scholarly journals Tunable genetic devices through simultaneous control of transcription and translation

2019 ◽  
Author(s):  
Vittorio Bartoli ◽  
Grace A. Meaker ◽  
Mario di Bernardo ◽  
Thomas E. Gorochowski
Keyword(s):  
Host Cell ◽  
Environmental Conditions ◽  
Logic Gates ◽  
Living Cells ◽  
Fold Change ◽  
Response Functions ◽  
Genetic Circuits ◽  
Regulatory Motif ◽  
Trade Offs ◽  
Simultaneous Control

AbstractSynthetic genetic circuits allow us to modify the behavior of living cells. However, changes in environmental conditions and unforeseen interactions with the host cell can cause deviations from a desired function, resulting in the need for time-consuming reassembly to fix these issues. Here, we use a regulatory motif that controls transcription and translation to create genetic devices whose response functions can be dynamically tuned. This allows us, after construction, to shift the on and off states of a sensor by 4.5- and 28-fold, respectively, and modify genetic NOT and NOR logic gates to allow their transitions between states to be varied over a >6-fold range. In all cases, tuning leads to trade-offs in the fold-change and the ability to distinguish cellular states. This work lays the foundation for adaptive genetic circuits that can be tuned after their physical assembly to maintain functionality across diverse environments and design contexts.

scholarly journals Multilevel Gene Regulation Using Switchable Transcription Terminator and Toehold Switch in Escherichia coli

2021 ◽  
Vol 11 (10) ◽  
pp. 4532
Author(s):  
Seongho Hong ◽  
Jeongwon Kim ◽  
Jongmin Kim
Keyword(s):  
Escherichia Coli ◽  
Dynamic Range ◽  
Scale Up ◽  
Logic Gates ◽  
Living Cells ◽  
Genetic Circuit ◽  
Seamless Integration ◽  
Library Size ◽  
Transcription Terminator ◽  
Simultaneous Control

Nucleic acid-based regulatory components provide a promising toolbox for constructing synthetic biological circuits due to their design flexibility and seamless integration towards complex systems. In particular, small-transcriptional activating RNA (STAR) and toehold switch as regulators of transcription and translation steps have shown a large library size and a wide dynamic range, meeting the criteria to scale up genetic circuit construction. Still, there are limited attempts to integrate the heterogeneous regulatory components for multilevel regulatory circuits in living cells. In this work, inspired by the design principle of STAR, we designed several switchable transcription terminators starting from natural and synthetic terminators. These switchable terminators could be designed to respond to specific RNA triggers with minimal sequence constraints. When combined with toehold switches, the switchable terminators allow simultaneous control of transcription and translation processes to minimize leakage in Escherichia coli. Further, we demonstrated a set of logic gates implementing 2-input AND circuits and multiplexing capabilities to control two different output proteins. This study shows the potential of novel switchable terminator designs that can be computationally designed and seamlessly integrated with other regulatory components, promising to help scale up the complexity of synthetic gene circuits in living cells.

scholarly journals dCas9 regulator to neutralize competition in CRISPRi circuits

2020 ◽  
Author(s):  
Hsin-Ho Huang ◽  
Massimo Bellato ◽  
Yili Qian ◽  
Pablo Cárdenas ◽  
Lorenzo Pasotti ◽  
...  
Keyword(s):  
Logic Gates ◽  
Fold Change ◽  
Single Stage ◽  
Gene Repression ◽  
Input Output ◽  
Independent Control ◽  
E Coli ◽  
Output Response ◽  
Multiple Genes ◽  
Simultaneous Control

AbstractCRISPRi-mediated gene repression allows simultaneous control of many genes. However, despite highly specific sgRNA-promoter binding, multiple sgRNAs still interfere with one another by competing for dCas9. We created a dCas9 regulator that adjusts dCas9 concentration based on sgRNAs’ demand, mitigating competition in CRISPRi-based logic gates. The regulator’s performance is demonstrated on both single-stage and layered CRISPRi logic gates and in two common E. coli strains. When a competitor sgRNA causes between two and ~25 fold-change in a logic gate’s input/output response without dCas9 regulator, the response is essentially unchanged when the regulator is used. The dCas9 regulator thus enables concurrent and independent operation of multiple sgRNAs, thereby supporting independent control of multiple genes.

scholarly journals Multifunctionality and Robustness Trade-Offs in Model Genetic Circuits

2008 ◽  
Vol 94 (8) ◽  
pp. 2927-2937 ◽  
Author(s):  
Olivier C. Martin ◽  
Andreas Wagner
Keyword(s):  
Genetic Circuits ◽  
Trade Offs

Ciprofloxacin as chemosensor for simultaneous recognition of Al3+ and Cu2+ by Logic Gates supported fluorescence: Application to bio-imaging for living cells

2017 ◽  
Vol 248 ◽  
pp. 447-459 ◽  
Author(s):  
Yessenia Scarlette García-Gutiérrez ◽  
Carlos Alberto Huerta-Aguilar ◽  
Pandiyan Thangarasu ◽  
Jorge Manuel Vázquez-Ramos
Keyword(s):  
Logic Gates ◽  
Living Cells ◽  
Bio Imaging

scholarly journals Single-Molecule Analysis of Human Immunodeficiency Virus Type 1 gp120-Receptor Interactions in Living Cells

2005 ◽  
Vol 79 (23) ◽  
pp. 14748-14755 ◽  
Author(s):  
Melissa I. Chang ◽  
Porntula Panorchan ◽  
Terrence M. Dobrowsky ◽  
Yiider Tseng ◽  
Denis Wirtz
Keyword(s):  
Human Immunodeficiency Virus ◽  
Host Cell ◽  
Single Molecule ◽  
Quantitative Description ◽  
Living Cells ◽  
Viral Envelope ◽  
Binding Interactions ◽  
Immunodeficiency Virus ◽  
Single Molecule Analysis

ABSTRACT A quantitative description of the binding interactions between human immunodeficiency virus (HIV) type 1 envelope glycoproteins and their host cell surface receptors remains incomplete. Here, we introduce a single-molecule analysis that directly probes the binding interactions between an individual viral subunit gp120 and a single receptor CD4 and/or chemokine coreceptor CCR5 in living cells. This analysis differentiates single-molecule binding from multimolecule avidity and shows that, while the presence of CD4 is required for gp120 binding to CCR5, the force required to rupture a single gp120-coreceptor bond is significantly higher and its lifetime is much longer than those of a single gp120-receptor bond. The lifetimes of these bonds are themselves shorter than those of the P-selectin/PSGL-1 bond involved in leukocyte attachment to the endothelium bonds during an inflammation response. These results suggest an amended model of HIV entry in which, immediately after the association of gp120 to its receptor, gp120 seeks its coreceptor to rapidly form a new bond. This “bond transfer” occurs only if CCR5 is in close proximity to CD4 and CD4 is still attached to gp120. The analysis presented here may serve as a general framework to study mechanisms of receptor-mediated interactions between viral envelope proteins and host cell receptors at the single-molecule level in living cells.

scholarly journals Primordial mimicry induces morphological change in Escherichia coli

2022 ◽  
Vol 5 (1) ◽  
Author(s):  
Hui Lu ◽  
Honoka Aida ◽  
Masaomi Kurokawa ◽  
Feng Chen ◽  
Yang Xia ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Morphological Change ◽  
Energy Resource ◽  
Volume Ratio ◽  
Living Cells ◽  
Carbon Utilization ◽  
Spherical Form ◽  
Trade Offs ◽  
Prebiotic Earth

AbstractThe morphology of primitive cells has been the subject of extensive research. A spherical form was commonly presumed in prebiotic studies but lacked experimental evidence in living cells. Whether and how the shape of living cells changed are unclear. Here we exposed the rod-shaped bacterium Escherichia coli to a resource utilization regime mimicking a primordial environment. Oleate was given as an easy-to-use model prebiotic nutrient, as fatty acid vesicles were likely present on the prebiotic Earth and might have been used as an energy resource. Six evolutionary lineages were generated under glucose-free but oleic acid vesicle (OAV)-rich conditions. Intriguingly, fitness increase was commonly associated with the morphological change from rod to sphere and the decreases in both the size and the area-to-volume ratio of the cell. The changed cell shape was conserved in either OAVs or glucose, regardless of the trade-offs in carbon utilization and protein abundance. Highly differentiated mutations present in the genome revealed two distinct strategies of adaption to OAV-rich conditions, i.e., either directly targeting the cell wall or not. The change in cell morphology of Escherichia coli for adapting to fatty acid availability supports the assumption of the primitive spherical form.

scholarly journals Red hot maples: Acer rubrum first-year phenology and growth responses to soil warming

2017 ◽  
Vol 47 (2) ◽  
pp. 159-165 ◽  
Author(s):  
J.A. Wheeler ◽  
N.M. Gonzalez ◽  
K.A. Stinson
Keyword(s):  
Biomass Allocation ◽  
Environmental Conditions ◽  
Acer Rubrum ◽  
Growing Season ◽  
Tree Seedlings ◽  
Red Maple ◽  
Growth Responses ◽  
Soil Warming ◽  
Leaf Production ◽  
Trade Offs

Microhabitat environmental conditions are an important filter for seedling establishment, controlling the availability of optimal recruitment sites. Understanding how tree seedlings respond to warming soil temperature is critical for predicting population recruitment in the future hardwood forests of northeastern North America, particularly as environmental conditions and thus optimal microhabitat availabilities change. We examined the effect of soil warming of 5 °C during the first growing season on germination, survival, phenology, growth, and stem and root biomass allocation in Acer rubrum L. (red maple) seedlings. While there was no effect of soil warming on germination or survival, seedlings growing in warmer soils demonstrated significantly accelerated leaf expansion, delayed autumn leaf senescence, and an extended leaf production period. Further, seedlings growing in warmer soils showed larger leaf area and stem and root structures at the end of the first growing season, with no evidence of biomass allocation trade-offs. Results suggest that A. rubrum seedlings can capitalize on soil warming by adjusting leaf phenology and leaf production, resulting in a longer period of carbon uptake and leading to higher overall biomass. The absence of growth allocation trade-offs suggests that A. rubrum will respond positively to increasing soil temperatures in northeastern forests, at least in the early life stages.

scholarly journals Realizing ‘integral control’ in living cells: how to overcome leaky integration due to dilution?

2018 ◽  
Vol 15 (139) ◽  
pp. 20170902 ◽  
Author(s):  
Yili Qian ◽  
Domitilla Del Vecchio
Keyword(s):  
Gene Expression ◽  
General Principle ◽  
Design Principle ◽  
Living Cells ◽  
Genetic Circuits ◽  
Memory Element ◽  
Integral Control ◽  
Leaky Integration ◽  
Integral Controller ◽  
Unique Ability

A major problem in the design of synthetic genetic circuits is robustness to perturbations and uncertainty. Because of this, there have been significant efforts in recent years in finding approaches to implement integral control in genetic circuits. Integral controllers have the unique ability to make the output of a process adapt perfectly to disturbances. However, implementing an integral controller is challenging in living cells. This is because a key aspect of any integral controller is a ‘memory’ element that stores the accumulation (integral) of the error between the output and its desired set-point. The ability to realize such a memory element in living cells is fundamentally challenged by the fact that all biomolecules dilute as cells grow, resulting in a ‘leaky’ memory that gradually fades away. As a consequence, the adaptation property is lost. Here, we propose a general principle for designing integral controllers such that the performance is practically unaffected by dilution. In particular, we mathematically prove that if the reactions implementing the integral controller are all much faster than dilution, then the adaptation error due to integration leakiness becomes negligible. We exemplify this design principle with two synthetic genetic circuits aimed at reaching adaptation of gene expression to fluctuations in cellular resources. Our results provide concrete guidance on the biomolecular processes that are most appropriate for implementing integral controllers in living cells.

scholarly journals High and dry: Trade-off in arboreal calling in a treefrog mediated by local environment

2019 ◽  
Author(s):  
Amanda S Cicchino ◽  
Nicholas A Cairns ◽  
Grégory Bulté ◽  
Stephen C Lougheed
Keyword(s):  
Environmental Conditions ◽  
Ground Cover ◽  
Local Environment ◽  
Environmental Data ◽  
Calling Behavior ◽  
Vegetation Density ◽  
Trade Off ◽  
Trade Offs ◽  
Mating Call ◽  
The Cost

Abstract Trade-offs shaping behavioral variation are often influenced by the environment. We investigated the role that the environment plays in mediating trade-offs using a widespread frog with a conspicuous mating display, Pseudacris crucifer. We first demonstrated, using playback and desiccation experiments, that calling site selection involves a trade-off between sound transmission and desiccation. We then determined the influence of local environmental conditions on the intensity of the trade-off by examining range-wide behavioral and environmental data. We showed that the benefit of improved call transmission is positively influenced by vegetation density and ground cover. Behavioral data are consistent with this relationship: sites with a greater transmission benefit have increased prevalence of arboreally calling males. We also found that the prevalence of arboreal calling behavior increases with relative humidity and air temperature, suggesting an influence of these environmental variables on the desiccation cost of arboreal calling. This study provides a clear example of the role of the environment in mediating trade-off intensities and shaping critical behavioral traits. Local environment mediates the intensity of a trade-off associated with arboreal calling behavior in a treefrog. Combining observational and experimental approaches, we show that arboreal calling behavior increases the transmission of a mating call while potentially subjecting individuals to a rate of desiccation six times greater than terrestrial calling. Local environmental conditions influence both the benefit and the cost of this trade-off, subjecting different populations to varying trade-off intensities and shaping arboreal calling behavior.

scholarly journals Numerical operations in living cells by programmable RNA devices

2019 ◽  
Vol 5 (8) ◽  
pp. eaax0835 ◽  
Author(s):  
Kei Endo ◽  
Karin Hayashi ◽  
Hirohide Saito
Keyword(s):  
Messenger Rna ◽  
Design Principle ◽  
Logic Gates ◽  
Living Cells ◽  
Multiple Biomarkers ◽  
Intracellular Changes ◽  
And Function ◽  
Single Living Cells ◽  
Single Living ◽  
Numerical Operations

Integrated bioengineering systems can make executable decisions according to the cell state. To sense the state, multiple biomarkers are detected and processed via logic gates with synthetic biological devices. However, numerical operations have not been achieved. Here, we show a design principle for messenger RNA (mRNA) devices that recapitulates intracellular information by multivariate calculations in single living cells. On the basis of this principle and the collected profiles of multiple microRNA activities, we demonstrate that rationally programmed mRNA sets classify living human cells and track their change during differentiation. Our mRNA devices automatically perform multivariate calculation and function as a decision-maker in response to dynamic intracellular changes in living cells.

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