scholarly journals De novo design of programmable inducible promoters

2019 ◽  
Vol 47 (19) ◽  
pp. 10452-10463 ◽  
Author(s):  
Xiangyang Liu ◽  
Sanjan T P Gupta ◽  
Devesh Bhimsaria ◽  
Jennifer L Reed ◽  
José A Rodríguez-Martínez ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Throughput Screening ◽  
De Novo ◽  
Vital Role ◽  
Control Of Gene Expression ◽  
Core Motif ◽  
Expression Levels ◽  
Synthetic Promoters ◽  
The Core ◽  
Rich Diversity

Abstract Ligand-responsive allosteric transcription factors (aTF) play a vital role in genetic circuits and high-throughput screening because they transduce biochemical signals into gene expression changes. Programmable control of gene expression from aTF-regulated promoter is important because different downstream effector genes function optimally at different expression levels. However, tuning gene expression of native promoters is difficult due to complex layers of homeostatic regulation encoded within them. We engineered synthetic promoters de novo by embedding operator sites with varying affinities and radically reshaped binding preferences within a minimal, constitutive Escherichia coli promoter. Multiplexed cell-based screening of promoters for three TetR-like aTFs generated with this approach gave rich diversity of gene expression levels, dynamic ranges and ligand sensitivities and were 50- to 100-fold more active over their respective native promoters. Machine learning on our dataset revealed that relative position of the core motif and bases flanking the core motif play an important role in modulating induction response. Our generalized approach yields customizable and programmable aTF-regulated promoters for engineering cellular pathways and enables the discovery of new small molecule biosensors.

scholarly journals Heat shock protein gene expression is higher and more variable with thermal stress and mutation accumulation in Daphnia

2021 ◽  
Author(s):  
Henry Scheffer ◽  
Jeremy Coate ◽  
Eddie K. H. Ho ◽  
Sarah Schaack
Keyword(s):  
Gene Expression ◽  
Thermal Stress ◽  
Heat Shock ◽  
De Novo ◽  
Global Climate ◽  
Mutation Accumulation ◽  
Integrative Approach ◽  
Expression Levels ◽  
Shock Proteins ◽  
Quantify Gene Expression

AbstractUnderstanding the genetic architecture of the stress response and its ability to evolve in response to different stressors requires an integrative approach. Here we quantify gene expression changes in response to two stressors associated with global climate change and habitat loss—heat shock and mutation accumulation. We measure expression levels for two Heat Shock Proteins (HSP90 and HSP60)—members of an important family of conserved molecular chaperones that have been shown to play numerous roles in the cell. While HSP90 assists with protein folding, stabilization, and degradation throughout the cell, HSP60 primarily localizes to the mitochondria and mediates de novo folding and stress-induced refolding of proteins. We perform these assays in Daphnia magna originally collected from multiple genotypes and populations along a latitudinal gradient, which differ in their annual mean, maximum, and range of temperatures. We find significant differences in overall expression between loci (10-fold), in response to thermal stress (~6x increase) and with mutation accumulation (~4x increase). Importantly, stressors interact synergistically to increase gene expression levels when more than one is applied (increasing, on average, >20x). While there is no evidence for differences among the three populations assayed, individual genotypes vary considerably in HSP90 expression. Overall, our results support previous proposals that HSP90 may act as an important buffer against not only heat, but also mutation, and expands this hypothesis to include another member of the gene family acting in a different domain of the cell.

scholarly journals Supervised generative design of regulatory DNA for gene expression control

2021 ◽  
Author(s):  
Jan Zrimec ◽  
Xiaozhi Fu ◽  
Azam Sheikh Muhammad ◽  
Christos Skrekas ◽  
Vykintas Jauniskis ◽  
...  
Keyword(s):  
Gene Expression ◽  
De Novo ◽  
Regulatory Region ◽  
Genomic Data ◽  
Mrna Levels ◽  
Expression Levels ◽  
Expression Control ◽  
Gene Expression Control ◽  
Gene Regulatory ◽  
Regulatory Dna

In order to control gene expression, regulatory DNA variants are commonly designed using random synthetic approaches with mutagenesis and screening. This however limits the size of the designed DNA to span merely a part of a single regulatory region, whereas the whole gene regulatory structure including the coding and adjacent non-coding regions is involved in controlling gene expression. Here, we prototype a deep neural network strategy that models whole gene regulatory structures and generates de novo functional regulatory DNA with prespecified expression levels. By learning directly from natural genomic data, without the need for large synthetic DNA libraries, our ExpressionGAN can traverse the whole sequence-expression landscape to produce sequence variants with target mRNA levels as well as natural-like properties, including over 30% dissimilarity to any natural sequence. We experimentally demonstrate that this generative strategy is more efficient than a mutational one when using purely natural genomic data, as 57% of the newly-generated highly-expressed sequences surpass the expression levels of natural controls. We foresee this as a lucrative strategy to expand our knowledge of gene expression regulation as well as increase expression control in any desired organism for synthetic biology and metabolic engineering applications.

scholarly journals Characterization of five purine riboswitches in cellular and cell-free expression systems

2021 ◽  
Author(s):  
Milca Rachel da Costa Ribeiro Lins ◽  
Graciely Gomes Correa ◽  
Laura Araujo da Silva Amorim ◽  
Rafael Augusto Lopes Franco ◽  
Nathan Vinicius Ribeiro ◽  
...  
Keyword(s):  
Gene Expression ◽  
De Novo ◽  
In Vitro Transcription ◽  
Vitro Assay ◽  
Control Of Gene Expression ◽  
Transcription Terminator ◽  
Variable Expression ◽  
Expression Platform

Bacillus subtilis employs five purine riboswitches for the control of purine de novo synthesis and transport at the transcription level. All of them are formed by a structurally conserved aptamer, and a variable expression platform harboring a rho-independent transcription terminator. In this study, we characterized all five purine riboswitches under the context of active gene expression processes both in vitro and in vivo. We identified transcription pause sites located in the expression platform upstream of the terminator of each riboswitch. Moreover, we defined a correlation between in vitro transcription readthrough and in vivo gene expression. Our in vitro assay demonstrated that the riboswitches operate in the micromolar range of concentration for the cognate metabolite. Our in vivo assay showed the dynamics of control of gene expression by each riboswitch. This study deepens the knowledge of the regulatory mechanism of purine riboswitches.

Synthesis of trans N-Substituted Pyrrolidine Derivatives Bearing 1,2,4-triazole Ring

2021 ◽  
Vol 19 ◽  
Author(s):  
Tangella Nagendra Prasad ◽  
Yeruva Pavankumar Reddy ◽  
Poorna Chandrasekhar Settipalli ◽  
Vadiga Shanthi Kumar ◽  
Eeda Koti Reddy ◽  
...  
Keyword(s):  
Ring Opening ◽  
Biological Activities ◽  
Triazole Ring ◽  
Vital Role ◽  
Epoxide Ring ◽  
Core Motif ◽  
Epoxide Ring Opening ◽  
The Core ◽  
Clinical Drugs ◽  
Benzoyl Chlorides

Background: 1,2,4-triazoles scaffolds display significant biological activities due to hydrogen bonding, solubility, dipole character, and rigidity Objective: The core motif of 1,2,4-triazoles plays a vital role in clinical drugs such as Rizatriptan (anti-migraine), Ribavirin (antiviral), anastrozole (anticancer), etizolam (anxiolytic), estazolam (anticonvulsant), alprazolam (anti-hypnotic), letrozole (aromatase inhibitor), loreclezole (anticonvulsant), trazadone (antidepressant) etc Method: Epoxide ring opening of tert-butyl 6-oxa-3-azabicyclo [3.1.0] hexane-3-carboxylate followed by methylation under basic conditions and de-protection gave the corresponding trans 1-(4-methoxypyrrolidin-3-yl)-1H-1,2,4-triazole hydrochloride salt as the precursor. This precursor on reaction with substituted benzoyl chlorides and benzyl bromides gave the desired amide and amine products Results: A library of 14 N-substituted pyrrolidine derivatives i.e. trans3-methoxy-4-(1H-1,2,4-triazol-1-yl) pyrrolidin-1-yl) (phenyl)methanone and trans 1-benzyl-4-methoxypyrrolidin-3-yl)-1H-1,2,4-triazole were prepared Conclusion: Eight novel amides (6a-h) and six amines (8a-f) derivatives were synthesized using 1-(4-methoxypyrrolidin-3-yl)-1H-1,2,4-triazole 4 salt with substituted benzoyl chlorides and benzyl bromides.

scholarly journals Optimized gene expression from bacterial chromosome by high-throughput integration and screening

2021 ◽  
Vol 7 (7) ◽  
pp. eabe1767
Author(s):  
Tatyana E. Saleski ◽  
Meng Ting Chung ◽  
David N. Carruthers ◽  
Azzaya Khasbaatar ◽  
Katsuo Kurabayashi ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Throughput ◽  
High Throughput Screening ◽  
Efficient Production ◽  
Expression Levels ◽  
Pathway Gene ◽  
Pathway Expression ◽  
Wide Range ◽  
Cell To Cell Variability ◽  
Gene Expression Levels

Chromosomal integration of recombinant genes is desirable compared with expression from plasmids due to increased stability, reduced cell-to-cell variability, and elimination of the need for antibiotics for plasmid maintenance. Here, we present a new approach for tuning pathway gene expression levels via random integration and high-throughput screening. We demonstrate multiplexed gene integration and expression-level optimization for isobutanol production in Escherichia coli. The integrated strains could, with far lower expression levels than plasmid-based expression, produce high titers (10.0 ± 0.9 g/liter isobutanol in 48 hours) and yields (69% of the theoretical maximum). Close examination of pathway expression in the top-performing, as well as other isolates, reveals the complexity of cellular metabolism and regulation, underscoring the need for precise optimization while integrating pathway genes into the chromosome. We expect this method for pathway integration and optimization can be readily extended to a wide range of pathways and chassis to create robust and efficient production strains.

scholarly journals De novo Design of Translational RNA Repressors

2018 ◽  
Author(s):  
Paul D. Carlson ◽  
Cameron J. Glasscock ◽  
Julius B. Lucks
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
De Novo ◽  
Genetic Circuit ◽  
Bacterial Cells ◽  
Control Of Gene Expression ◽  
Sequence Characterization ◽  
Mrna Targets ◽  
Modern Drug ◽  
And Function

ABSTRACTA central goal of synthetic biology is the development of methods for the predictable control of gene expression. RNA is an attractive substrate by which to achieve this goal because the relationship between its sequence, structure, and function is being uncovered with increasing depth. In addition, design approaches that use this relationship are becoming increasingly effective, as evidenced by significant progress in the de novo design of RNA-based gene regulatory mechanisms that activate transcription and translation in bacterial cells. However, the design of synthetic RNA mechanisms that are efficient and versatile repressors of gene expression has lagged, despite their importance for gene regulation and genetic circuit construction. We address this gap by developing two new classes of RNA regulators, toehold repressors and looped antisense oligonucleotides (LASOs), that repress translation of a downstream gene in response to an arbitrary input RNA sequence. Characterization studies show that these designed RNAs robustly repress translation, are highly orthogonal, and can be multiplexed with translational activators. We show that our LASO design can repress endogenous mRNA targets and distinguish between closely-related genes with a high degree of specificity and predictability. These results demonstrate significant yet easy-to-implement improvements in the design of synthetic RNA repressors for synthetic biology, and point more broadly to design principles for repressive RNA interactions relevant to modern drug design.

scholarly journals Restoration of the GTPase activity of Gαo mutants by Zn2+ in GNAO1 encephalopathy models

2021 ◽  
Author(s):  
Vladimir Katanaev ◽  
Yonika Larasati ◽  
Mikhail Savitsky ◽  
Alexey Koval ◽  
Gonzalo Solis
Keyword(s):  
G Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
De Novo ◽  
Motor Dysfunction ◽  
Cellular Interactions ◽  
Gtpase Activity ◽  
Gtp Hydrolysis ◽  
The Core ◽  
Gtp Binding

Abstract GNAO1 encephalopathy is a rare pediatric disease characterized by motor dysfunction, developmental delay, and epileptic seizures1-3. De novo point mutations in the gene encoding Gαo, the major neuronal G protein, lie at the core of this dominant genetic malady4. Half of the clinical case mutations fall on codons Gly203, Arg209, or Glu246 near the GTP binding/hydrolysis pocket of Gαo1-3. We here show that these pathologic mutations strongly speed up GTP uptake and inactivate GTP hydrolysis by Gαo, resulting in constitutive GTP binding by the G protein. Molecular dynamics simulations indicate that the mutations cause displacement of Gln205, the key to GTP hydrolysis. Decreased interactions with cellular partners including RGS19 suggest that despite the enhanced GTP residence, the mutants fail to fully adopt the activated conformation and thus transmit the signal. Through a high-throughput screening of approved drugs aiming at correction of this core biochemical dysfunction, we identify zinc pyrithione and Zn2+ ions as agents restoring the active conformation, GTPase activity, and cellular interactions of the encephalopathy mutants, with a negligible effect on wild type Gαo. We describe a Drosophila model of GNAO1 encephalopathy and show that dietary zinc supplementation restores the motor function and longevity of the mutant flies. With zinc supplements frequently recommended for diverse human neurological conditions, our work spanning from identification of the core biochemical defect in Gαo mutants and cellular interactions analysis to high-throughput screening and animal validation of the deficiency-correcting drug defines the potential therapy for GNAO1 encephalopathy patients.

scholarly journals Orthogonal Control of Gene Expression in Plants Using Synthetic Promoters and CRISPR-Based Transcription Factors

2022 ◽  
Author(s):  
Shaunak Kar ◽  
Yogendra Bordiya ◽  
Nestor Rodriguez ◽  
Junghyun Kim ◽  
Elizabeth C Gardner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
System Level ◽  
Plant Signaling ◽  
Promoter Elements ◽  
Control Of Gene Expression ◽  
Form Complex ◽  
Pol Ii ◽  
Synthetic Promoters ◽  
Pol Iii

Abstract Background: The construction and application of synthetic genetic circuits is frequently improved if gene expression can be orthogonally controlled, relative to the host. In plants, orthogonality can be achieved via the use of CRISPR-based transcription factors that are programmed to act on natural or synthetic promoters. The construction of complex gene circuits can require multiple, orthogonal regulatory interactions, and this in turn requires that the full programmability of CRISPR elements be adapted to non-natural and non-standard promoters that have few constraints on their design. Therefore, we have developed synthetic promoter elements in which regions upstream of the minimal 35S CaMV promoter are designed from scratch to interact via programmed gRNAs with dCas9 fusions that allow activation of gene expression. Results: A panel of three, mutually orthogonal promoters that can be acted on by artificial gRNAs bound by CRISPR regulators were designed. Guide RNA expression targeting these promoters was in turn controlled by either Pol III (U6) or ethylene-inducible Pol II promoters, implementing for the first time a fully artificial Orthogonal Control System (OCS). Following demonstration of the complete orthogonality of the designs, the OCS was tied to cellular metabolism by putting gRNA expression under the control of an endogenous plant signaling molecule, ethylene. The ability to form complex circuitry was demonstrated via the ethylene-driven, ratiometric expression of fluorescent proteins in single plants. Conclusions: The design of synthetic promoters is highly generalizable to large tracts of sequence space, allowing Orthogonal Control Systems of increasing complexity to potentially be generated at will. The ability to tie in several different basal features of plant molecular biology (Pol II and Pol III promoters, ethylene regulation) to the OCS demonstrates multiple opportunities for engineering at the system level. Moreover, given the fungibility of the core 35S CaMV promoter elements, the derived synthetic promoters can potentially be utilized across a variety of plant species.

scholarly journals Orthogonal control of gene expression in plants using synthetic promoters and CRISPR-based transcription factors

2021 ◽  
Author(s):  
Shaunak Kar ◽  
Yogendra Bordiya ◽  
Nestor Rodriguez ◽  
Jungyun Kim ◽  
Elizabeth C Gardner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
System Level ◽  
Plant Signaling ◽  
Promoter Elements ◽  
Control Of Gene Expression ◽  
Form Complex ◽  
Pol Ii ◽  
Synthetic Promoters ◽  
Pol Iii

Background: The construction and application of synthetic genetic circuits is frequently improved if gene expression can be orthogonally controlled, relative to the host. In plants, orthogonality can be achieved via the use of CRISPR-based transcription factors that are programmed to act on natural or synthetic promoters. The construction of complex gene circuits can require multiple, orthogonal regulatory interactions, and this in turn requires that the full programmability of CRISPR elements be adapted to non-natural and non-standard promoters that have few constraints on their design. Therefore, we have developed synthetic promoter elements in which regions upstream of the minimal 35S CaMV promoter are designed from scratch to interact via programmed gRNAs with dCas9 fusions that allow activation of gene expression. Results: A panel of three, mutually orthogonal promoters that can be acted on by artificial gRNAs bound by CRISPR regulators were designed. Guide RNA expression targeting these promoters was in turn controlled by either Pol III (U6) or ethylene-inducible Pol II promoters, implementing for the first time a fully artificial Orthogonal Control System (OCS). Following demonstration of the complete orthogonality of the designs, the OCS was tied to cellular metabolism by putting gRNA expression under the control of an endogenous plant signaling molecule, ethylene. The ability to form complex circuitry was demonstrated via the ethylene-driven, ratiometric expression of fluorescent proteins in single plants. Conclusions: The design of synthetic promoters is highly generalizable to large tracts of sequence space, allowing Orthogonal Control Systems of increasing complexity to potentially be generated at will. The ability to tie in several different basal features of plant molecular biology (Pol II and Pol III promoters, ethylene regulation) to the OCS demonstrates multiple opportunities for engineering at the system level. Moreover, given the fungibility of the core 35S CaMV promoter elements, the derived synthetic promoters can potentially be utilized across a variety of plant species.

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