scholarly journals Robust digital logic circuits in eukaryotic cells with CRISPR/dCas9 NOR gates

2016 ◽  
Author(s):  
Miles W. Gander ◽  
Justin D. Vrana ◽  
William E. Voje ◽  
James M. Carothers ◽  
Eric Kalvins
Keyword(s):  
Logic Circuits ◽  
Genetic Circuits ◽  
Gene Circuits ◽  
Guide Rna ◽  
Signal Degradation ◽  
Genetic Components ◽  
Input Signals ◽  
Eukaryotic Gene ◽  
Hill Coefficients ◽  
Arbitrary Connectivity

SummaryNatural genetic circuits enable cells to make sophisticated digital decisions. Building equally complex synthetic circuits in eukaryotes remains difficult, however, because commonly used genetic components leak transcriptionally, do not allow arbitrary interconnections, or do not have digital responses. Here, we designed a new dCas9-Mxi1 based NOR gate architecture in S. cerevisiae that allows arbitrary connectivity and large genetic circuits. Because we used the strong chromatin remodeler Mxi1, our system showed very little leak and exhibits a highly digital response. In particular, we built a combinatorial library of NOR gates that each directly convert guide RNA (gRNA) input signals into gRNA output signals, enabling NOR gates to be “wired” together. We constructed and characterized logic circuits with up to seven independent gRNAs, including repression cascades with up to seven layers. Modeling predicted that the NOR gates have Hill Coefficients of approximately 1.71 ± 0.09, explaining the minimal signal degradation we observed in these deeply layered circuits. Our approach enables the construction of the largest, eukaryotic gene circuits to date and will form the basis for large, synthetic, decision making systems in living cells.

scholarly journals Conditional guide RNA through two intermediate hairpins for programmable CRISPR/Cas9 function: building regulatory connections between endogenous RNA expressions

2020 ◽  
Vol 48 (20) ◽  
pp. 11773-11784
Author(s):  
Jiao Lin ◽  
Yan Liu ◽  
Peidong Lai ◽  
Huixia Ye ◽  
Liang Xu
Keyword(s):  
Nucleic Acid ◽  
Genetic Regulation ◽  
Binding Activity ◽  
Strand Displacement ◽  
Genetic Circuits ◽  
Reporter System ◽  
Gene Expressions ◽  
Guide Rna ◽  
Naturally Occurring ◽  
Novel Approach

Abstract A variety of nanodevices developed for nucleic acid computation provide great opportunities to construct versatile synthetic circuits for manipulation of gene expressions. In our study, by employing a two-hairpin mediated nucleic acid strand displacement as a processing joint for conditional guide RNA, we aim to build artificial connections between naturally occurring RNA expressions through programmable CRISPR/Cas9 function. This two-hairpin joint possesses a sequence-switching machinery, in which a random trigger strand can be processed to release an unconstrained sequence-independent strand and consequently activate the self-inhibitory guide RNA for conditional gene regulation. This intermediate processor was characterized by the fluorescence reporter system and applied for regulation of the CRISPR/Cas9 binding activity. Using plasmids to generate this sequence-switching machinery in situ, we achieved the autonomous genetic regulation of endogenous RNA expressions controlled by other unrelated endogenous RNAs in both E. coli and human cells. Unlike previously reported strand-displacement genetic circuits, this advanced nucleic acid nanomachine provides a novel approach that can establish regulatory connections between naturally occurring endogenous RNAs. In addition to CRISPR systems, we anticipate this two-hairpin machine can serve as a general processing joint for wide applications in the development of other RNA-based genetic circuits.

scholarly journals Rational Design of RNA Structures that Predictably Tune Eukaryotic Gene Expression

2017 ◽  
Author(s):  
Tim Weenink ◽  
Robert M. McKiernan ◽  
Tom Ellis
Keyword(s):  
Gene Expression ◽  
Rational Design ◽  
Rna Structures ◽  
Genetic Circuits ◽  
Protein Coding ◽  
Eukaryotic Gene Expression ◽  
Eukaryotic Gene ◽  
Engineering Projects ◽  
Fine Tune ◽  
Different Levels

AbstractPredictable tuning of gene expression is essential for engineering genetic circuits and for optimising enzyme levels in metabolic engineering projects. In bacteria, gene expression can be tuned at the stage of transcription, by exchanging the promoter, or at stage of translation by altering the ribosome binding site sequence. In eukaryotes, however, only promoter exchange is regularly used, as the tools to modulate translation are lacking. Working in S. cerevisiae yeast, we here describe how hairpin RNA structures inserted into the 5’ untranslated region (5’UTR) of mRNAs can be used to tune expression levels by altering the efficiency of translation initiation. We demonstrate a direct link between the calculated free energy of folding in the 5’UTR and protein abundance, and show that this enables rational design of hairpin libraries that give predicted expression outputs. Our approach is modular, working with different promoters and protein coding sequences, and it outperforms promoter mutation as a way to predictably generate a library where a protein is induced to express at a range of different levels. With this tool, computational RNA sequence design can be used to predictably fine-tune protein production, providing a new way to modulate gene expression in eukaryotes.

scholarly journals Synthetic Biology Approaches in the Development of Engineered Therapeutic Microbes

2020 ◽  
Vol 21 (22) ◽  
pp. 8744
Author(s):  
Minjeong Kang ◽  
Donghui Choe ◽  
Kangsan Kim ◽  
Byung-Kwan Cho ◽  
Suhyung Cho
Keyword(s):  
Synthetic Biology ◽  
Physiological State ◽  
Therapeutic Agents ◽  
Logic Circuits ◽  
Form Complex ◽  
Bacterial Proteins ◽  
Genetic Components ◽  
Disease Occurrence ◽  
Wide Range ◽  
Therapeutic Molecules

Since the intimate relationship between microbes and human health has been uncovered, microbes have been in the spotlight as therapeutic targets for several diseases. Microbes contribute to a wide range of diseases, such as gastrointestinal disorders, diabetes and cancer. However, as host-microbiome interactions have not been fully elucidated, treatments such as probiotic administration and fecal transplantations that are used to modulate the microbial community often cause nonspecific results with serious safety concerns. As an alternative, synthetic biology can be used to rewire microbial networks such that the microbes can function as therapeutic agents. Genetic sensors can be transformed to detect biomarkers associated with disease occurrence and progression. Moreover, microbes can be reprogrammed to produce various therapeutic molecules from the host and bacterial proteins, such as cytokines, enzymes and signaling molecules, in response to a disturbed physiological state of the host. These therapeutic treatment systems are composed of several genetic parts, either identified in bacterial endogenous regulation systems or developed through synthetic design. Such genetic components are connected to form complex genetic logic circuits for sophisticated therapy. In this review, we discussed the synthetic biology strategies that can be used to construct engineered therapeutic microbes for improved microbiome-based treatment.

scholarly journals Contextual dependencies expand the re-usability of genetic inverters

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Huseyin Tas ◽  
Lewis Grozinger ◽  
Ruud Stoof ◽  
Victor de Lorenzo ◽  
Ángel Goñi-Moreno
Keyword(s):  
Escherichia Coli ◽  
Synthetic Biology ◽  
Pseudomonas Putida ◽  
Genetic Background ◽  
Design Parameter ◽  
Logic Gates ◽  
Logic Circuits ◽  
Boolean Logic ◽  
Genetic Components ◽  
Fundamental Design

AbstractThe implementation of Boolean logic circuits in cells have become a very active field within synthetic biology. Although these are mostly focussed on the genetic components alone, the context in which the circuit performs is crucial for its outcome. We characterise 20 genetic NOT logic gates in up to 7 bacterial-based contexts each, to generate 135 different functions. The contexts we focus on are combinations of four plasmid backbones and three hosts, two Escherichia coli and one Pseudomonas putida strains. Each gate shows seven different dynamic behaviours, depending on the context. That is, gates can be fine-tuned by changing only contextual parameters, thus improving the compatibility between gates. Finally, we analyse portability by measuring, scoring, and comparing gate performance across contexts. Rather than being a limitation, we argue that the effect of the genetic background on synthetic constructs expands functionality, and advocate for considering context as a fundamental design parameter.

scholarly journals A New Method for Low Power Design of Two-Level Logic Circuits

VLSI Design ◽  
1999 ◽  
Vol 9 (2) ◽  
pp. 147-157
Author(s):  
G. Theodoridis ◽  
S. Theoharis ◽  
D. Soudris ◽  
C. Goutis
Keyword(s):  
Low Power Design ◽  
Logic Circuits ◽  
New Method ◽  
Covering Problem ◽  
Switching Activity ◽  
Network Nodes ◽  
Input Signals ◽  
Input Variables ◽  
Comparison Of The Results ◽  
Switching Activity Reduction

A new method for implementing two-level logic circuits, which exhibit minimal power dissipation, is introduced. Switching activity reduction of the logic network nodes is achieved by adding extra input signals to specific gates. Employing the statistic properties of the primary inputs, a new concept for grouping the input variables with similar features is introduced. Appropriate input variables are chosen for reducing the switching activity of a logic circuit. For that purpose, an efficient synthesis algorithm, which generates the set of all groups of the variables and solves the minimum covering problem for each group is developed. The comparison of the results, produced by the proposed method, and those from ESPRESSO shows that a substantial power reduction can be achieved.

scholarly journals Enhanced regulation of prokaryotic gene expression by a eukaryotic transcriptional activator

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
I. Cody MacDonald ◽  
Travis R. Seamons ◽  
Jonathan C. Emmons ◽  
Shwan B. Javdan ◽  
Tara L. Deans
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcriptional Activation ◽  
Dynamic Range ◽  
Current Trend ◽  
Medical Science ◽  
Genetic Circuits ◽  
Eukaryotic Transcription ◽  
Complex Functions ◽  
Eukaryotic Gene

AbstractExpanding the genetic toolbox for prokaryotic synthetic biology is a promising strategy for enhancing the dynamic range of gene expression and enabling new engineered applications for research and biomedicine. Here, we reverse the current trend of moving genetic parts from prokaryotes to eukaryotes and demonstrate that the activating eukaryotic transcription factor QF and its corresponding DNA-binding sequence can be moved to E. coli to introduce transcriptional activation, in addition to tight off states. We further demonstrate that the QF transcription factor can be used in genetic devices that respond to low input levels with robust and sustained output signals. Collectively, we show that eukaryotic gene regulator elements are functional in prokaryotes and establish a versatile and broadly applicable approach for constructing genetic circuits with complex functions. These genetic tools hold the potential to improve biotechnology applications for medical science and research.

scholarly journals Contextual dependencies expand the re-usability of genetic inverters

2020 ◽  
Author(s):  
Huseyin Tas ◽  
Lewis Grozinger ◽  
Ruud Stoof ◽  
Victor de Lorenzo ◽  
Angel Goñi-Moreno
Keyword(s):  
Synthetic Biology ◽  
Computational Analysis ◽  
Logic Gate ◽  
Logic Gates ◽  
Boolean Logic ◽  
Regulatory Proteins ◽  
Genetic Circuits ◽  
Genetic Construct ◽  
Genetic Components ◽  
Logic Functions

The design and implementation of Boolean logic functions in living cells has become a very active field within synthetic biology. By controlling networks of regulatory proteins, novel genetic circuits are engineered to generate predefined output responses. Although many current implementations focus solely on the genetic components of the circuit, the host context in which the circuit performs is crucial for its outcome. Here, we characterise 20 genetic NOT logic gates (inverters) in up to 7 bacterial-based contexts each, to finally generate 135 different functions. The contexts we focus on are particular combinations of four plasmid backbones and three hosts, two Escherichia coli and one Pseudomonas putida strains. Each NOT logic gate shows seven different logic behaviours, depending on the context. That is, gates can be reconfigured to fit response requirements by changing only contextual parameters. Computational analysis shows that this range of behaviours improves the compatibility between gates, because there are considerably more possibilities for combination than when considering a unique function per genetic construct. Finally, we address the issue of interoperability and portability by measuring, scoring, and comparing gate performance across contexts. Rather than being a limitation, we argue that the effect of the genetic background on synthetic constructs expand the scope of the functions that can be engineered in complex cellular environments, and advocate for considering context as a fundamental design parameter for synthetic biology.

scholarly journals A MODULAR SYNTHETIC DEVICE TO CALIBRATE PROMOTERS

2012 ◽  
Vol 20 (01) ◽  
pp. 37-55
Author(s):  
D. GAMERMANN ◽  
A. MONTAGUD ◽  
P. APARICIO ◽  
E. NAVARRO ◽  
J. TRIANA ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Fast Response ◽  
Relative Strength ◽  
Genetic Circuit ◽  
Model Parameters ◽  
Genetic Circuits ◽  
New Family ◽  
Hill Coefficients ◽  
And Performance ◽  
Critical Model

In this contribution, a design of a synthetic calibration genetic circuit to characterize the relative strength of different sensing promoters is proposed and its specifications and performance are analyzed via an effective mathematical model. Our calibrator device possesses certain novel and useful features like modularity (and thus the possibility of being used in many different biological contexts), simplicity, being based on a single cell, high sensitivity and fast response. To uncover the critical model parameters and the corresponding parameter domain at which the calibrator performance will be optimal, a sensitivity analysis of the model parameters was carried out over a given range of sensing protein concentrations (acting as input). Our analysis suggests that the half saturation constants for repression, sensing and difference in binding cooperativity (Hill coefficients) for repression are the key to the performance of the proposed device. They furthermore are determinant for the sensing speed of the device, showing that it is possible to produce detectable differences in the repression protein concentrations and in turn in the corresponding fluorescence in less than two hours. This analysis paves the way for the design, experimental construction and validation of a new family of functional genetic circuits for the purpose of calibrating promoters.

scholarly journals Single cell characterization of a synthetic bacterial clock with a hybrid feedback loop containing dCas9-sgRNA

2020 ◽  
Author(s):  
John Henningsen ◽  
Matthaeus Schwarz-Schilling ◽  
Andreas Leibl ◽  
Joaquin A. M. Guttierez ◽  
Sandra Sagredo ◽  
...  
Keyword(s):  
Single Cell ◽  
Transcriptional Repression ◽  
Time Course ◽  
Cellular Growth ◽  
Gene Circuits ◽  
Rna Molecules ◽  
Guide Rna ◽  
Oscillatory Dynamics ◽  
Cellular Processes ◽  
Wide Range

AbstractGenetic networks that generate oscillations in gene expression activity are found in a wide range of organisms throughout all kingdoms of life. Oscillatory dynamics facilitates the temporal orchestration of metabolic and growth processes inside cells and organisms, as well as the synchronization of such processes with periodically occurring changes in the environment. Synthetic oscillator gene circuits such as the ‘repressilator’ can perform similar functions in bacteria. Until recently, such circuits were mainly based on a relatively small set of well-characterized transcriptional repressors and activators. A promising, sequence-programmable alternative for gene regulation is given by CRISPR interference (CRISPRi), which enables transcriptional repression of nearly arbitrary gene targets directed by short guide RNA molecules. In order to demonstrate the use of CRISPRi in the context of dynamic gene circuits, we here replaced one of the nodes of a repressilator circuit by the RNA-guided dCas9 protein. Using single cell experiments in microfluidic reactors we show that this system displays robust relaxation oscillations over multiple periods and over the time course of several days. Through statistical analysis of the single cell data, the potential for the circuit to act as a synthetic pacemaker for cellular processes is evaluated. The use of CRISPRi in the context of an oscillator circuit is found to have profound effects on its dynamics. Specifically, irreversible binding of dCas9-sgRNA appears to prolong the period of the oscillator. Further, we demonstrate that the oscillator affects cellular growth, leading to variations in growth rate with the oscillator’s frequency.

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