scholarly journals CRISPR-based gene expression control for synthetic gene circuits

2020 ◽  
Vol 48 (5) ◽  
pp. 1979-1993
Author(s):  
Javier Santos-Moreno ◽  
Yolanda Schaerli
Keyword(s):  
Circuit Design ◽  
Living Cells ◽  
Synthetic Gene ◽  
Cell Behavior ◽  
Gene Circuits ◽  
Synthetic Gene Circuits ◽  
Expression Control ◽  
Gene Expression Control ◽  
Synthetic Circuit ◽  
Forward Engineering

Synthetic gene circuits allow us to govern cell behavior in a programmable manner, which is central to almost any application aiming to harness engineered living cells for user-defined tasks. Transcription factors (TFs) constitute the ‘classic’ tool for synthetic circuit construction but some of their inherent constraints, such as insufficient modularity, orthogonality and programmability, limit progress in such forward-engineering endeavors. Here we review how CRISPR (clustered regularly interspaced short palindromic repeats) technology offers new and powerful possibilities for synthetic circuit design. CRISPR systems offer superior characteristics over TFs in many aspects relevant to a modular, predictable and standardized circuit design. Thus, the choice of CRISPR technology as a framework for synthetic circuit design constitutes a valid alternative to complement or replace TFs in synthetic circuits and promises the realization of more ambitious designs.

scholarly journals Multistable and dynamic CRISPRi-based synthetic circuits

2019 ◽  
Author(s):  
Javier Santos-Moreno ◽  
Eve Tasiudi ◽  
Joerg Stelling ◽  
Yolanda Schaerli
Keyword(s):  
Context Dependency ◽  
Synthetic Gene ◽  
Toggle Switch ◽  
Stripe Pattern ◽  
Gene Circuits ◽  
Synthetic Gene Circuits ◽  
Gene Expression Control ◽  
Unspecific Binding ◽  
High Predictability ◽  
Pattern Forming

AbstractGene expression control based on CRISPRi (clustered regularly interspaced short palindromic repeats interference) has emerged as a powerful tool for creating synthetic gene circuits, both in prokaryotes and in eukaryotes; yet, its lack of cooperativity has been pointed out as a potential obstacle for dynamic or multistable circuit construction. Here we use CRISPRi to build prominent synthetic gene circuits in Escherichia coli. We report the first-ever CRISPRi oscillator (“CRISPRlator”), bistable network (toggle switch) and stripe pattern-forming incoherent feed-forward loop (IFFL). Our circuit designs, conceived to feature high predictability and orthogonality, as well as low metabolic burden and context-dependency, allowed us to achieve robust circuit behaviors. Mathematical modeling suggests that unspecific binding in CRISPRi is essential to establish multistability. Our work demonstrates the wide applicability of CRISPRi in synthetic circuits and paves the way for future efforts towards engineering more complex synthetic networks, boosted by the advantages of CRISPR technology.

scholarly journals Engineering Synthetic Gene Circuits in Living Cells with CRISPR Technology

2016 ◽  
Vol 34 (7) ◽  
pp. 535-547 ◽  
Author(s):  
Barbara Jusiak ◽  
Sara Cleto ◽  
Pablo Perez-Piñera ◽  
Timothy K. Lu
Keyword(s):  
Living Cells ◽  
Synthetic Gene ◽  
Gene Circuits ◽  
Synthetic Gene Circuits

Synthetic Gene Circuits: Design, Implement, and Apply

2021 ◽  
pp. 1-18
Author(s):  
Andrew Lezia ◽  
Arianna Miano ◽  
Jeff Hasty
Keyword(s):  
Synthetic Gene ◽  
Gene Circuits ◽  
Synthetic Gene Circuits

Synthetic Gene Circuits

2014 ◽  
pp. 1-56 ◽  
Author(s):  
Barbara Jusiak ◽  
Ramiz Daniel ◽  
Fahim Farzadfard ◽  
Lior Nissim ◽  
Oliver Purcell ◽  
...  
Keyword(s):  
Synthetic Gene ◽  
Gene Circuits ◽  
Synthetic Gene Circuits

scholarly journals A fluoride-responsive genetic circuit enables in vivo biofluorination in engineered Pseudomonas putida

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Patricia Calero ◽  
Daniel C. Volke ◽  
Phillip T. Lowe ◽  
Charlotte H. Gotfredsen ◽  
David O’Hagan ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Building Blocks ◽  
T7 Rna Polymerase ◽  
Genetic Circuit ◽  
Gene Circuits ◽  
Fluorinated Building Blocks ◽  
Synthetic Gene Circuits ◽  
Cell Factories ◽  
Synthetic Circuit

Abstract Fluorine is a key element in the synthesis of molecules broadly used in medicine, agriculture and materials. Addition of fluorine to organic structures represents a unique strategy for tuning molecular properties, yet this atom is rarely found in Nature and approaches to integrate fluorometabolites into the biochemistry of living cells are scarce. In this work, synthetic gene circuits for organofluorine biosynthesis are implemented in the platform bacterium Pseudomonas putida. By harnessing fluoride-responsive riboswitches and the orthogonal T7 RNA polymerase, biochemical reactions needed for in vivo biofluorination are wired to the presence of fluoride (i.e. circumventing the need of feeding expensive additives). Biosynthesis of fluoronucleotides and fluorosugars in engineered P. putida is demonstrated with mineral fluoride both as only fluorine source (i.e. substrate of the pathway) and as inducer of the synthetic circuit. This approach expands the chemical landscape of cell factories by providing alternative biosynthetic strategies towards fluorinated building-blocks.

scholarly journals Synthetic gene-regulatory networks in the opportunistic human pathogenStreptococcus pneumoniae

2020 ◽  
Vol 117 (44) ◽  
pp. 27608-27619 ◽  
Author(s):  
Robin A. Sorg ◽  
Clement Gallay ◽  
Laurye Van Maele ◽  
Jean-Claude Sirard ◽  
Jan-Willem Veening
Keyword(s):  
Gene Expression ◽  
Virulence Factor ◽  
Gene Regulatory Networks ◽  
Regulatory Networks ◽  
Combinatorial Libraries ◽  
Regulatory Elements ◽  
Synthetic Gene ◽  
Expression Control ◽  
Gene Expression Control ◽  
Gene Regulatory

Streptococcus pneumoniaecan cause disease in various human tissues and organs, including the ear, the brain, the blood, and the lung, and thus in highly diverse and dynamic environments. It is challenging to study how pneumococci control virulence factor expression, because cues of natural environments and the presence of an immune system are difficult to simulate in vitro. Here, we apply synthetic biology methods to reverse-engineer gene expression control inS. pneumoniae. A selection platform is described that allows for straightforward identification of transcriptional regulatory elements out of combinatorial libraries. We present TetR- and LacI-regulated promoters that show expression ranges of four orders of magnitude. Based on these promoters, regulatory networks of higher complexity are assembled, such as logic AND gates and IMPLY gates. We demonstrate single-copy genome-integrated toggle switches that give rise to bimodal population distributions. The tools described here can be used to mimic complex expression patterns, such as the ones found for pneumococcal virulence factors. Indeed, we were able to rewire gene expression of the capsule operon, the main pneumococcal virulence factor, to be externally inducible (YES gate) or to act as an IMPLY gate (only expressed in absence of inducer). Importantly, we demonstrate that these synthetic gene-regulatory networks are functional in an influenza A virus superinfection murine model of pneumonia, paving the way for in vivo investigations of the importance of gene expression control on the pathogenicity ofS. pneumoniae.

scholarly journals Two cellular resource‐based models linking growth and parts characteristics aids the study and optimisation of synthetic gene circuits

2017 ◽  
Vol 1 (1) ◽  
pp. 30-39 ◽  
Author(s):  
Huijuan Wang ◽  
Maurice H.T. Ling ◽  
Tze Kwang Chua ◽  
Chueh Loo Poh
Keyword(s):  
Synthetic Gene ◽  
Gene Circuits ◽  
Synthetic Gene Circuits
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