scholarly journals A reversible memory switch for plant synthetic biology based on the phage PhiC31 integration system

2019 ◽  
Author(s):  
Bernabé-Orts Joan Miquel ◽  
Quijano-Rubio Alfredo ◽  
Mancheño-Bonillo Javier ◽  
Moles-Casas Victor ◽  
Selma Sara ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Cell Fate ◽  
Regulatory Element ◽  
Living Organism ◽  
Inducible Promoter ◽  
Chemical Stimulus ◽  
Toggle Switch ◽  
Genetic Switch ◽  
Whole Plants ◽  
Plant Synthetic Biology

ABSTRACTPlant synthetic biology aims to contribute to global food security by engineering plants with new or improved functionalities. In recent years, synthetic biology has rapidly advanced from the setup of basic genetic devices to the design of increasingly complex gene circuits able to provide organisms with novel functions. While many bacterial, fungal and mammalian unicellular chassis have been extensively engineered, this progress has been delayed in plants due to their complex multicellular nature and the lack of reliable DNA devices that allow an accurate design of more sophisticated biological circuits. Among these basic devices, gene switches are crucial to deploying new layers of regulation into the engineered organisms. Of special interest are bistable genetic toggle switches, which allow a living organism to exist in two alternative states and switch between them with a minimal metabolic burden. Naturally occurring toggle switches control important decision-making processes such as cell fate and developmental events. We sought to engineer whole plants with an orthogonal genetic toggle switch to be able to regulate artificial functions with minimal interference with their natural pathways. Here we report a bistable toggle memory switch for whole plants based on the phage PhiC31 serine integrase and its cognate recombination directionality factor (RDF). This genetic device was designed to control the transcription of two genes of interest by inversion of a central DNA regulatory element. Each state of the device is defined by one transcriptionally active gene of interest, while the other one remains inactive. The state of the switch can be reversibly modified by the action of the recombination actuators, which were administered externally (e.g. via agroinfiltration), or produced internally in response to an inducible chemical stimulus. We extensively characterized the kinetics, memory, and reversibility of this genetic switch in Nicotiana benthamiana through transient and stable transformation experiments using transgenic plants and hairy roots. Finally, we coupled the integrase expression to an estradiol-inducible promoter as a proof of principle of inducible activation of the switch.

scholarly journals A memory switch for plant synthetic biology based on the phage ϕC31 integration system

2020 ◽  
Vol 48 (6) ◽  
pp. 3379-3394 ◽  
Author(s):  
Joan Miquel Bernabé-Orts ◽  
Alfredo Quijano-Rubio ◽  
Marta Vazquez-Vilar ◽  
Javier Mancheño-Bonillo ◽  
Victor Moles-Casas ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Regulatory Element ◽  
Alternative States ◽  
Gene Circuits ◽  
Site Specific ◽  
Precise Control ◽  
Bacteriophage Φc31 ◽  
Whole Plants ◽  
Transcriptional State

Abstract Synthetic biology has advanced from the setup of basic genetic devices to the design of increasingly complex gene circuits to provide organisms with new functions. While many bacterial, fungal and mammalian unicellular chassis have been extensively engineered, this progress has been delayed in plants due to the lack of reliable DNA parts and devices that enable precise control over these new synthetic functions. In particular, memory switches based on DNA site-specific recombination have been the tool of choice to build long-term and stable synthetic memory in other organisms, because they enable a shift between two alternative states registering the information at the DNA level. Here we report a memory switch for whole plants based on the bacteriophage ϕC31 site-specific integrase. The switch was built as a modular device made of standard DNA parts, designed to control the transcriptional state (on or off) of two genes of interest by alternative inversion of a central DNA regulatory element. The state of the switch can be externally operated by action of the ϕC31 integrase (Int), and its recombination directionality factor (RDF). The kinetics, memory, and reversibility of the switch were extensively characterized in Nicotiana benthamiana plants.

GondenBraid2.0: A comprehensive toolkit for plant synthetic biology

Planta Medica ◽  
2013 ◽  
Vol 79 (13) ◽  
Author(s):  
A Sarrion-Perdigones ◽  
M Vazquez-Vilar ◽  
J Palaci ◽  
A Granell ◽  
D Orzáez
Keyword(s):  
Synthetic Biology ◽  
Plant Synthetic Biology

Enabling plant synthetic biology through genome engineering

2015 ◽  
Vol 33 (2) ◽  
pp. 120-131 ◽  
Author(s):  
Nicholas J. Baltes ◽  
Daniel F. Voytas
Keyword(s):  
Synthetic Biology ◽  
Genome Engineering ◽  
Plant Synthetic Biology

scholarly journals Jagged–Delta asymmetry in Notch signaling can give rise to a Sender/Receiver hybrid phenotype

2015 ◽  
Vol 112 (5) ◽  
pp. E402-E409 ◽  
Author(s):  
Marcelo Boareto ◽  
Mohit Kumar Jolly ◽  
Mingyang Lu ◽  
José N. Onuchic ◽  
Cecilia Clementi ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Cell Fate ◽  
Target Genes ◽  
Tumor Stroma ◽  
Notch Receptor ◽  
Toggle Switch ◽  
Cell Fate Determination ◽  
Asymmetric Effect ◽  
Fate Determination

Notch signaling pathway mediates cell-fate determination during embryonic development, wound healing, and tumorigenesis. This pathway is activated when the ligand Delta or the ligand Jagged of one cell interacts with the Notch receptor of its neighboring cell, releasing the Notch Intracellular Domain (NICD) that activates many downstream target genes. NICD affects ligand production asymmetrically––it represses Delta, but activates Jagged. Although the dynamical role of Notch–Jagged signaling remains elusive, it is widely recognized that Notch–Delta signaling behaves as an intercellular toggle switch, giving rise to two distinct fates that neighboring cells adopt––Sender (high ligand, low receptor) and Receiver (low ligand, high receptor). Here, we devise a specific theoretical framework that incorporates both Delta and Jagged in Notch signaling circuit to explore the functional role of Jagged in cell-fate determination. We find that the asymmetric effect of NICD renders the circuit to behave as a three-way switch, giving rise to an additional state––a hybrid Sender/Receiver (medium ligand, medium receptor). This phenotype allows neighboring cells to both send and receive signals, thereby attaining similar fates. We also show that due to the asymmetric effect of the glycosyltransferase Fringe, different outcomes are generated depending on which ligand is dominant: Delta-mediated signaling drives neighboring cells to have an opposite fate; Jagged-mediated signaling drives the cell to maintain a similar fate to that of its neighbor. We elucidate the role of Jagged in cell-fate determination and discuss its possible implications in understanding tumor–stroma cross-talk, which frequently entails Notch–Jagged communication.

scholarly journals Cell-free genetic devices confer autonomic and adaptive properties to hydrogels

2019 ◽  
Author(s):  
Colette J. Whitfield ◽  
Alice M. Banks ◽  
Gema Dura ◽  
John Love ◽  
Jonathan E. Fieldsend ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Information Processing ◽  
Synthetic Biology ◽  
Smart Materials ◽  
Living Organism ◽  
Chemical Diversity ◽  
Biological Information ◽  
Self Healing ◽  
Wide Range

AbstractSmart materials are able to alter one or more of their properties in response to defined stimuli. Our ability to design and create such materials, however, does not match the diversity and specificity of responses seen within the biological domain. We propose that relocation of molecular phenomena from living cells into hydrogels can be used to confer smart functionality to materials. We establish that cell-free protein synthesis can be conducted in agarose hydrogels, that gene expression occurs throughout the material and that co-expression of genes is possible. We demonstrate that gene expression can be controlled transcriptionally (using in gel gene interactions) and translationally in response to small molecule and nucleic acid triggers. We use this system to design and build a genetic device that can alter the structural property of its chassis material in response to exogenous stimuli. Importantly, we establish that a wide range of hydrogels are appropriate chassis for cell-free synthetic biology, meaning a designer may alter both the genetic and hydrogel components according to the requirements of a given application. We probe the relationship between the physical structure of the gel and in gel protein synthesis and reveal that the material itself may act as a macromolecular crowder enhancing protein synthesis. Given the extensive range of genetically encoded information processing networks in the living kingdom and the structural and chemical diversity of hydrogels, this work establishes a model by which cell-free synthetic biology can be used to create autonomic and adaptive materials.Significance statementSmart materials have the ability to change one or more of their properties (e.g. structure, shape or function) in response to specific triggers. They have applications ranging from light-sensitive sunglasses and drug delivery systems to shape-memory alloys and self-healing coatings. The ability to programme such materials, however, is basic compared to the ability of a living organism to observe, understand and respond to its environment. Here we demonstrate the relocation of biological information processing systems from cells to materials. We achieved this by operating small, programmable genetic devices outside the confines of a living cell and inside hydrogel matrices. These results establish a method for developing materials functionally enhanced with molecular machinery from biological systems.

scholarly journals The Multiplanetary Future of Plant Synthetic Biology

Genes ◽  
2018 ◽  
Vol 9 (7) ◽  
pp. 348 ◽  
Author(s):  
Briardo Llorente ◽  
Thomas Williams ◽  
Hugh Goold
Keyword(s):  
Synthetic Biology ◽  
Plant Synthetic Biology

A molecular basis for transdetermination in Drosophila imaginal discs: interactions between wingless and decapentaplegic signaling

Development ◽  
1998 ◽  
Vol 125 (1) ◽  
pp. 115-124
Author(s):  
L. Maves ◽  
G. Schubiger
Keyword(s):  
Cell Fate ◽  
Molecular Basis ◽  
Regulatory Element ◽  
Ectopic Expression ◽  
Wing Development ◽  
Downstream Target ◽  
Leg Development ◽  
Disc Cells ◽  
Targeted Expression ◽  
Necessary And Sufficient

We are investigating how Drosophila imaginal disc cells establish and maintain their appendage-specific determined states. We have previously shown that ectopic expression of wingless (wg) induces leg disc cells to activate expression of the wing marker Vestigial (Vg) and transdetermine to wing cells. Here we show that ectopic wg expression non-cell-autonomously induces Vg expression in leg discs and that activated Armadillo, a cytosolic transducer of the Wg signal, cell-autonomously induces Vg expression in leg discs, indicating that this Vg expression is directly activated by Wg signaling. We find that ubiquitous expression of wg in leg discs can induce only dorsal leg disc cells to express Vg and transdetermine to wing. Dorsal leg disc cells normally express high levels of decapentaplegic (dpp) and its downstream target, optomotor-blind (omb). We find that high levels of dpp expression, which are both necessary and sufficient for dorsal leg development, are required for wg-induced transdetermination. We show that dorsalization of ventral leg disc cells, through targeted expression of either dpp or omb, is sufficient to allow wg to induce Vg expression and wing fate. Thus, dpp and omb promote both dorsal leg cell fate as well as transdetermination-competent leg disc cells. Taken together, our results show that the Wg and Dpp signaling pathways cooperate to induce Vg expression and leg-towing transdetermination. We also show that a specific vg regulatory element, the vg boundary enhancer, is required for transdetermination. We propose that an interaction between Wg and Dpp signaling can explain why leg disc cells transdetermine to wing and that our results have implications for normal leg and wing development.

scholarly journals Streamlined Construction of the Cyanobacterial CO2-Fixing Organelle via Protein Domain Fusions for Use in Plant Synthetic Biology

2015 ◽  
Vol 27 (9) ◽  
pp. 2637-2644 ◽  
Author(s):  
C. Raul Gonzalez-Esquer ◽  
Tyler B. Shubitowski ◽  
Cheryl A. Kerfeld
Keyword(s):  
Synthetic Biology ◽  
Protein Domain ◽  
Plant Synthetic Biology

scholarly journals Standards for plant synthetic biology: a common syntax for exchange ofDNAparts

2015 ◽  
Vol 208 (1) ◽  
pp. 13-19 ◽  
Author(s):  
Nicola J. Patron ◽  
Diego Orzaez ◽  
Sylvestre Marillonnet ◽  
Heribert Warzecha ◽  
Colette Matthewman ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Plant Synthetic Biology
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