A tight cold-inducible switch built by coupling thermosensitive transcriptional and proteolytic regulatory parts

Abstract Natural organisms have evolved intricate regulatory mechanisms that sense and respond to fluctuating environmental temperatures in a heat- or cold-inducible fashion. Unlike dominant heat-inducible switches, very few cold-inducible genetic switches are available in either natural or engineered systems. Moreover, the available cold-inducible switches still have many shortcomings, including high leaky gene expression, small dynamic range (<10-fold) or broad transition temperature (>10°C). To address these problems, a high-performance cold-inducible switch that can tightly control target gene expression is highly desired. Here, we introduce a tight and fast cold-inducible switch that couples two evolved thermosensitive variants, TFts and TEVts, as well as an additional Mycoplasma florum Lon protease (mf-Lon) to effectively turn-off target gene expression via transcriptional and proteolytic mechanisms. We validated the function of the switch in different culture media and various Escherichia coli strains and demonstrated its tightness by regulating two morphogenetic bacterial genes and expressing three heat-unstable recombinant proteins, respectively. Moreover, the additional protease module enabled the cold-inducible switch to actively remove the pre-existing proteins in slow-growing cells. This work establishes a high-performance cold-inducible system for tight and fast control of gene expression which has great potential for basic research, as well as industrial and biomedical applications.

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E2f1, E2f2, and E2f3 Control E2F Target Expression and Cellular Proliferation via a p53-Dependent Negative Feedback Loop

Molecular and Cellular Biology ◽

10.1128/mcb.02147-05 ◽

2007 ◽

Vol 27 (1) ◽

pp. 65-78 ◽

Cited By ~ 59

Author(s):

Cynthia Timmers ◽

Nidhi Sharma ◽

Rene Opavsky ◽

Baidehi Maiti ◽

Lizhao Wu ◽

...

Keyword(s):

Gene Expression ◽

Target Gene ◽

Target Genes ◽

Cellular Proliferation ◽

Kinase Activity ◽

Cyclin Dependent Kinase ◽

Control Of Gene Expression ◽

Target Gene Expression ◽

Rb Phosphorylation ◽

P53 Target Genes

ABSTRACT E2F-mediated control of gene expression is believed to have an essential role in the control of cellular proliferation. Using a conditional gene-targeting approach, we show that the targeted disruption of the entire E2F activator subclass composed of E2f1, E2f2, and E2f3 in mouse embryonic fibroblasts leads to the activation of p53 and the induction of p53 target genes, including p21 CIP1 . Consequently, cyclin-dependent kinase activity and retinoblastoma (Rb) phosphorylation are dramatically inhibited, leading to Rb/E2F-mediated repression of E2F target gene expression and a severe block in cellular proliferation. Inactivation of p53 in E2f1-, E2f2-, and E2f3-deficient cells, either by spontaneous mutation or by conditional gene ablation, prevented the induction of p21 CIP1 and many other p53 target genes. As a result, cyclin-dependent kinase activity, Rb phosphorylation, and E2F target gene expression were restored to nearly normal levels, rendering cells responsive to normal growth signals. These findings suggest that a critical function of the E2F1, E2F2, and E2F3 activators is in the control of a p53-dependent axis that indirectly regulates E2F-mediated transcriptional repression and cellular proliferation.

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

10.1101/747121 ◽

2019 ◽

Cited By ~ 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 ◽

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.

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Redesigned TetR-Aptamer System To Control Gene Expression in Plasmodium falciparum

mSphere ◽

10.1128/msphere.00457-20 ◽

2020 ◽

Vol 5 (4) ◽

Author(s):

Krithika Rajaram ◽

Hans B. Liu ◽

Sean T. Prigge

Keyword(s):

Gene Expression ◽

Plasmodium Falciparum ◽

Target Gene ◽

Essential Genes ◽

Untranslated Regions ◽

Dependent Manner ◽

Control Of Gene Expression ◽

Content Type ◽

Target Gene Expression ◽

The Impact

ABSTRACT One of the most powerful approaches to understanding gene function involves turning genes on and off at will and measuring the impact at the cellular or organismal level. This particularly applies to the cohort of essential genes where traditional gene knockouts are inviable. In Plasmodium falciparum, conditional control of gene expression has been achieved by using multicomponent systems in which individual modules interact with each other to regulate DNA recombination, transcription, or posttranscriptional processes. The recently devised TetR-DOZI aptamer system relies on the ligand-regulatable interaction of a protein module with synthetic RNA aptamers to control the translation of a target gene. This technique has been successfully employed to study essential genes in P. falciparum and involves the insertion of several aptamer copies into the 3′ untranslated regions (UTRs), which provide control over mRNA fate. However, aptamer repeats are prone to recombination and one or more copies can be lost from the system, resulting in a loss of control over target gene expression. We rectified this issue by redesigning the aptamer array to minimize recombination while preserving the control elements. As proof of concept, we compared the original and modified arrays for their ability to knock down the levels of a putative essential apicoplast protein (PF3D7_0815700) and demonstrated that the modified array is highly stable and efficient. This redesign will enhance the utility of a tool that is quickly becoming a favored strategy for genetic studies in P. falciparum. IMPORTANCE Malaria elimination efforts have been repeatedly hindered by the evolution and spread of multidrug-resistant strains of Plasmodium falciparum. The absence of a commercially available vaccine emphasizes the need for a better understanding of Plasmodium biology in order to further translational research. This has been partly facilitated by targeted gene deletion strategies for the functional analysis of parasite genes. However, genes that are essential for parasite replication in erythrocytes are refractory to such methods, and require conditional knockdown or knockout approaches to dissect their function. One such approach is the TetR-DOZI system that employs multiple synthetic aptamers in the untranslated regions of target genes to control their expression in a tetracycline-dependent manner. Maintaining modified parasites with intact aptamer copies has been challenging since these repeats can be lost by recombination. By interspacing the aptamers with unique sequences, we created a stable genetic system that remains effective at controlling target gene expression.

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RNA interference in biology and disease

Blood ◽

10.1182/blood-2004-12-4643 ◽

2005 ◽

Vol 106 (3) ◽

pp. 787-794 ◽

Cited By ~ 93

Author(s):

Carol A. Sledz ◽

Bryan R. G. Williams

Keyword(s):

Gene Expression ◽

Rna Interference ◽

Large Scale ◽

Target Gene ◽

Therapeutic Potential ◽

Research Effort ◽

Leukemia Cell ◽

Basic Research ◽

Target Gene Expression ◽

Biologic Response

Abstract RNA interference (RNAi) is a conserved biologic response to double-stranded RNA that results in the sequence-specific silencing of target gene expression. Over the past 5 years, an intensive research effort has facilitated the rapid movement of RNAi from a relatively obscure biologic phenomenon to a valuable tool used to silence target gene expression and perform large-scale functional genomic screens. In fact, recent studies reported in this journal and others have demonstrated success using RNAi to address the role of oncogene expression in leukemia cell lines and to validate the therapeutic potential of RNAi for treating these blood disorders. In order to advance these applications and gain an appreciation for the future of RNAi both in basic research and in the treatment of diseases caused by aberrant gene expression, it is important to have an understanding of the process of RNAi and its limitations.

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Suitable reference genes selection for reliable normalization of gene expression in melanoma: implementation of an innovative GenExpA software

10.1101/2021.05.10.443386 ◽

2021 ◽

Author(s):

Dorota Hoja-Lukowicz ◽

Dawid Maciazek ◽

Marcelina Elzbieta Janik

Keyword(s):

Gene Expression ◽

Experimental Model ◽

Target Gene ◽

Basic Research ◽

Stable Gene ◽

Target Gene Expression ◽

Promising Tool ◽

Reliable Assessment ◽

Reference Selection ◽

Suitable Reference

Melanoma is characterized by a high mutation rate and the proper reference selection is a serious problem. The algorithms commonly used to select the best stable reference gene or pair of genes in RT-qPCR data analysis have their limitations which affects the interpretation of the results and drawing conclusions. For reliable assessment of the B4GALT genes expression changes in melanoma and comparing them to their expression levels in melanocytes, we implemented an innovative GenExpA software. We proposed selection of the best reference by combining the NormFinder algorithm with progressive removal of the least stable gene from the candidate genes in a given experimental model and the set of daughter models assigned to it. The reliability of references is validated by normalizing of target gene expression level through all models and is described by the coherence score. The software performs statistical analyses and generates results in the form of ready-to-use graphs and tables. GenExpA is a promising tool for basic research; it produces analyses of target gene expression in a manner independent of the experimental model and the normalizer. GenExpA and its manual is freely available at https://drive.google.com/drive/folders/1FcpBtEc_bKPvEnM5GcPZerBb_Tw_fCkt?usp=sharing or https://www.sciencemarket.pl/baza-programow-open-source#oferty. Supplemental materials are deposited under DOI number: 10.5281/zenodo.4544296.

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A redesigned TetR-aptamer system to control gene expression in Plasmodium falciparum

10.1101/2020.05.19.105411 ◽

2020 ◽

Author(s):

Krithika Rajaram ◽

Hans B. Liu ◽

Sean T. Prigge

Keyword(s):

Gene Expression ◽

Plasmodium Falciparum ◽

Target Gene ◽

Dna Recombination ◽

Essential Genes ◽

Untranslated Regions ◽

Dependent Manner ◽

Control Of Gene Expression ◽

Target Gene Expression ◽

The Impact

AbstractOne of the most powerful approaches to understanding gene function involves turning genes on and off at will and measuring the impact at the cellular or organismal level. This particularly applies to the cohort of essential genes where traditional gene knockouts are inviable. In Plasmodium falciparum, conditional control of gene expression has been achieved by using multi-component systems in which individual modules interact with each other to regulate DNA recombination, transcription or posttranscriptional processes. The recently devised TetR-DOZI aptamer system relies on the ligand-regulatable interaction of a protein module with synthetic RNA aptamers to control the translation of a target gene. This technique has been successfully employed to study essential genes in P. falciparum and involves the insertion of several aptamer copies into their 3’ untranslated regions (UTRs) which provide control over mRNA fate. However, aptamer repeats are prone to recombination and one or more copies can be lost from the system, resulting in a loss of control over target gene expression. We rectified this issue by redesigning the aptamer array to minimize recombination while preserving the control elements. As proof of concept, we compared the original and modified arrays for their ability to knock down the levels of a putative essential apicoplast protein (PF3D7_0815700) and demonstrated that the modified array is highly stable and efficient. This redesign will enhance the utility of a tool that is quickly becoming a favored strategy for genetic studies in P. falciparum.ImportanceMalaria elimination efforts have been repeatedly hindered by the evolution and spread of multidrug-resistant strains of Plasmodium falciparum. The absence of a commercially available vaccine emphasizes the need for a better understanding of Plasmodium biology in order to further translational research. This has been partly facilitated by targeted gene deletion strategies for the functional analysis of parasite genes. However, genes that are essential for parasite replication in erythrocytes are refractory to such methods, and require conditional knockdown or knockout approaches to dissect their function. One such approach is the TetR-DOZI system that employs multiple synthetic aptamers in the untranslated regions of target genes to control their expression in a tetracycline-dependent manner. Maintaining modified parasites with intact aptamer copies has been challenging since these repeats are frequently lost by recombination. By interspacing the aptamers with unique sequences, we created a stable genetic system that remains effective at controlling target gene expression.

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2049-P: The Chd4 Helicase of the Nucleosome Remodeling and Deacetylase Complex Modulates Pdx1 Target Gene Expression In Vitro

Diabetes ◽

10.2337/db20-2049-p ◽

2020 ◽

Vol 69 (Supplement 1) ◽

pp. 2049-P

Author(s):

REBECCA K. DAVIDSON ◽

NOLAN CASEY ◽

JASON SPAETH

Keyword(s):

Gene Expression ◽

Target Gene ◽

Nucleosome Remodeling ◽

Target Gene Expression

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On the way toward regulatable expression systems in acetic acid bacteria: target gene expression and use cases

Applied Microbiology and Biotechnology ◽

10.1007/s00253-021-11269-z ◽

2021 ◽

Author(s):

Philipp Moritz Fricke ◽

Angelika Klemm ◽

Michael Bott ◽

Tino Polen

Keyword(s):

Gene Expression ◽

Acetic Acid ◽

Literature Search ◽

Target Gene ◽

Target Genes ◽

Recombinant Dna ◽

Acetic Acid Bacteria ◽

Homoserine Lactone ◽

Expression Systems ◽

Target Gene Expression

Abstract Acetic acid bacteria (AAB) are valuable biocatalysts for which there is growing interest in understanding their basics including physiology and biochemistry. This is accompanied by growing demands for metabolic engineering of AAB to take advantage of their properties and to improve their biomanufacturing efficiencies. Controlled expression of target genes is key to fundamental and applied microbiological research. In order to get an overview of expression systems and their applications in AAB, we carried out a comprehensive literature search using the Web of Science Core Collection database. The Acetobacteraceae family currently comprises 49 genera. We found overall 6097 publications related to one or more AAB genera since 1973, when the first successful recombinant DNA experiments in Escherichia coli have been published. The use of plasmids in AAB began in 1985 and till today was reported for only nine out of the 49 AAB genera currently described. We found at least five major expression plasmid lineages and a multitude of further expression plasmids, almost all enabling only constitutive target gene expression. Only recently, two regulatable expression systems became available for AAB, an N-acyl homoserine lactone (AHL)-inducible system for Komagataeibacter rhaeticus and an l-arabinose-inducible system for Gluconobacter oxydans. Thus, after 35 years of constitutive target gene expression in AAB, we now have the first regulatable expression systems for AAB in hand and further regulatable expression systems for AAB can be expected. Key points • Literature search revealed developments and usage of expression systems in AAB. • Only recently 2 regulatable plasmid systems became available for only 2 AAB genera. • Further regulatable expression systems for AAB are in sight.

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

Nature Communications ◽

10.1038/s41467-019-12800-7 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 9

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.

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