A synthetic circuit for buffering gene dosage variation between individual mammalian cells

AbstractPrecise control of gene expression is critical for biological research and biotechnology. However, transient plasmid transfections in mammalian cells produce a wide distribution of copy numbers per cell, and consequently, high expression heterogeneity. Here, we report plasmid-based synthetic circuits – Equalizers – that buffer copy-number variation at the single-cell level. Equalizers couple a transcriptional negative feedback loop with post-transcriptional incoherent feedforward control. Computational modeling suggests that the combination of these two topologies enables Equalizers to operate over a wide range of plasmid copy numbers. We demonstrate experimentally that Equalizers outperform other gene dosage compensation topologies and produce as low cell-to-cell variation as chromosomally integrated genes. We also show that episome-encoded Equalizers enable the rapid generation of extrachromosomal cell lines with stable and uniform expression. Overall, Equalizers are simple and versatile devices for homogeneous gene expression and can facilitate the engineering of synthetic circuits that function reliably in every cell.

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Photoregulated Gene Expression in Human Cells With Light-Inducible Engineered Transcription Factors

ASME 2012 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2012-80573 ◽

2012 ◽

Author(s):

Lauren R. Polstein ◽

Charles A. Gersbach

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Mammalian Cells ◽

Regulatory System ◽

Human Cells ◽

Biological Research ◽

Specific Gene ◽

Spatial Control ◽

Wide Range ◽

Gene Regulatory

Systems for controlling gene expression in mammalian cells have a wide range of applications in medicine, biotechnology and basic science. An ideal gene regulatory system would allow for precise and specific control over the magnitude and kinetics of gene expression in space and time, while also exerting minimal influence on other genes and cellular components. Several gene regulatory systems have been developed in which orthogonal transcription machinery from prokaryotes or insects has been imported into mammalian cells and used to control the expression of a specific gene. Despite the transformative impact of these systems in biomedical and biological research, several limitations of these technologies restrict the scope of possible applications. For example, gene expression in these systems is controlled by a freely diffusible small molecule, such as an antibiotic or steroid. Consequently, it is not possible to achieve spatial control over gene expression within cell culture, tissues, or whole organisms. This is in contrast to natural mechanisms of biological regulation in which spatial control is critical, such as developmental patterning and tissue morphogenesis. Second, dynamic gene regulation requires the removal of these small molecules, which may be slow, laborious, and/or impractical for a particular application. To overcome these limitations, we have engineered an optogenetic system in which the magnitude of gene expression in human cells can be finely tuned by photoregulated synthetic transcription factors.

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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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Computational Strategies for Analyzing Data in Gene Expression Microarray Experiments

Journal of Bioinformatics and Computational Biology ◽

10.1142/s0219720003000319 ◽

2003 ◽

Vol 01 (03) ◽

pp. 541-586 ◽

Cited By ~ 33

Author(s):

Tero Aittokallio ◽

Markus Kurki ◽

Olli Nevalainen ◽

Tuomas Nikula ◽

Anne West ◽

...

Keyword(s):

Gene Expression ◽

Data Analysis ◽

Microarray Data ◽

Microarray Data Analysis ◽

Biological Research ◽

Microarray Experiments ◽

Dna Microarray Data ◽

Open Questions ◽

Analysis Technique ◽

Wide Range

Microarray analysis has become a widely used method for generating gene expression data on a genomic scale. Microarrays have been enthusiastically applied in many fields of biological research, even though several open questions remain about the analysis of such data. A wide range of approaches are available for computational analysis, but no general consensus exists as to standard for microarray data analysis protocol. Consequently, the choice of data analysis technique is a crucial element depending both on the data and on the goals of the experiment. Therefore, basic understanding of bioinformatics is required for optimal experimental design and meaningful interpretation of the results. This review summarizes some of the common themes in DNA microarray data analysis, including data normalization and detection of differential expression. Algorithms are demonstrated by analyzing cDNA microarray data from an experiment monitoring gene expression in T helper cells. Several computational biology strategies, along with their relative merits, are overviewed and potential areas for additional research discussed. The goal of the review is to provide a computational framework for applying and evaluating such bioinformatics strategies. Solid knowledge of microarray informatics contributes to the implementation of more efficient computational protocols for the given data obtained through microarray experiments.

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Artificial control of gene expression in mammalian cells by modulating RNA interference through aptamer-small molecule interaction

RNA ◽

10.1261/rna.2299306 ◽

2006 ◽

Vol 12 (5) ◽

pp. 710-716 ◽

Cited By ~ 107

Author(s):

C.-I. An

Keyword(s):

Gene Expression ◽

Rna Interference ◽

Small Molecule ◽

Mammalian Cells ◽

Control Of Gene Expression ◽

Expression In Mammalian Cells ◽

Molecule Interaction ◽

Artificial Control

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Establishing Probiotic Saccharomyces boulardii as a Model Organism for Synthesis and Delivery of Biomolecules

10.1101/2020.01.22.915389 ◽

2020 ◽

Author(s):

Deniz Durmusoglu ◽

Ibrahim Al’Abri ◽

Scott P. Collins ◽

Chase Beisel ◽

Nathan Crook

Keyword(s):

Gene Expression ◽

Copy Number ◽

Model Organism ◽

Saccharomyces Boulardii ◽

Gastrointestinal Disorders ◽

Control Of Gene Expression ◽

Germ Free ◽

Gut Colonization ◽

Wide Range ◽

Dosing Strategy

AbstractSaccharomyces boulardii is a widely used yeast probiotic which can counteract various gastrointestinal disorders1. As a relative of Saccharomyces cerevisiae, S. boulardii exhibits rapid growth and is easy to transform2 and thus represents a promising chassis for the engineered secretion of biomolecules. To establish S. boulardii as a platform for delivery of biomolecules to the mammalian gut, we measured the amount and variance in protein expression enabled by promoters, terminators, selective markers, and copy number control elements in this organism. These genetic elements were characterized in plasmidic and genomic contexts, revealing strategies for tunable control of gene expression and CRISPR-mediated genome editing in this strain. We then leveraged this set of genetic parts to combinatorially assemble pathways enabling a wide range of drug and vitamin titers. Finally, we measured S. boulardii’s residence time in the gastrointestinal tracts of germ-free and antibiotic-treated mice, revealing the relationships between dosing strategy and colonization level. This work establishes S. boulardii as a genetically tractable commensal fungus and provides a set of strategies for engineering S. boulardii to synthesize and deliver biomolecules during gut colonization.

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Dynamics in Copy Numbers of Five Plasmids of a DairyLactococcus lactisStrain under Dairy-Related Conditions Including Near-Zero Growth Rates

Applied and Environmental Microbiology ◽

10.1128/aem.00314-18 ◽

2018 ◽

Vol 84 (11) ◽

Cited By ~ 1

Author(s):

Oscar van Mastrigt ◽

Marcel M. A. N. Lommers ◽

Yorick C. de Vries ◽

Tjakko Abee ◽

Eddy J. Smid

Keyword(s):

Nutrient Limitation ◽

Lactococcus Lactis ◽

Growth Rates ◽

Copy Number ◽

Plasmid Copy ◽

Content Type ◽

Copy Numbers ◽

Wide Range ◽

Zero Growth ◽

The Impact

ABSTRACTLactic acid bacteria can carry multiple plasmids affecting their performance in dairy fermentations. The expression of plasmid-borne genes and the activity of the corresponding proteins are severely affected by changes in the numbers of plasmid copies. We studied the impact of growth rate on the dynamics of plasmid copy numbers at high growth rates in chemostat cultures and down to near-zero growth rates in retentostat cultures. Five plasmids of the dairy strainLactococcus lactisFM03-V1 were selected, and these varied in size (3 to 39 kb), in replication mechanism (theta or rolling circle), and in putative (dairy-associated) functions. The copy numbers ranged from 1.5 to 40.5, and the copy number of theta-type replicating plasmids was negatively correlated to the plasmid size. Despite the extremely wide range of growth rates (0.0003 h−1to 0.6 h−1), the copy numbers of the five plasmids were stable and only slightly increased at near-zero growth rates, showing that the plasmid replication rate was strictly controlled. One low-copy-number plasmid, carrying a large exopolysaccharide gene cluster, was segregationally unstable during retentostat cultivations, reflected in a complete loss of the plasmid in one of the retentostat cultures. The copy number of the five plasmids was also hardly affected by varying the pH value, nutrient limitation, or the presence of citrate (maximum 2.2-fold), signifying the stability in copy number of the plasmids.IMPORTANCELactococcus lactisis extensively used in starter cultures for dairy fermentations. Important traits for the growth and survival ofL. lactisin dairy fermentations are encoded by genes located on plasmids, such as genes involved in lactose and citrate metabolism, protein degradation, oligopeptide uptake, and bacteriophage resistance. Because the number of plasmid copies could affect the expression of plasmid-borne genes, it is important to know the factors that influence the plasmid copy numbers. We monitored the plasmid copy numbers ofL. lactisat near-zero growth rates, characteristic for cheese ripening. Moreover, we analyzed the effects of pH, nutrient limitation, and the presence of citrate. This showed that the plasmid copy numbers were stable, giving insight into plasmid copy number dynamics in dairy fermentations.

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