scholarly journals Control of Gene Expression With Quercetin-Responsive Modular Circuits

2021 ◽  
Vol 9 ◽  
Author(s):  
Fernanda Miyuki Kashiwagi ◽  
Brenno Wendler Miranda ◽  
Fabio de Oliveira Pedrosa ◽  
Emanuel Maltempi de Souza ◽  
Marcelo Müller-Santos
Keyword(s):  
Gene Expression ◽  
Regulatory System ◽  
Genetic Circuits ◽  
Plant Metabolites ◽  
Control Of Gene Expression ◽  
E Coli ◽  
Ribosome Binding Sites ◽  
Flavonoid Quercetin ◽  
Quercetin Concentration ◽  
Different Levels

Control of gene expression is crucial for several biotechnological applications, especially for implementing predictable and controllable genetic circuits. Such circuits are often implemented with a transcriptional regulator activated by a specific signal. These regulators should work independently of the host machinery, with low gratuitous induction or crosstalk with host components. Moreover, the signal should also be orthogonal, recognized only by the regulator with minimal interference with the host operation. In this context, transcriptional regulators activated by plant metabolites as flavonoids emerge as candidates to control gene expression in bacteria. However, engineering novel circuits requires the characterization of the genetic parts (e.g., genes, promoters, ribosome binding sites, and terminators) in the host of interest. Therefore, we decomposed the QdoR regulatory system of B. subtilis, responsive to the flavonoid quercetin, and reassembled its parts into genetic circuits programmed to have different levels of gene expression and noise dependent on the concentration of quercetin. We showed that only one of the promoters regulated by QdoR worked well in E. coli, enabling the construction of other circuits induced by quercetin. The QdoR expression was modulated with constitutive promoters of different transcriptional strengths, leading to low expression levels when QdoR was highly expressed and vice versa. E. coli strains expressing high and low levels of QdoR were mixed and induced with the same quercetin concentration, resulting in two stable populations expressing different levels of their gene reporters. Besides, we demonstrated that the level of QdoR repression generated different noise levels in gene expression dependent on the concentration of quercetin. The circuits presented here can be exploited in applications requiring adjustment of gene expression and noise using a highly available and natural inducer as quercetin.

scholarly journals High-performance chemical- and light-inducible recombinases in mammalian cells and mice

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Benjamin H. Weinberg ◽  
Jang Hwan Cho ◽  
Yash Agarwal ◽  
N. T. Hang Pham ◽  
Leidy D. Caraballo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
High Performance ◽  
Genome Engineering ◽  
Genetic Circuits ◽  
Control Of Gene Expression ◽  
Expression Control ◽  
Gene Expression Control ◽  
High Performance Systems ◽  
Spatiotemporal Control

Abstract Site-specific DNA recombinases are important genome engineering tools. Chemical- and light-inducible recombinases, in particular, enable spatiotemporal control of gene expression. However, inducible recombinases are scarce due to the challenge of engineering high performance systems, thus constraining the sophistication of genetic circuits and animal models that can be created. Here we present a library of >20 orthogonal inducible split recombinases that can be activated by small molecules, light and temperature in mammalian cells and mice. Furthermore, we engineer inducible split Cre systems with better performance than existing systems. Using our orthogonal inducible recombinases, we create a genetic switchboard that can independently regulate the expression of 3 different cytokines in the same cell, a tripartite inducible Flp, and a 4-input AND gate. We quantitatively characterize the inducible recombinases for benchmarking their performances, including computation of distinguishability of outputs. This library expands capabilities for multiplexed mammalian gene expression control.

scholarly journals A modular toolset for electrogenetics

2021 ◽  
Author(s):  
Joshua M Lawrence ◽  
Yutong Yin ◽  
Paolo Bombelli ◽  
Alberto Scarampi ◽  
Marko Storch ◽  
...  
Keyword(s):  
Gene Expression ◽  
Industrial Applications ◽  
Electrochemical Activation ◽  
Genetic Circuits ◽  
Control Of Gene Expression ◽  
Promoter Library ◽  
Redox Responsive ◽  
Electrochemical Control ◽  
Redox Molecules ◽  
New Applications

Synthetic biology research and its industrial applications rely on the deterministic spatiotemporal control of gene expression. Recently, electrochemical control of gene expression has been demonstrated in electrogenetic systems (redox-responsive promoters used alongside redox inducers and an electrode), allowing for the direct integration of electronics with complex biological processes for a variety of new applications. However, the use of electrogenetic systems is limited by poor activity, tunability and standardisation. Here, we have developed a variety of genetic and electrochemical tools that facilitate the design and vastly improve the performance of electrogenetic systems. We developed a strong, unidirectional, redox-responsive promoter before deriving a mutant promoter library with a spectrum of strengths. We then constructed genetic circuits with these parts and demonstrated their activation by multiple classes of redox molecules. Finally, we demonstrated electrochemical activation of gene expression in aerobic conditions utilising a novel, modular bioelectrochemical device. This toolset provides researchers with all the elements needed to design and build optimised electrogenetic systems for specific applications.

scholarly journals Tailor-made sRNAs: a toolbox to control metabolic targets

2019 ◽  
Author(s):  
Patrícia Apura ◽  
Alexandra Peregrina ◽  
Margarida Saramago ◽  
Sandra C. Viegas ◽  
Sandra M. Carvalho ◽  
...  
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
Regulatory System ◽  
High Specificity ◽  
Reporter Genes ◽  
Genetic Circuits ◽  
Modulation Of Gene Expression ◽  
Industrial Level ◽  
Industrial Needs ◽  
Modular Composition

SummaryPseudomonas putida is a highly attractive production system for industrial needs. Modulation of gene expression is an urgent need to redesign P. putida metabolism for its improvement as biocatalyst at industrial level. We report the construction of a small RNA-based system with potential to be used for different purposes in synthetic biology. Due to their modular composition, design facilities and ability in tuning gene expression, sRNAs constitute a powerful tool in genetic and metabolic engineering. In the toolbox presented here, the synthetic sRNA is specifically directed to any region of a chosen target. The expression of the synthetic sRNAs is shown to differentially modulate the level of endogenous and reporter genes. The antisense interaction of the sRNA with the mRNA results in different outcomes. Depending on the particularity of each sRNA-target mRNA pair, we managed to demonstrate the duality of this system, able either to repress or overexpress a given gene. This system combines a high specificity with a wide applicability due to its ability to modulate the expression of virtually any given gene. By plugging-in and -out genetic circuits, this tailor-made regulatory system can be used to redesign P. putida metabolism, fulfilling an important industrial gap in synthetic biology.

scholarly journals Rational Design of RNA Structures that Predictably Tune Eukaryotic Gene Expression

2017 ◽  
Author(s):  
Tim Weenink ◽  
Robert M. McKiernan ◽  
Tom Ellis
Keyword(s):  
Gene Expression ◽  
Rational Design ◽  
Rna Structures ◽  
Genetic Circuits ◽  
Protein Coding ◽  
Eukaryotic Gene Expression ◽  
Eukaryotic Gene ◽  
Engineering Projects ◽  
Fine Tune ◽  
Different Levels

AbstractPredictable tuning of gene expression is essential for engineering genetic circuits and for optimising enzyme levels in metabolic engineering projects. In bacteria, gene expression can be tuned at the stage of transcription, by exchanging the promoter, or at stage of translation by altering the ribosome binding site sequence. In eukaryotes, however, only promoter exchange is regularly used, as the tools to modulate translation are lacking. Working in S. cerevisiae yeast, we here describe how hairpin RNA structures inserted into the 5’ untranslated region (5’UTR) of mRNAs can be used to tune expression levels by altering the efficiency of translation initiation. We demonstrate a direct link between the calculated free energy of folding in the 5’UTR and protein abundance, and show that this enables rational design of hairpin libraries that give predicted expression outputs. Our approach is modular, working with different promoters and protein coding sequences, and it outperforms promoter mutation as a way to predictably generate a library where a protein is induced to express at a range of different levels. With this tool, computational RNA sequence design can be used to predictably fine-tune protein production, providing a new way to modulate gene expression in eukaryotes.

Engineered protein switches for exogenous control of gene expression

2020 ◽  
Vol 48 (5) ◽  
pp. 2205-2212
Author(s):  
Shaun Spisak ◽  
Marc Ostermeier
Keyword(s):  
Gene Expression ◽  
Domain Swapping ◽  
Signaling Proteins ◽  
Genetic Circuits ◽  
Control Of Gene Expression ◽  
Regulate Gene Expression ◽  
Substrate Specificities ◽  
Large Pool ◽  
Domain Insertion ◽  
Exogenous Control

There is an ongoing need in the synthetic biology community for novel ways to regulate gene expression. Protein switches, which sense biological inputs and respond with functional outputs, represent one way to meet this need. Despite the fact that there is already a large pool of transcription factors and signaling proteins available, the pool of existing switches lacks the substrate specificities and activities required for certain applications. Therefore, a large number of techniques have been applied to engineer switches with novel properties. Here we discuss some of these techniques by broadly organizing them into three approaches. We show how novel switches can be created through mutagenesis, domain swapping, or domain insertion. We then briefly discuss their use as biosensors and in complex genetic circuits.

scholarly journals Intra- and Interspecies Regulation of Gene Expression by Actinobacillus actinomycetemcomitansLuxS

2001 ◽  
Vol 69 (12) ◽  
pp. 7625-7634 ◽  
Author(s):  
Karen P. Fong ◽  
Whasun O. Chung ◽  
Richard J. Lamont ◽  
Donald R. Demuth
Keyword(s):  
Gene Expression ◽  
Conditioned Medium ◽  
Iron Acquisition ◽  
Regulation Of Gene Expression ◽  
Fusobacterium Nucleatum ◽  
Homoserine Lactone ◽  
Periodontal Pathogen ◽  
Control Of Gene Expression ◽  
E Coli ◽  
Sterile Medium

ABSTRACT The cell density-dependent control of gene expression is employed by many bacteria for regulating a variety of physiological functions, including the generation of bioluminescence, sporulation, formation of biofilms, and the expression of virulence factors. Although periodontal organisms do not appear to secrete acyl-homoserine lactone signals, several species, e.g., Porphyromonas gingivalis,Prevotella intermedia, and Fusobacterium nucleatum, have recently been shown to secrete a signal related to the autoinducer II (AI-2) of the signal system 2 pathway inVibrio harveyi. Here, we report that the periodontal pathogen Actinobacillus actinomycetemcomitans expresses a homolog of V. harveyi luxS and secretes an AI-2-like signal. Cell-free conditioned medium from A. actinomycetemcomitans or from a recombinant Escherichia coli strain (E. coli AIS) expressing A. actinomycetemcomitans luxS induced luminescence in V. harveyi BB170 >200-fold over controls. AI-2 levels peaked in mid-exponential-phase cultures of A. actinomycetemcomitans and were significantly reduced in late-log- and stationary-phase cultures. Incubation of early-log-phaseA. actinomycetemcomitans cells with conditioned medium from A. actinomycetemcomitans or from E. coli AIS resulted in a threefold induction of leukotoxic activity and a concomitant increase in leukotoxin polypeptide. In contrast, no increase in leukotoxin expression occurred when cells were exposed to sterile medium or to conditioned broth from E. coli AIS−, a recombinant strain in whichluxS was insertionally inactivated. A. actinomycetemcomitans AI-2 also induced expression ofafuA, encoding a periplasmic iron transport protein, approximately eightfold, suggesting that LuxS-dependent signaling may play a role in the regulation of iron acquisition by A. actinomycetemcomitans. Finally, A. actinomycetemcomitans AI-2 added in transcomplemented a luxS knockout mutation in P. gingivalis by modulating the expression of theluxS-regulated genes uvrB andhasF in this organism. Together, these results suggest that LuxS-dependent signaling may modulate aspects of virulence and the uptake of iron by A. actinomycetemcomitans and induce responses in other periodontal organisms in mixed-species oral biofilm.

scholarly journals A Synthetic Transcription Platform for Programmable Gene Expression in Mammalian Cells

2020 ◽  
Author(s):  
William C.W. Chen ◽  
Leonid Gaidukov ◽  
Ming-Ru Wu ◽  
Jicong Cao ◽  
Gigi C.G. Choi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Interferon Gamma ◽  
Mammalian Cells ◽  
Cell Types ◽  
Regulatory Elements ◽  
Genetic Circuits ◽  
Control Of Gene Expression ◽  
Transcription System ◽  
Multiple Cell ◽  
Cellular Phenotypes

Precise, scalable, and sustainable control of genetic and cellular activities in mammalian cells is key to developing precision therapeutics and smart biomanufacturing. We created a highly tunable, modular, versatile CRISPR-based synthetic transcription system for the programmable control of gene expression and cellular phenotypes in mammalian cells. Genetic circuits consisting of well-characterized libraries of guide RNAs, binding motifs of synthetic operators, transcriptional activators, and additional genetic regulatory elements expressed mammalian genes in a highly predictable and tunable manner. We demonstrated the programmable control of reporter genes episomally and chromosomally, with up to 25-fold more EF1[alpha]; promoter activity, in multiple cell types. We used these circuits to program secretion of human monoclonal antibodies and to control T cell effector function marked by interferon-[gamma] production. Antibody titers and interferon-[gamma]; concentrations were significantly correlated with synthetic promoter strengths, providing a platform for programming gene expression and cellular function for biological, biomanufacturing, and biomedical applications.

scholarly journals High-performance chemical and light-inducible recombinases in mammalian cells and mice

2019 ◽  
Author(s):  
Benjamin H. Weinberg ◽  
Jang Hwan Cho ◽  
Yash Agarwal ◽  
N. T. Hang Pham ◽  
Leidy D. Caraballo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
High Performance ◽  
Genome Engineering ◽  
Dynamic Range ◽  
Signal To Noise Ratio ◽  
Genetic Circuits ◽  
Control Of Gene Expression ◽  
Gene Expression Control ◽  
High Performance Systems

ABSTRACTSite-specific DNA recombinases are some of the most powerful genome engineering tools in biology. Chemical and light-inducible recombinases, in particular, enable spatiotemporal control of gene expression. However, the availability of inducible recombinases is scarce due to the challenge of engineering high performance systems with low basal activity and sufficient dynamic range. This limitation constrains the sophistication of genetic circuits and animal models that can be created. To expand the number of available inducible recombinases, here we present a library of >20 orthogonal split recombinases that can be inducibly dimerized and activated by various small molecules, light, and temperature in mammalian cells and mice.Furthermore, we have engineered inducible split Cre systems with better performance than existing inducible Cre systems. Using our orthogonal inducible recombinases, we created a “genetic switchboard” that can independently regulate the expression of 3 different cytokines in the same cell. To demonstrate novel capability with our split recombinases, we created a tripartite inducible Flp and a 4-Input AND gate. We have performed extensive quantitative characterization of the inducible recombinases for benchmarking their performances, including computation of distinguishability of outputs in terms of signal-to-noise ratio (SNR). To facilitate sharing of this set of reagents, we have deposited our library to Addgene. This library thus significantly expands capabilities for precise and multiplexed mammalian gene expression control.

scholarly journals LED control of gene expression in a nanobiosystem composed of metallic nanoparticles and a genetically modified E. coli strain

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Hossein Alishah Aratboni ◽  
Nahid Rafiei ◽  
Larousse Khosravi Khorashad ◽  
Albert Isaac Lerma-Escalera ◽  
Francisco de Jesús Balderas-Cisneros ◽  
...  
Keyword(s):  
Gene Expression ◽  
Metallic Nanoparticles ◽  
Protein Production ◽  
Culture Media ◽  
Genetically Modified ◽  
External Control ◽  
Control Of Gene Expression ◽  
E Coli ◽  
Genetically Modified Microorganisms ◽  
Non Invasive

Abstract Background Within the last decade, genetic engineering and synthetic biology have revolutionized society´s ability to mass-produce complex biological products within genetically-modified microorganisms containing elegantly designed genetic circuitry. However, many challenges still exist in developing bioproduction processes involving genetically modified microorganisms with complex or multiple gene circuits. These challenges include the development of external gene expression regulation methods with the following characteristics: spatial–temporal control and scalability, while inducing minimal permanent or irreversible system-wide conditions. Different stimuli have been used to control gene expression and mitigate these challenges, and they can be characterized by the effect they produce in the culture media conditions. Invasive stimuli that cause permanent, irreversible changes (pH and chemical inducers), non-invasive stimuli that cause partially reversible changes (temperature), and non-invasive stimuli that cause reversible changes in the media conditions (ultrasound, magnetic fields, and light). Methods Opto-control of gene expression is a non-invasive external trigger that complies with most of the desired characteristics of an external control system. However, the disadvantage relies on the design of the biological photoreceptors and the necessity to design them to respond to a different wavelength for every bioprocess needed to be controlled or regulated in the microorganism. Therefore, this work proposes using biocompatible metallic nanoparticles as external controllers of gene expression, based on their ability to convert light into heat and the capacity of nanotechnology to easily design a wide array of nanostructures capable of absorbing light at different wavelengths and inducing plasmonic photothermal heating. Results Here, we designed a nanobiosystem that can be opto-thermally triggered using LED light. The nanobiosystem is composed of biocompatible gold nanoparticles and a genetically modified E. coli with a plasmid that allows mCherry fluorescent protein production at 37 °C in response to an RNA thermometer. Conclusions The LED-triggered photothermal protein production system here designed offers a new, cheaper, scalable switchable method, non-destructive for living organisms, and contribute toward the evolution of bioprocess production systems.

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