (0.2 mg/L), to achieve a 65-fold (12.9 mg/L) improvement in production. We perform this optimisation at the prototype level (using microtiter plate cultures) with E. coli DH10β, as a routine cloning host. The use of E. coli DH10β facilitates the Golden Gate cloning process for the screening of combinatorial libraries. In addition, we also newly establish a novel colour-based phenotypic screen to identify productive clones quickly from solid/liquid culture. Conclusions  Our findings provide a stable raspberry ketone pathway that relies upon a natural feedstock (L-tyrosine) and uses only constitutive promoters to control gene expression. In conclusion we demonstrate the capability of EcoFlex for fine-tuning a model fine chemical pathway and provide a range of newly characterised promoter tools gene expression in E. coli.

scholarly journals Cell-free genetic devices confer autonomic and adaptive properties to hydrogels

2019 ◽  
Author(s):  
Colette J. Whitfield ◽  
Alice M. Banks ◽  
Gema Dura ◽  
John Love ◽  
Jonathan E. Fieldsend ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Information Processing ◽  
Synthetic Biology ◽  
Smart Materials ◽  
Living Organism ◽  
Chemical Diversity ◽  
Biological Information ◽  
Self Healing ◽  
Wide Range

AbstractSmart materials are able to alter one or more of their properties in response to defined stimuli. Our ability to design and create such materials, however, does not match the diversity and specificity of responses seen within the biological domain. We propose that relocation of molecular phenomena from living cells into hydrogels can be used to confer smart functionality to materials. We establish that cell-free protein synthesis can be conducted in agarose hydrogels, that gene expression occurs throughout the material and that co-expression of genes is possible. We demonstrate that gene expression can be controlled transcriptionally (using in gel gene interactions) and translationally in response to small molecule and nucleic acid triggers. We use this system to design and build a genetic device that can alter the structural property of its chassis material in response to exogenous stimuli. Importantly, we establish that a wide range of hydrogels are appropriate chassis for cell-free synthetic biology, meaning a designer may alter both the genetic and hydrogel components according to the requirements of a given application. We probe the relationship between the physical structure of the gel and in gel protein synthesis and reveal that the material itself may act as a macromolecular crowder enhancing protein synthesis. Given the extensive range of genetically encoded information processing networks in the living kingdom and the structural and chemical diversity of hydrogels, this work establishes a model by which cell-free synthetic biology can be used to create autonomic and adaptive materials.Significance statementSmart materials have the ability to change one or more of their properties (e.g. structure, shape or function) in response to specific triggers. They have applications ranging from light-sensitive sunglasses and drug delivery systems to shape-memory alloys and self-healing coatings. The ability to programme such materials, however, is basic compared to the ability of a living organism to observe, understand and respond to its environment. Here we demonstrate the relocation of biological information processing systems from cells to materials. We achieved this by operating small, programmable genetic devices outside the confines of a living cell and inside hydrogel matrices. These results establish a method for developing materials functionally enhanced with molecular machinery from biological systems.

scholarly journals Blueprints for Biosensors: Design, Limitations, and Applications

Genes ◽  
2018 ◽  
Vol 9 (8) ◽  
pp. 375 ◽  
Author(s):  
Alexander Carpenter ◽  
Ian Paulsen ◽  
Thomas Williams
Keyword(s):  
Transcription Factors ◽  
Synthetic Biology ◽  
Nucleic Acids ◽  
Dynamic Range ◽  
Optimal Performance ◽  
Ease Of Use ◽  
Ligand Specificity ◽  
Biotechnological Application ◽  
Wide Range ◽  
Output Time

Biosensors are enabling major advances in the field of analytics that are both facilitating and being facilitated by advances in synthetic biology. The ability of biosensors to rapidly and specifically detect a wide range of molecules makes them highly relevant to a range of industrial, medical, ecological, and scientific applications. Approaches to biosensor design are as diverse as their applications, with major biosensor classes including nucleic acids, proteins, and transcription factors. Each of these biosensor types has advantages and limitations based on the intended application, and the parameters that are required for optimal performance. Specifically, the choice of biosensor design must consider factors such as the ligand specificity, sensitivity, dynamic range, functional range, mode of output, time of activation, ease of use, and ease of engineering. This review discusses the rationale for designing the major classes of biosensor in the context of their limitations and assesses their suitability to different areas of biotechnological application.

scholarly journals Multiple cytochrome P-450 genes are concomitantly regulated by vitamin A under steady-state conditions and by retinoic acid during hepatic first-pass metabolism

2011 ◽  
Vol 43 (1) ◽  
pp. 57-67 ◽  
Author(s):  
A. Catharine Ross ◽  
Christopher J. Cifelli ◽  
Reza Zolfaghari ◽  
Nan-qian Li
Keyword(s):  
Gene Expression ◽  
Retinoic Acid ◽  
Steady State ◽  
Vitamin A ◽  
Dynamic Range ◽  
Dynamic Change ◽  
The Body ◽  
Retinol Binding Protein ◽  
Dietary Vitamin ◽  
Wide Range

Vitamin A (retinol) is an essential precursor for the production of retinoic acid (RA), which in turn is a major regulator of gene expression, affecting cell differentiation throughout the body. Understanding how vitamin A nutritional status, as well as therapeutic retinoid treatment, regulates the expression of retinoid homeostatic genes is important for improvement of dietary recommendations and therapeutic strategies using retinoids. This study investigated genes central to processes of retinoid uptake and storage, release to plasma, and oxidation in the liver of rats under steady-state conditions after different exposures to dietary vitamin A (deficient, marginal, adequate, and supplemented) and acutely after administration of a therapeutic dose of all- trans-RA. Over a very wide range of dietary vitamin A, lecithin:retinol acyltransferase (LRAT) as well as multiple cytochrome P-450s (CYP26A1, CYP26B1, and CYP2C22) differed by diet and were highly correlated with one another and with vitamin A status assessed by liver retinol concentration (all correlations, P < 0.05). After acute treatment with RA, the same genes were rapidly and concomitantly induced, preceding retinoic acid receptor (RAR)β, a classical direct target of RA. CYP26A1 mRNA exhibited the greatest dynamic range (change of log 26 in 3 h). Moreover, CYP26A1 increased more rapidly in the liver of RA-primed rats than naive rats, evidenced by increased CYP26A1 gene expression and increased conversion of [3H]RA to polar metabolites. By in situ hybridization, CYP26A1 mRNA was strongly regulated within hepatocytes, closely resembling retinol-binding protein (RBP)4 in location. Overall, whether RA is produced endogenously from retinol or administered exogenously, changes in retinoid homeostatic gene expression simultaneously favor both retinol esterification and RA oxidation, with CYP26A1 exhibiting the greatest dynamic change.

scholarly journals Translational gene expression control in Chlamydia trachomatis.

2021 ◽  
Author(s):  
Nicole A Grieshaber ◽  
Travis J Chiarelli ◽  
Cody R Appa ◽  
Grace Neiswanger ◽  
Kristina Peretti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chlamydia Trachomatis ◽  
Translational Control ◽  
Regulatory Protein ◽  
Inducible Promoter ◽  
Cloning And Expression ◽  
Developmental Cycle ◽  
Expression Control ◽  
Gene Expression Control ◽  
Wide Range

The human pathogen Chlamydia trachomatis proceeds through a multi phenotypic developmental cycle with each cell form specialized for different roles in pathogenesis. Understanding the mechanisms regulating this complex cycle has historically been hampered by limited genetic tools. In an effort to address this issue, we developed a translational control system to regulate gene expression in Chlamydia using a synthetic riboswitch. Here we demonstrate that translational control via a riboswitch can be used in combination with a wide range of promoters in C. trachomatis. The synthetic riboswitch E, inducible with theophylline, was used to replace the ribosome binding site of the synthetic promoter T5-lac, the native chlamydial promoter of the pgp4plasmid gene and an anhydrotetracycline responsive promoter. In all cases the riboswitch inhibited translation, and high levels of protein expression was induced with theophylline. Combining the Tet transcriptional inducible promoter with the translational control of the riboswitch resulted in strong repression and allowed for the cloning and expression of the potent chlamydial regulatory protein, HctB. The ability to control the timing and strength of gene expression independently from promoter specificity is a new and important tool for studying chlamydial regulatory and virulence genes.

scholarly journals Transcriptome Analysis of Alternative Splicing Events Induced by Arbuscular Mycorrhizal Fungi (Rhizophagus irregularis) in Pea (Pisum sativum L.) Roots

Plants ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1700
Author(s):  
Evgeny A. Zorin ◽  
Alexey M. Afonin ◽  
Olga A. Kulaeva ◽  
Emma S. Gribchenko ◽  
Oksana Y. Shtark ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Pisum Sativum ◽  
Mycorrhizal Fungi ◽  
Intron Retention ◽  
Protein A ◽  
Fine Tuning ◽  
Mrna Isoforms ◽  
Pisum Sativum L ◽  
Mycorrhizal Roots

Alternative splicing (AS), a process that enables formation of different mRNA isoforms due to alternative ways of pre-mRNA processing, is one of the mechanisms for fine-tuning gene expression. Currently, the role of AS in symbioses formed by plants with soil microorganisms is not fully understood. In this work, a comprehensive analysis of the transcriptome of garden pea (Pisum sativum L.) roots in symbiosis with arbuscular mycorrhiza was performed using RNAseq and following bioinformatic analysis. AS profiles of mycorrhizal and control roots were highly similar, intron retention accounting for a large proportion of the observed AS types (67%). Using three different tools (SUPPA2, DRIMSeq and IsoformSwitchAnalyzeR), eight genes with AS events specific for mycorrhizal roots of pea were identified, among which four were annotated as encoding an apoptosis inhibitor protein, a serine/threonine-protein kinase, a dehydrodolichyl diphosphate synthase, and a pre-mRNA-splicing factor ATP-dependent RNA helicase DEAH1. In pea mycorrhizal roots, the isoforms of these four genes with preliminary stop codons leading to a truncated ORFs were up-regulated. Interestingly, two of these four genes demonstrating mycorrhiza-specific AS are related to the process of splicing, thus forming parts of the feedback loops involved in fine-tuning of gene expression during mycorrhization.

scholarly journals Activity-dependent aberrations in gene expression and alternative splicing in a mouse model of Rett syndrome

2018 ◽  
Vol 115 (23) ◽  
pp. E5363-E5372 ◽  
Author(s):  
Sivan Osenberg ◽  
Ariel Karten ◽  
Jialin Sun ◽  
Jin Li ◽  
Shaun Charkowick ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Mouse Model ◽  
Rett Syndrome ◽  
Neuronal Activity ◽  
Intron Retention ◽  
Neurodevelopmental Disorder ◽  
Fine Tuning ◽  
Global Pattern ◽  
Activity Dependent

Rett syndrome (RTT) is a severe neurodevelopmental disorder that affects about 1 in 10,000 female live births. The underlying cause of RTT is mutations in the X-linked gene, methyl-CpG-binding protein 2 (MECP2); however, the molecular mechanism by which these mutations mediate the RTT neuropathology remains enigmatic. Specifically, although MeCP2 is known to act as a transcriptional repressor, analyses of the RTT brain at steady-state conditions detected numerous differentially expressed genes, while the changes in transcript levels were mostly subtle. Here we reveal an aberrant global pattern of gene expression, characterized predominantly by higher levels of expression of activity-dependent genes, and anomalous alternative splicing events, specifically in response to neuronal activity in a mouse model for RTT. Notably, the specific splicing modalities of intron retention and exon skipping displayed a significant bias toward increased retained introns and skipped exons, respectively, in the RTT brain compared with the WT brain. Furthermore, these aberrations occur in conjunction with higher seizure susceptibility in response to neuronal activity in RTT mice. Our findings advance the concept that normal MeCP2 functioning is required for fine-tuning the robust and immediate changes in gene transcription and for proper regulation of alternative splicing induced in response to neuronal stimulation.

scholarly journals Positive-feedback, ratiometric biosensor expression improves high-throughput metabolite-producer screening efficiency in yeast

2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Thomas C. Williams ◽  
Xin Xu ◽  
Martin Ostrowski ◽  
Isak S. Pretorius ◽  
Ian T. Paulsen
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
High Throughput ◽  
Positive Feedback ◽  
High Throughput Screening ◽  
Transcriptional Regulator ◽  
Gfp Expression ◽  
Dynamic Regulation ◽  
Wide Range ◽  
Screening Efficiency

Biosensors are valuable and versatile tools in synthetic biology that are used to modulate gene expression in response to a wide range of stimuli. Ligand responsive transcription factors are a class of biosensor that can be used to couple intracellular metabolite concentration with gene expression to enable dynamic regulation and high-throughput metabolite producer screening. We have established the Saccharomyces cerevisiae WAR1 transcriptional regulator and PDR12 promoter as an organic acid biosensor that can be used to detect varying levels of para-hydroxybenzoic acid (PHBA) production from the shikimate pathway and output green fluorescent protein (GFP) expression in response. The dynamic range of GFP expression in response to PHBA was dramatically increased by engineering positive-feedback expression of the WAR1 transcriptional regulator from its target PDR12 promoter. In addition, the noise in GFP expression at the population-level was controlled by normalising GFP fluorescence to constitutively expressed mCherry fluorescence within each cell. These biosensor modifications increased the high-throughput screening efficiency of yeast cells engineered to produce PHBA by 5,000-fold, enabling accurate fluorescence activated cell sorting isolation of producer cells that were mixed at a ratio of 1 in 10,000 with non-producers. Positive-feedback, ratiometric transcriptional regulator expression is likely applicable to many other transcription-factor/promoter pairs used in synthetic biology and metabolic engineering for both dynamic regulation and high-throughput screening applications.

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