scholarly journals Engineering a circular riboregulator inEscherichia coli

2014 ◽  
Author(s):  
William Rostain ◽  
Shensi Shen ◽  
Teresa Cordero ◽  
Guillermo Rodrigo ◽  
Alfonso Jaramillo
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Dynamic Range ◽  
Circular Rnas ◽  
Antibiotic Resistance Marker ◽  
Fluorescent Reporter ◽  
Group I ◽  
Circular Molecule ◽  
Self Splicing ◽  
Activate Gene

Circular RNAs have recently been shown to be important gene expression regulators in mammalian cells. However, their role in prokaryotes remains elusive. Here, we engineered a synthetic riboregulator that self-splice to produce a circular molecule, exploiting group I permuted intron-exon (PIE) sequences. We demonstrated that the resulting circular riboregulator can activate gene expression, showing increased dynamic range compared to the linear form. We characterized the system with a fluorescent reporter and with an antibiotic resistance marker. Thanks to the increased regulatory activity by higher stability, isolation due to self-splicing, and modularity of PIE, we envisage engineered circular riboregulators in further synthetic biology applications.

scholarly journals Engineering a Circular Riboregulator in Escherichia coli

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
William Rostain ◽  
Shensi Shen ◽  
Teresa Cordero ◽  
Guillermo Rodrigo ◽  
Alfonso Jaramillo
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Messenger Rna ◽  
Noncoding Rna ◽  
Circular Rna ◽  
Circular Rnas ◽  
Fluorescent Reporter ◽  
Small Noncoding Rna ◽  
E Coli ◽  
Group I

RNAs of different shapes and sizes, natural or synthetic, can regulate gene expression in prokaryotes and eukaryotes. Circular RNAs have recently appeared to be more widespread than previously thought, but their role in prokaryotes remains elusive. Here, by inserting a riboregulatory sequence within a group I permuted intron-exon ribozyme, we created a small noncoding RNA that self-splices to produce a circular riboregulator in Escherichia coli. We showed that the resulting riboregulator can trans-activate gene expression by interacting with a cis-repressed messenger RNA. We characterized the system with a fluorescent reporter and with an antibiotic resistance marker, and we modeled this novel posttranscriptional mechanism. This first reported example of a circular RNA regulating gene expression in E. coli adds to an increasing repertoire of RNA synthetic biology parts, and it highlights that topological molecules can play a role in the case of prokaryotic regulation.

scholarly journals Evaluating Group I Intron Catalytic Efficiency in Mammalian Cells

1999 ◽  
Vol 19 (10) ◽  
pp. 6479-6487 ◽  
Author(s):  
Meredith B. Long ◽  
Bruce A. Sullenger
Keyword(s):  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Catalytic Efficiency ◽  
Human Cells ◽  
The Self ◽  
Group I Introns ◽  
Cellular Environment ◽  
Group I ◽  
Self Splicing ◽  
The Impact

ABSTRACT Recent reports have demonstrated that the group I ribozyme fromTetrahymena thermophila can performtrans-splicing reactions to repair mutant RNAs. For therapeutic use, such ribozymes must function efficiently when transcribed from genes delivered to human cells, yet it is unclear how group I splicing reactions are influenced by intracellular expression of the ribozyme. Here we evaluate the self-splicing efficiency of group I introns from transcripts expressed by RNA polymerase II in human cells to directly measure ribozyme catalysis in a therapeutically relevant setting. Intron-containing expression cassettes were transfected into a human cell line, and RNA transcripts were analyzed for intron removal. The percentage of transcripts that underwent self-splicing ranged from 0 to 50%, depending on the construct being tested. Thus, self-splicing activity is supported in the mammalian cellular environment. However, we find that the extent of self-splicing is greatly influenced by sequences flanking the intron and presumably reflects differences in the intron’s ability to fold into an active conformation inside the cell. In support of this hypothesis, we show that the ability of the intron to fold and self-splice from cellular transcripts in vitro correlates well with the catalytic efficiency observed from the same transcripts expressed inside cells. These results underscore the importance of evaluating the impact of sequence context on the activity of therapeutic group I ribozymes. The self-splicing system that we describe should facilitate these efforts as well as aid in efforts at enhancing in vivo ribozyme activity for various applications of RNA repair.

scholarly journals Characterizing the Non-Normal Distribution of Flow Cytometry Measurements from Transiently Expressed Constructs in Mammalian Cells

2016 ◽  
Author(s):  
Peter F. McLean ◽  
Christina D. Smolke ◽  
Marc Salit
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
Normal Distribution ◽  
Mammalian Cells ◽  
Fluorescent Proteins ◽  
Transient Gene Expression ◽  
Single Copy ◽  
Asymmetric Distribution ◽  
Experimental Conditions ◽  
Fluorescent Reporter

AbstractIn mammalian cells, transient gene expression (TGE) is a rapid, minimal-investment alternative to single-copy integrations for testing of transgenic constructs. However, transient gene expression, as measured by flow cytometry with a fluorescent reporter, typically displays a broad, asymmetric distribution with a left-tail that is convolved with background signal. Common approaches for deriving a summary statistic for transiently expressed gene products impose a normal distribution on gated or ungated data. Summary statistics derived from these models are heavily biased by experimental conditions and instrument settings that are difficult to replicate and insufficient to accurately describe the underlying data. Here, we present a convolved gamma distribution as a superior model for TGE datasets. The 4-6 parameters of this model are sufficient to accurately describe the entire, ungated distribution of transiently transfected HEK cells expressing monomeric fluorescent proteins, that operates consistently across a range of transfection conditions and instrument settings. Based on these observations, a convolved gamma model of TGE distributions has the potential to significantly improve the accuracy and reproducibility of genetic device characterization in mammalian cells.

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.

Sequencing metabolically labeled transcripts in single cells reveals mRNA turnover strategies

Science ◽  
2020 ◽  
Vol 367 (6482) ◽  
pp. 1151-1156 ◽  
Author(s):  
Nico Battich ◽  
Joep Beumer ◽  
Buys de Barbanson ◽  
Lenno Krenning ◽  
Chloé S. Baron ◽  
...  
Keyword(s):  
Gene Expression ◽  
Messenger Rna ◽  
Mammalian Cells ◽  
Dynamic Range ◽  
Single Cells ◽  
Regulatory Strategies ◽  
Degradation Rates ◽  
Gene Expression Dynamics ◽  
Homogeneous Cell ◽  
Synthesis And Degradation

The regulation of messenger RNA levels in mammalian cells can be achieved by the modulation of synthesis and degradation rates. Metabolic RNA-labeling experiments in bulk have quantified these rates using relatively homogeneous cell populations. However, to determine these rates during complex dynamical processes, for instance during cellular differentiation, single-cell resolution is required. Therefore, we developed a method that simultaneously quantifies metabolically labeled and preexisting unlabeled transcripts in thousands of individual cells. We determined synthesis and degradation rates during the cell cycle and during differentiation of intestinal stem cells, revealing major regulatory strategies. These strategies have distinct consequences for controlling the dynamic range and precision of gene expression. These findings advance our understanding of how individual cells in heterogeneous populations shape their gene expression dynamics.

scholarly journals Controlling gene expression in mammalian cells using multiplexed conditional guide RNAs for Cas12a

2021 ◽  
Author(s):  
Lukas Oesinghaus ◽  
Friedrich C. Simmel
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Basic Research ◽  
Dependent Manner ◽  
Multiple Control ◽  
Guide Rnas ◽  
Single Transcript ◽  
Spatiotemporal Control ◽  
Expression In Mammalian Cells ◽  
Activate Gene

AbstractSpatiotemporal control of the activity of Cas proteins is of considerable interest for both basic research and therapeutics. Only few mechanisms have been demonstrated for regulating the activity of guide RNAs (gRNAs) for Cas12a in mammalian cells, however, and combining and compactly integrating multiple control instances on single transcripts has not been possible so far. Here, we show that conditional processing of the 3’ tail is a viable general approach towards switchable Pol II-transcribed Cas12a gRNAs that can activate gene expression in mammalian cells in an input-dependent manner. Processing of the 3’ tail can be achieved using microRNA and short hairpin RNA as inputs, via a guanine-responsive ribozyme, and also using an RNA strand displacement mechanism. We further show that Cas12a along with several independently switchable gRNAs can be integrated on a single transcript using stabilizing RNA triplexes, providing a route towards compact Cas12a-based gene regulation constructs with multi-input switching capabilities.

scholarly journals A tunable dual-input system for on-demand dynamic gene expression regulation

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Elisa Pedone ◽  
Lorena Postiglione ◽  
Francesco Aulicino ◽  
Dan L. Rocca ◽  
Sandra Montes-Olivas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Stability ◽  
Mammalian Cells ◽  
Dynamic Range ◽  
Embryonic Stem ◽  
Cellular Systems ◽  
Control Of Gene Expression ◽  
Cell Functions ◽  
Input System ◽  
Gene Expression Control

Abstract Cellular systems have evolved numerous mechanisms to adapt to environmental stimuli, underpinned by dynamic patterns of gene expression. In addition to gene transcription regulation, modulation of protein levels, dynamics and localization are essential checkpoints governing cell functions. The introduction of inducible promoters has allowed gene expression control using orthogonal molecules, facilitating its rapid and reversible manipulation to study gene function. However, differing protein stabilities hinder the generation of protein temporal profiles seen in vivo. Here, we improve the Tet-On system integrating conditional destabilising elements at the post-translational level and permitting simultaneous control of gene expression and protein stability. We show, in mammalian cells, that adding protein stability control allows faster response times, fully tunable and enhanced dynamic range, and improved in silico feedback control of gene expression. Finally, we highlight the effectiveness of our dual-input system to modulate levels of signalling pathway components in mouse Embryonic Stem Cells.

scholarly journals Activity of Hoechst 33258 against Pneumocystis carinii f. sp. muris, Candida albicans, and Candida dubliniensis

2005 ◽  
Vol 49 (4) ◽  
pp. 1326-1330 ◽  
Author(s):  
Matthew D. Disney ◽  
Ruth Stephenson ◽  
Terry W. Wright ◽  
Constantine G. Haidaris ◽  
Douglas H. Turner ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Nucleic Acids ◽  
Mammalian Cells ◽  
Pneumocystis Carinii ◽  
Candida Dubliniensis ◽  
Group I Introns ◽  
Hoechst 33258 ◽  
Group I ◽  
Self Splicing

ABSTRACT Hoechst 33258 is a compound that binds nucleic acids. We report that Hoechst 33258 exhibits antimicrobial activity against Pneumocystis carinii f. sp. muris in a mouse model for P. carinii pneumonia and against Candida albicans and Candida dubliniensis in vitro. Relative to saline treatment, a 14-day, daily treatment of mice with 37.5 mg of Hoechst 33258/kg of body weight after inoculation with P. carinii reduced by about 100-fold the number of P. carinii organisms detected by either PCR or by microscopy after silver staining. For comparison, treatment based on a dose of 15 to 20 mg of the trimethoprim component in trimethoprim-sulfamethoxazole/kg reduced the number of P. carinii by about fourfold. In vitro inhibition of P. carinii group I intron splicing was observed with a 50% inhibitory concentration (IC50)of 30 μM in 2 or 4 mM Mg2+, suggesting RNA as a possible target. However, Hoechst 33258 inhibits growth of Candida strains with and without group I introns. IC50s ranged from 1 to 9 μM for strains with group I introns and were 12 and 32 μM for two strains without group I introns. These studies demonstrate that compounds that bind fungal nucleic acids have the potential to be developed as new therapeutics for Pneumocystis and possibly other fungi, especially if they could be directed to structures that are not present in mammalian cells, such as self-splicing introns.

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