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 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.

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 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 Development and Characterization of a Subtilin-Regulated Expression System in Bacillus subtilis: Strict Control of Gene Expression by Addition of Subtilin

2005 ◽  
Vol 71 (12) ◽  
pp. 8818-8824 ◽  
Author(s):  
Roger S. Bongers ◽  
Jan-Willem Veening ◽  
Maarten Van Wieringen ◽  
Oscar P. Kuipers ◽  
Michiel Kleerebezem
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Fluorescent Protein ◽  
Expression System ◽  
Functional Characterization ◽  
Industrial Applications ◽  
Wild Type ◽  
Control Of Gene Expression ◽  
Regulated Gene Expression

ABSTRACT A system for subtilin-regulated gene expression (SURE) in Bacillus subtilis that is based on the regulatory module involved in cell-density-dependent control of the production of subtilin is described. An integration vector for introduction of the essential sensor-regulator couple spaRK into the amyE locus of the B. subtilis chromosome and a B. subtilis 168-derived production host in which the spaRK genes were functionally introduced were constructed. Furthermore, several expression plasmids harboring the subtilin-inducible wild-type spaS promoter or a mutated derivative of this promoter were constructed, which facilitated both transcriptional and translational promoter-gene fusions. Functional characterization of both spaS promoters and the cognate expression host could be performed by controlled overproduction of the β-glucuronidase (GusA) and green fluorescent protein (GFP) reporters. Both spaS promoters exhibited very low levels of basal expression, while extremely high levels of expression were observed upon induction with subtilin. Moreover, the level of expression depended directly on the amount of inducer (subtilin) used. The wild-type spaS promoter appeared to be more strictly controlled by the addition of subtilin, while the highest levels of expression were obtained when the mutated spaS promoter was used. Induction by subtilin led to 110- and 80-fold increases in GusA activity for the spaS promoter and its mutant derivative, respectively. Since the SURE system has attractive functional characteristics, including promoter silence under noninducing conditions and a controlled and high level of expression upon induction, and since it is not subject to catabolite control, we anticipate that it can provide a suitable expression system for various scientific and industrial applications.

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 GAPPromoter Library for Fine-Tuning of Gene Expression in Pichia pastoris

2011 ◽  
Vol 77 (11) ◽  
pp. 3600-3608 ◽  
Author(s):  
Xiulin Qin ◽  
Jiangchao Qian ◽  
Gaofeng Yao ◽  
Yingping Zhuang ◽  
Siliang Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pichia Pastoris ◽  
Fine Tuning ◽  
Promoter Strength ◽  
Control Of Gene Expression ◽  
Content Type ◽  
Expression Levels ◽  
Precise Control ◽  
Promoter Library

ABSTRACTA library of engineered promoters of various strengths is a useful genetic tool that enables the fine-tuning and precise control of gene expression across a continuum of broad expression levels. The methylotrophic yeastPichia pastorisis a well-established expression host with a large academic and industrial user base. To facilitate manipulation of gene expression spanning a wide dynamic range inP. pastoris, we created a functional promoter library through mutagenesis of the constitutiveGAPpromoter. Using yeast-enhanced green fluorescent protein (yEGFP) as the reporter, 33 mutants were chosen to form the functional promoter library. The 33 mutants spanned an activity range between ∼0.6% and 19.6-fold of the wild-type promoter activity with an almost linear fluorescence intensity distribution. After an extensive characterization of the library, the broader applicability of the results obtained with the yEGFP reporter was confirmed using two additional reporters (β-galactosidase and methionine adenosyltransferase [MAT]) at the transcription and enzyme activity levels. Furthermore, the utility of the promoter library was tested by investigating the influence of heterologous MAT gene expression levels on cell growth andS-adenosylmethionine (SAM) production. The extensive characterization of the promoter strength enabled identification of the optimal MAT activity (around 1.05 U/mg of protein) to obtain maximal volumetric SAM production. The promoter library permits precise control of gene expression and quantitative assessment that correlates gene expression level with physiologic parameters. Thus, it is a useful toolbox for both basic and applied research inP. pastoris.

scholarly journals Fungal Light-Oxygen-Voltage Domains for Optogenetic Control of Gene Expression and Flocculation in Yeast

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Francisco Salinas ◽  
Vicente Rojas ◽  
Verónica Delgado ◽  
Javiera López ◽  
Eduardo Agosin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Heterologous Protein ◽  
Dynamic Range ◽  
Industrial Applications ◽  
Yeast Cells ◽  
Heterologous Protein Expression ◽  
Control Of Gene Expression ◽  
Content Type ◽  
Chemical Inducers

ABSTRACT Optogenetic switches permit accurate control of gene expression upon light stimulation. These synthetic switches have become a powerful tool for gene regulation, allowing modulation of customized phenotypes, overcoming the obstacles of chemical inducers, and replacing their use by an inexpensive resource: light. In this work, we implemented FUN-LOV, an optogenetic switch based on the photon-regulated interaction of WC-1 and VVD, two LOV (light-oxygen-voltage) blue-light photoreceptors from the fungus Neurospora crassa. When tested in yeast, FUN-LOV yields light-controlled gene expression with exquisite temporal resolution and a broad dynamic range of over 1,300-fold, as measured by a luciferase reporter. We also tested the FUN-LOV switch for heterologous protein expression in Saccharomyces cerevisiae, where Western blot analysis confirmed strong induction upon light stimulation, surpassing by 2.5 times the levels achieved with a classic GAL4/galactose chemical-inducible system. Additionally, we utilized FUN-LOV to control the ability of yeast cells to flocculate. Light-controlled expression of the flocculin-encoding gene FLO1, by the FUN-LOV switch, yielded flocculation in light (FIL), whereas the light-controlled expression of the corepressor TUP1 provided flocculation in darkness (FID). Altogether, the results reveal the potential of the FUN-LOV optogenetic switch to control two biotechnologically relevant phenotypes such as heterologous protein expression and flocculation, paving the road for the engineering of new yeast strains for industrial applications. Importantly, FUN-LOV’s ability to accurately manipulate gene expression, with a high temporal dynamic range, can be exploited in the analysis of diverse biological processes in various organisms. IMPORTANCE Optogenetic switches are molecular devices which allow the control of different cellular processes by light, such as gene expression, providing a versatile alternative to chemical inducers. Here, we report a novel optogenetic switch (FUN-LOV) based on the LOV domain interaction of two blue-light photoreceptors (WC-1 and VVD) from the fungus N. crassa. In yeast cells, FUN-LOV allowed tight regulation of gene expression, with low background in darkness and a highly dynamic and potent control by light. We used FUN-LOV to optogenetically manipulate, in yeast, two biotechnologically relevant phenotypes, heterologous protein expression and flocculation, resulting in strains with potential industrial applications. Importantly, FUN-LOV can be implemented in diverse biological platforms to orthogonally control a multitude of cellular processes.

scholarly journals FUN-LOV: Fungal LOV domains for optogenetic control of gene expression and flocculation in yeast

2018 ◽  
Author(s):  
Francisco Salinas ◽  
Vicente Rojas ◽  
Verónica Delgado ◽  
Javiera López ◽  
Eduardo Agosin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Heterologous Protein ◽  
Dynamic Range ◽  
Industrial Applications ◽  
Yeast Cells ◽  
Heterologous Protein Expression ◽  
Control Of Gene Expression ◽  
Chemical Inducers ◽  
Cellular Processes

AbstractOptogenetic switches permit accurate control of gene expression upon light stimulation. These synthetic switches have become a powerful tool for gene regulation, allowing modulation of customized phenotypes, overcoming the obstacles of chemical inducers and replacing their use by an inexpensive resource: light. In this work, we implemented FUN-LOV; an optogenetic switch based on the photon-regulated interaction of WC-1 and VVD, two LOV (Light Oxygen Voltage) blue-light photoreceptors from the fungus Neurospora crassa. When tested in yeast, FUN-LOV yields light-controlled gene expression with exquisite temporal resolution, and a broad dynamic range of over 1300-fold, as measured by a luciferase reporter. We also tested the FUN-LOV switch for heterologous protein expression in Saccharomyces cerevisiae, where Western blot analysis confirmed strong induction upon light stimulation, surpassing by 2.5 times the levels achieved with a classic GAL4/galactose chemical inducible system. Additionally, we utilized FUN-LOV to control the ability of yeast cells to flocculate. Light-controlled expression of the flocculin encoding gene FLO1, by the FUN-LOV switch, yielded Flocculation in Light (FIL), whereas the light-controlled expression of the co-repressor TUP1 provided Flocculation in Darkness (FID). Overall, the results revealed the potential of the FUN-LOV optogenetic switch to control two biotechnologically relevant phenotypes such as heterologous protein expression and flocculation, paving the road for the engineering of new yeast strains for industrial applications. Importantly, FUN-LOV’ s ability to accurately manipulate gene expression, with a high-temporal dynamic range, can be exploited in the analysis of diverse biological processes in various organisms.ImportanceOptogenetic switches are molecular devices which allow the control of different cellular processes by light, such as gene expression, providing a versatile alternative to chemical inducers. Herein, we report a novel optogenetic switch (FUN-LOV) based on the LOV-domain interaction of two blue-light photoreceptors (WC-1 and VVD) from the fungus N. crassa. In yeast cells, FUN-LOV allowed tight regulation of gene expression, with low background in darkness and a highly dynamic and potent control by light. We used FUN-LOV to optogenetically manipulate, in yeast, two biotechnologically relevant phenotypes: heterologous protein expression and flocculation, resulting in strains with potential industrial applications. Importantly, FUN-LOV can be implemented in diverse biological platforms to orthogonally control a multitude of cellular processes.

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