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 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 Optimizing the Production of Recombinant Hydroperoxide Lyase in Escherichia coli Using Statistical Design

Catalysts ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 176
Author(s):  
Sophie Vincenti ◽  
Magali Mariani ◽  
Jessica Croce ◽  
Eva Faillace ◽  
Virginie Brunini-Bronzini de Caraffa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Expression System ◽  
Scale Up ◽  
Food Additives ◽  
Statistical Design ◽  
Green Leaf Volatiles ◽  
Hydroperoxide Lyase ◽  
Soluble Enzyme ◽  
Leaf Volatiles ◽  
Conversion Rates

Hydroperoxide lyase (HPL) catalyzes the synthesis of volatiles C6 or C9 aldehydes from fatty acid hydroperoxides. These short carbon chain aldehydes, known as green leaf volatiles (GLV), are widely used in cosmetic industries and as food additives because of their “fresh green” aroma. To meet the growing demand for natural GLVs, the use of recombinant HPL as a biocatalyst in enzyme-catalyzed processes appears to be an interesting application. Previously, we cloned and expressed a 13-HPL from olive fruit in Escherichia coli and showed high conversion rates (up to 94%) during the synthesis of C6 aldehydes. To consider a scale-up of this process, optimization of the recombinant enzyme production is necessary. In this study, four host-vector combinations were tested. Experimental design and response surface methodology (RSM) were used to optimize the expression conditions. Three factors were considered, i.e., temperature, inducer concentration and induction duration. The Box–Behnken design consisted of 45 assays for each expression system performed in deep-well microplates. The regression models were built and fitted well to the experimental data (R2 coefficient > 97%). The best response (production level of the soluble enzyme) was obtained with E. coli BL21 DE3 cells. Using the optimal conditions, 2277 U L−1of culture of the soluble enzyme was produced in microliter plates and 21,920 U L−1of culture in an Erlenmeyer flask, which represents a 79-fold increase compared to the production levels previously reported.

scholarly journals CRIMoClo plasmids for modular assembly and orthogonal chromosomal integration of synthetic circuits in Escherichia coli

2019 ◽  
Vol 13 (1) ◽  
Author(s):  
Stefano Vecchione ◽  
Georg Fritz
Keyword(s):  
Escherichia Coli ◽  
Synthetic Biology ◽  
Integrated Circuits ◽  
High Efficiency ◽  
Genetic Circuit ◽  
Dna Assembly ◽  
Chromosomal Integration ◽  
Golden Gate ◽  
Genetic Circuits ◽  
E Coli

Abstract Background Synthetic biology heavily depends on rapid and simple techniques for DNA engineering, such as Ligase Cycling Reaction (LCR), Gibson assembly and Golden Gate assembly, all of which allow for fast, multi-fragment DNA assembly. A major enhancement of Golden Gate assembly is represented by the Modular Cloning (MoClo) system that allows for simple library propagation and combinatorial construction of genetic circuits from reusable parts. Yet, one limitation of the MoClo system is that all circuits are assembled in low- and medium copy plasmids, while a rapid route to chromosomal integration is lacking. To overcome this bottleneck, here we took advantage of the conditional-replication, integration, and modular (CRIM) plasmids, which can be integrated in single copies into the chromosome of Escherichia coli and related bacteria by site-specific recombination at different phage attachment (att) sites. Results By combining the modularity of the MoClo system with the CRIM plasmids features we created a set of 32 novel CRIMoClo plasmids and benchmarked their suitability for synthetic biology applications. Using CRIMoClo plasmids we assembled and integrated a given genetic circuit into four selected phage attachment sites. Analyzing the behavior of these circuits we found essentially identical expression levels, indicating orthogonality of the loci. Using CRIMoClo plasmids and four different reporter systems, we illustrated a framework that allows for a fast and reliable sequential integration at the four selected att sites. Taking advantage of four resistance cassettes the procedure did not require recombination events between each round of integration. Finally, we assembled and genomically integrated synthetic ECF σ factor/anti-σ switches with high efficiency, showing that the growth defects observed for circuits encoded on medium-copy plasmids were alleviated. Conclusions The CRIMoClo system enables the generation of genetic circuits from reusable, MoClo-compatible parts and their integration into 4 orthogonal att sites into the genome of E. coli. Utilizing four different resistance modules the CRIMoClo system allows for easy, fast, and reliable multiple integrations. Moreover, utilizing CRIMoClo plasmids and MoClo reusable parts, we efficiently integrated and alleviated the toxicity of plasmid-borne circuits. Finally, since CRIMoClo framework allows for high flexibility, it is possible to utilize plasmid-borne and chromosomally integrated circuits simultaneously. This increases our ability to permute multiple genetic modules and allows for an easier design of complex synthetic metabolic pathways in E. coli.

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 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 Construction of a Nanosensor for Non-Invasive Imaging of Hydrogen Peroxide Levels in Living Cells

Biology ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 430
Author(s):  
Amreen ◽  
Hayssam M. Ali ◽  
Mohammad Ahmad ◽  
Mohamed Z. M. Salem ◽  
Altaf Ahmad
Keyword(s):  
Hydrogen Peroxide ◽  
Real Time ◽  
Mammalian Cells ◽  
Dynamic Range ◽  
Resonance Energy ◽  
Living Cells ◽  
Stress Conditions ◽  
Live Cells ◽  
Cell Physiology ◽  
Broad Dynamic Range

Hydrogen peroxide (H2O2) serves fundamental regulatory functions in metabolism beyond the role as damage signal. During stress conditions, the level of H2O2 increases in the cells and causes oxidative stress, which interferes with normal cell growth in plants and animals. The H2O2 also acts as a central signaling molecule and regulates numerous pathways in living cells. To better understand the generation of H2O2 in environmental responses and its role in cellular signaling, there is a need to study the flux of H2O2 at high spatio–temporal resolution in a real-time fashion. Herein, we developed a genetically encoded Fluorescence Resonance Energy Transfer (FRET)-based nanosensor (FLIP-H2O2) by sandwiching the regulatory domain (RD) of OxyR between two fluorescent moieties, namely ECFP and mVenus. This nanosensor was pH stable, highly selective to H2O2, and showed insensitivity to other oxidants like superoxide anions, nitric oxide, and peroxynitrite. The FLIP-H2O2 demonstrated a broad dynamic range and having a binding affinity (Kd) of 247 µM. Expression of sensor protein in living bacterial, yeast, and mammalian cells showed the localization of the sensor in the cytosol. The flux of H2O2 was measured in these live cells using the FLIP-H2O2 under stress conditions or by externally providing the ligand. Time-dependent FRET-ratio changes were recorded, which correspond to the presence of H2O2. Using this sensor, real-time information of the H2O2 level can be obtained non-invasively. Thus, this nanosensor would help to understand the adverse effect of H2O2 on cell physiology and its role in redox signaling.

scholarly journals Adaptation by stochastic switching of a monostable genetic circuit in Escherichia coli

2011 ◽  
Vol 7 (1) ◽  
pp. 493 ◽  
Author(s):  
Saburo Tsuru ◽  
Nao Yasuda ◽  
Yoshie Murakami ◽  
Junya Ushioda ◽  
Akiko Kashiwagi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genetic Circuit ◽  
Stochastic Switching
Sign in / Sign up

Export Citation Format

Share Document