scholarly journals Engineering a Functional small RNA Negative Autoregulation Network with Model-guided Design

2017 ◽  
Author(s):  
Chelsea Y. Hu ◽  
Melissa K. Takahashi ◽  
Yan Zhang ◽  
Julius B. Lucks
Keyword(s):  
Steady State ◽  
Response Time ◽  
Gene Networks ◽  
Genetic Networks ◽  
Synthetic Gene Networks ◽  
Network Response ◽  
Simple Set ◽  
Negative Autoregulation ◽  
Network Response Time

AbstractRNA regulators are powerful components of the synthetic biology toolbox. Here, we expand the repertoire of synthetic gene networks built from these regulators by constructing a transcriptional negative autoregulation (NAR) network out of small RNAs (sRNAs). NAR network motifs are core motifs of natural genetic networks, and are known for reducing network response time and steady state signal noise. Here we use cell-free transcription-translation (TX-TL) reactions and a computational model to design and prototype sRNA NAR constructs. Using parameter sensitivity analysis, we design a simple set of experiments that allow us to accurately predict NAR function in TX-TL. We transfer successful network designs in vivo and show that our sRNA transcriptional network reduces both network response time and noise in steady-state gene expression. This work broadens our ability to construct increasingly sophisticated RNA genetic networks with predictable function.

scholarly journals Characterizing and Prototyping Genetic Networks with Cell-Free Transcription-Translation Reactions

2015 ◽  
Author(s):  
Melissa K Takahashi ◽  
Clarmyra A. Hayes ◽  
James Chappell ◽  
Zachary Z. Sun ◽  
Richard M Murray ◽  
...  
Keyword(s):  
Gene Networks ◽  
Genetic Networks ◽  
Cellular Behavior ◽  
Network Function ◽  
Design Build ◽  
A Cell ◽  
In Vivo Characterization ◽  
Large Repertoire

A central goal of synthetic biology is to engineer cellular behavior by engineering synthetic gene networks for a variety of biotechnology and medical applications. The process of engineering gene networks often involves an iterative ‘design-build-test’ cycle, whereby the parts and connections that make up the network are built, characterized and varied until the desired network function is reached. Many advances have been made in the design and build portions of this cycle. However, the slow process of in vivo characterization of network function often limits the timescale of the testing step. Cell-free transcription-translation (TX-TL) systems offer a simple and fast alternative to performing these characterizations in cells. Here we provide an overview of a cell-free TX-TL system that utilizes the native Escherichia coli TX-TL machinery, thereby allowing a large repertoire of parts and networks to be characterized. As a way to demonstrate the utility of cell-free TX-TL, we illustrate the characterization of two genetic networks: an RNA transcriptional cascade and a protein regulated incoherent feed-forward loop. We also provide guidelines for designing TX-TL experiments to characterize new genetic networks. We end with a discussion of current and emerging applications of cell free systems.

scholarly journals In-Vivo Real-Time Control of Protein Expression from Endogenous and Synthetic Gene Networks

2014 ◽  
Vol 10 (5) ◽  
pp. e1003625 ◽  
Author(s):  
Filippo Menolascina ◽  
Gianfranco Fiore ◽  
Emanuele Orabona ◽  
Luca De Stefano ◽  
Mike Ferry ◽  
...  
Keyword(s):  
Protein Expression ◽  
Real Time ◽  
Gene Networks ◽  
Synthetic Gene ◽  
Real Time Control ◽  
Synthetic Gene Networks ◽  
Time Control

scholarly journals Two new plasmid post-segregational killing mechanisms for the implementation of synthetic gene networks inE. coli

2018 ◽  
Author(s):  
Alex J H Fedorec ◽  
Tanel Ozdemir ◽  
Anjali Doshi ◽  
Luca Rosa ◽  
Oscar Velazquez ◽  
...  
Keyword(s):  
Real World ◽  
Gene Networks ◽  
Plasmid Stability ◽  
Industrial Biotechnology ◽  
Bacterial Cells ◽  
Two Component System ◽  
Synthetic Gene Networks ◽  
Therapeutic Setting

AbstractPlasmids are the workhorse of both industrial biotechnology and synthetic biology, but ensuring they remain in bacterial cells is a challenge. Antibiotic selection, commonly used in the laboratory, cannot be used to stabilise plasmids in most real-world applications, and inserting dynamical gene networks into the genome is difficult. Plasmids have evolved several mechanisms for stability, one of which, post-segregational killing (PSK), ensures that plasmid-free cells do not grow or survive. Here we demonstrate the plasmid-stabilising capabilities of the axe/txe two component system and the microcin-V system in the probiotic bacteriaEscherichia coliNissle 1917 and show they can outperform the hok/sok system commonly used in biotechnological applications. Using plasmid stability assays, automated flow cytometry analysis, mathematical models and Bayesian statistics we quantified plasmid stabilityin vitro. Further, we used anin vivomouse cancer model to demonstrate plasmid stability in a real-world therapeutic setting. These new PSK systems, plus the developed Bayesian methodology, will have wide applicability in clinical and industrial biotechnology.

scholarly journals Inducible ablation of mouse Langerhans cells diminishes but fails to abrogate contact hypersensitivity

2005 ◽  
Vol 169 (4) ◽  
pp. 569-576 ◽  
Author(s):  
Clare L. Bennett ◽  
Erwin van Rijn ◽  
Steffen Jung ◽  
Kayo Inaba ◽  
Ralph M. Steinman ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Steady State ◽  
Diphtheria Toxin ◽  
Langerhans Cells ◽  
Contact Hypersensitivity ◽  
Relative Importance ◽  
Skin Immunity

Langerhans cells (LC) form a unique subset of dendritic cells (DC) in the epidermis but so far their in vivo functions in skin immunity and tolerance could not be determined, in particular in relation to dermal DC (dDC). Here, we exploit a novel diphtheria toxin (DT) receptor (DTR)/DT-based system to achieve inducible ablation of LC without affecting the skin environment. Within 24 h after intra-peritoneal injection of DT into Langerin-DTR mice LC are completely depleted from the epidermis and only begin to return 4 wk later. LC deletion occurs by apoptosis in the absence of inflammation and, in particular, the dDC compartment is not affected. In LC-depleted mice contact hypersensitivity (CHS) responses are significantly decreased, although ear swelling still occurs indicating that dDC can mediate CHS when necessary. Our results establish Langerin-DTR mice as a unique tool to study LC function in the steady state and to explore their relative importance compared with dDC in orchestrating skin immunity and tolerance.

Synthetic gene networks in mammalian cells

2010 ◽  
Vol 21 (5) ◽  
pp. 690-696 ◽  
Author(s):  
Wilfried Weber ◽  
Martin Fussenegger
Keyword(s):  
Mammalian Cells ◽  
Gene Networks ◽  
Synthetic Gene ◽  
Synthetic Gene Networks
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