scholarly journals In Silico Design and Analysis of Genetic Circuit-Based Whole-Cell Biosensors for Detecting Groundwater Contaminants

2020 ◽  
Author(s):  
Samuel Fajemilua ◽  
Solomon Bada ◽  
M. Ahsanul Islam
Keyword(s):  
Salicylic Acid ◽  
Continuous Monitoring ◽  
Analytical Techniques ◽  
Genetic Circuit ◽  
Genetic Circuits ◽  
Natural Habitats ◽  
Whole Cell ◽  
Mathematical Simulations ◽  
Harmful Effects ◽  
Biology Research

AbstractContaminants of emerging concern (CEC) such as tetracycline, erythromycin, and salicylic acid in groundwater can seriously endanger the environment and human health due to their widespread and everlasting harmful effects. Thus, continuous monitoring of various CEC concentrations in groundwater is essential to ensure the safety, security, and biodiversity of natural habitats. CECs can be detected using whole-cell biosensors for environmental surveillance and monitoring purposes, as they provide a cheaper and more robust alternative to traditional and expensive analytical techniques. In this study, various genetic circuit designs are considered to model three biosensors using the genetic design automation (GDA) software, iBioSim. The genetic circuits were designed to detect multiple CECs, including atrazine, salicylic acid, and tetracycline simultaneously to produce quantitative fluorescent outputs. The biosensor responses and the viability of the genetic circuit designs were further analysed using ODE-based mathematical simulations in iBioSim. The designed circuits and subsequent biosensor modelling presented here, thus, not only show the usefulness and importance of GDA tools, but also highlight their limitations and shortcomings that need to overcome in the future; thereby, providing a practical guidance for further improvement of such tools, so that they can be more effectively and routinely used in synthetic biology research.

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.

Synthesis, Characterization, Applications, and Toxicity of Green Synthesized Nanoparticles

2021 ◽  
Vol 22 ◽  
Author(s):  
João Marcos Pereira Galúcio ◽  
Sorrel Godinho Barbosa de Souza ◽  
Arthur Abinader Vasconcelos ◽  
Alan Kelbis Oliveira Lima ◽  
Kauê Santana da Costa ◽  
...  
Keyword(s):  
Green Synthesis ◽  
Low Cost ◽  
Chemical Properties ◽  
Analytical Techniques ◽  
Industrial Applications ◽  
Synthesis Methods ◽  
Methods Of Synthesis ◽  
Food Applications ◽  
Living Organisms ◽  
Harmful Effects

: Nanotechnology is a cutting-edge area with numerous industrial applications. Nanoparticles are structures that have dimensions ranging from 1–100 nm which exhibit significantly different mechanical, optical, electrical, and chemical properties when compared with their larger counterparts. Synthetic routes that use natural sources, such as plant extracts, honey, and microorganisms are environmentally friendly and low-cost methods that can be used to obtain nanoparticles. These methods of synthesis generate products that are more stable and less toxic than those obtained using conventional methods. Nanoparticles formed by titanium dioxide, zinc oxide, silver, gold, and copper, as well as cellulose nanocrystals are among the nanostructures obtained by green synthesis that have shown interesting applications in several technological industries. Several analytical techniques have also been used to analyze the size, morphology, hydrodynamics, diameter, and chemical functional groups involved in the stabilization of the nanoparticles as well as to quantify and evaluate their formation. Despite their pharmaceutical, biotechnological, cosmetic, and food applications, studies have detected their harmful effects on human health and the environment; and thus, caution must be taken in uses involving living organisms. The present review aims to present an overview of the applications, the structural properties, and the green synthesis methods that are used to obtain nanoparticles, and special attention is given to those obtained from metal ions. The review also presents the analytical methods used to analyze, quantify, and characterize these nanostructures.

Multimaterial bioprinting—minus the printer: Synthetic bacterial patterning with UV-responsive genetic circuits

2020 ◽  
pp. 147807712096337
Author(s):  
Gizem Gumuskaya
Keyword(s):  
Industrial Revolution ◽  
Vibrio Fischeri ◽  
Raw Material ◽  
Genetic Circuit ◽  
Elastic Tissue ◽  
Genetic Circuits ◽  
Bacterial Colony ◽  
Synthetic Dna ◽  
Macro Scale ◽  
Bacterial Lawn

In this paper, we argue that synthetic biology can help us employ living systems’ unique capacity for self-construction and biomaterial production toward developing novel architectural fabrication paradigms, in which both the raw material production and its refinement into a target structure can be merged into a single computational process embedded in the living structure itself. To demonstrate, here we introduce bioPheme, a novel biofabrication method for engineering bacteria to build biomaterial(s) of designer’s choice into arbitrary 2D geometries specified via transient UV tracing. To this end, we present the design, construction, and testing of the enabling synthetic DNA circuit, which, once inserted into a bacterial colony, allows the bacteria to execute spatial computation by interacting with one another based on the if-then rules encoded in this circuit. At the heart of this genetic circuit is a pair of UV sensor – actuator, and a pair of cell-to-cell signal transmitter – receptor modules, created with genes extracted from the virus λ Phage and marine bacterium Vibrio fischeri, respectively. These modules are wired together to help designers engineer bacteria to build macro-scale structures with seamlessly integrated biomaterials, thereby bridge the molecular and architectural scales. In this way, a bacterial lawn can be programmed to produce different objects with complementary biomaterial compositions, such as a biomineralized superstructure and an elastic tissue filling in-between. In summary, this paper focuses on how scientists’ increasing ability to harness the innate computational capacity of living cells can help designers create self-constructing structures for architectural biofabrication. Through the discussions in this paper, we aim to initiate a shift in today’s biodesign practices toward a greater appreciation and adoption of bottom-up governance of living structures. We are confident that such a paradigm shift will allow for more efficient and sustainable biofabrication systems in the 4th industrial revolution and beyond.

Interaction of salicylic acid with zirconium diphosphate and its reactivity toward uranium (VI)

2018 ◽  
Vol 106 (4) ◽  
pp. 291-300
Author(s):  
Nidia García-González ◽  
Eduardo Ordoñez-Regil ◽  
María Guadalupe Almazán-Torres ◽  
Eric Simoni
Keyword(s):  
Salicylic Acid ◽  
Photoelectron Spectroscopy ◽  
Room Temperature ◽  
Analytical Techniques ◽  
Batch Method ◽  
Sorption Data ◽  
X Ray ◽  
Force Microscopy ◽  
Atomic Force ◽  
Surface Modified

AbstractThe interaction of salicylic acid with zirconium diphosphate surface and its reactivity toward uranium (VI) was investigated. The interaction of salicylic acid with zirconium diphosphate was firstly studied using several analytical techniques including atomic force microscopy, scanning electron microscopy and X-ray photoelectron spectroscopy. The sorption of uranium (VI) onto surface-modified zirconium diphosphate was evaluated by the classical batch method at room temperature. This study showed that the uranium (VI) sorption onto zirconium diphosphate is influenced by the presence of salicylic acid. A fluorescence spectroscopy study revealed the presence of a uranyl specie onto the modified solid surface. The spectroscopy results were then used to restrain the modeling of experimental sorption data, which are interpreted in terms of a constant capacitance model using the FITEQL code. The results indicated that interaction between the uranium (VI) and the surface of zirconium diphosphate modified with salicylic acid leads to the formation of a ternary surface complex.

scholarly journals Carbonates’ precipitation by halophilic bacteria as a potential biosignature for the search for life on Mars

2018 ◽  
Author(s):  
Bianca de Freitas Brenha ◽  
Douglas Galante ◽  
Monica Sánchez Róman ◽  
Rob van Spanning ◽  
Murilo de Carvalho ◽  
...  
Keyword(s):  
High Salinity ◽  
Halophilic Bacteria ◽  
Analytical Techniques ◽  
Hypersaline Lake ◽  
Mineral Formation ◽  
Sedimentary Environments ◽  
Life On Mars ◽  
Protection Mechanisms ◽  
Hypersaline Lagoon ◽  
Harmful Effects

The mechanisms of Mg-carbonate precipitation in the Earth's modern sedimentary environments has not yet been completely elucidated. However, it is known that the microbial activity is significant to facilitate or induce mineral formation. The organic EPS matrix secreted by microorganisms provide an ideal physicochemical environment to the mineral nucleation. Sediments and water samples were collected at Lagoa Vermelha, Araruama (RJ), Brazil. This lagoon is characterized of being a hypersaline lake, where we can find a range of microorganisms known as halophilic and halotolerant extremophiles that have adaptation strategies to compensate for the harmful effects of high salinity, such as EPS biosynthesis, which is one of the most common protection mechanisms in bacteria, helping to maintain the integrity of their cells’membrane. In the present project, the bacterial capability of bioprecipitation using bacterial isolates from a hypersaline lagoon was investigated by combining microbiological, microscopic and geochemical analytical techniques. The isolates were evaluated for their ability to produce bioprecipitates using this multi-technique approach that includes Scanning Electron Microscopy (SEM) with EDS, XRD and Raman spectroscopy. It was possible to characterize the carbonates formed by the bacteria isolated. Finally, these carbonates could represent a potential target for astrobiological studies of potential potential biosignatures for the search for life beyond Earth.

scholarly journals Genetic Circuits Combined with Machine Learning Provides Fast Responding Living Sensors

2020 ◽  
Author(s):  
Behide Saltepe ◽  
Eray Ulaş Bozkurt ◽  
Murat Alp Güngen ◽  
A. Ercüment Çiçek ◽  
Urartu Özgür Şafak Şeker
Keyword(s):  
Response Time ◽  
Short Term Memory ◽  
Cost Effective ◽  
Genetic Circuit ◽  
Analyte Concentration ◽  
Genetic Circuits ◽  
Wide Range ◽  
Biomedical Diagnostics ◽  
Long Short Term Memory ◽  
Increasing Demand

AbstractWhole cell biosensors (WCBs) have become prominent in many fields from environmental analysis to biomedical diagnostics thanks to advanced genetic circuit design principles. Despite increasing demand on cost effective and easy-to-use assessment methods, a considerable amount of WCBs retains certain drawbacks such as long response time, low precision and accuracy. Furthermore, the output signal level does not correspond to a specific analyte concentration value but shows comparative quantification. Here, we utilized a neural network-based architecture to improve the aforementioned features of WCBs and engineered a gold sensing WCB which has a long response time (18 h). Two Long-Short Term-Memory (LSTM)-based networks were integrated to assess both ON/OFF and concentration dependent states of the sensor output, respectively. We demonstrated that binary (ON/OFF) network was able to distinguish between ON/OFF states as early as 30 min with 78% accuracy and over 98% in 3 h. Furthermore, when analyzed in analog manner, we demonstrated that network can classify the raw fluorescence data into pre-defined analyte concentration groups with high precision (82%) in 3 h. This approach can be applied to a wide range of WCBs and improve rapidness, simplicity and accuracy which are the main challenges in synthetic biology enabled biosensing.

scholarly journals Trade-offs in Robustness to Perturbations of Bacterial Population Controllers

2020 ◽  
Author(s):  
Cameron McBride ◽  
Domitilla Del Vecchio
Keyword(s):  
Population Size ◽  
Cell Population ◽  
Resource Sharing ◽  
Genetic Circuit ◽  
Genetic Circuits ◽  
Genetic Population ◽  
Considerable Difficulty ◽  
Trade Offs ◽  
Multiple Cell ◽  
Circuit Components

AbstractSynthetic biology applications have the potential to have lasting impact; however, there is considerable difficulty in scaling up engineered genetic circuits. One of the current hurdles is resource sharing, where different circuit components become implicitly coupled through the host cell’s pool of resources, which may destroy circuit function. One potential solution around this problem is to distribute genetic circuit components across multiple cell strains and control the cell population size using a population controller. In these situations, perturbations in the availability of cellular resources, such as due to resource sharing, will affect the performance of the population controller. In this work, we model a genetic population controller implemented by a genetic circuit while considering perturbations in the availability of cellular resources. We analyze how these intracellular perturbations and extracellular disturbances to cell growth affect cell population size. We find that it is not possible to tune the population controller’s gain such that the population density is robust to both extracellular disturbances and perturbations to the pool of available resources.

scholarly journals Synthetic metabolic computation in a bioluminescence-sensing system

2019 ◽  
Vol 47 (19) ◽  
pp. 10464-10474 ◽  
Author(s):  
Natalia Barger ◽  
Phyana Litovco ◽  
Ximing Li ◽  
Mouna Habib ◽  
Ramez Daniel
Keyword(s):  
Protein Interactions ◽  
Detection Threshold ◽  
Living Cells ◽  
Genetic Circuit ◽  
Biological Research ◽  
Protein Protein Interactions ◽  
Genetic Circuits ◽  
Logic Function ◽  
Single Input ◽  
Logic Functions

Abstract Bioluminescence is visible light produced and emitted by living cells using various biological systems (e.g. luxCDABE cassette). Today, this phenomenon is widely exploited in biological research, biotechnology and medical applications as a quantitative technique for the detection of biological signals. However, this technique has mostly been used to detect a single input only. In this work, we re-engineered the complex genetic structure of luxCDABE cassette to build a biological unit that can detect multi-inputs, process the cellular information and report the computation results. We first split the luxCDABE operon into several parts to create a genetic circuit that can compute a soft minimum in living cells. Then, we used the new design to implement an AND logic function with better performance as compared to AND logic functions based on protein-protein interactions. Furthermore, by controlling the reverse reaction of the luxCDABE cassette independently from the forward reaction, we built a comparator with a programmable detection threshold. Finally, we applied the redesigned cassette to build an incoherent feedforward loop that reduced the unwanted crosstalk between stress-responsive promoters (recA, katG). This work demonstrates the construction of genetic circuits that combine regulations of gene expression with metabolic pathways, for sensing and computing in living cells.

scholarly journals Rosa26 docking sites for investigating genetic circuit silencing in stem cells

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Michael Fitzgerald ◽  
Mark Livingston ◽  
Chelsea Gibbs ◽  
Tara L Deans
Keyword(s):  
Stem Cells ◽  
Synthetic Biology ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Genetic Circuit ◽  
Genetic Circuits ◽  
Immortalized Cell Lines ◽  
Site Specific Integration ◽  
Docking Sites ◽  
Immortalized Cell

Abstract Approaches in mammalian synthetic biology have transformed how cells can be programmed to have reliable and predictable behavior, however, the majority of mammalian synthetic biology has been accomplished using immortalized cell lines that are easy to grow and easy to transfect. Genetic circuits that integrate into the genome of these immortalized cell lines remain functional for many generations, often for the lifetime of the cells, yet when genetic circuits are integrated into the genome of stem cells gene silencing is observed within a few generations. To investigate the reactivation of silenced genetic circuits in stem cells, the Rosa26 locus of mouse pluripotent stem cells was modified to contain docking sites for site-specific integration of genetic circuits. We show that the silencing of genetic circuits can be reversed with the addition of sodium butyrate, a histone deacetylase inhibitor. These findings demonstrate an approach to reactivate the function of genetic circuits in pluripotent stem cells to ensure robust function over many generations. Altogether, this work introduces an approach to overcome the silencing of genetic circuits in pluripotent stem cells that may enable the use of genetic circuits in pluripotent stem cells for long-term function.

An Autonomous In Vivo Dual Selection Protocol for Boolean Genetic Circuits

2015 ◽  
Vol 21 (2) ◽  
pp. 247-260 ◽  
Author(s):  
David Beneš ◽  
Petr Sosík ◽  
Alfonso Rodríguez-Patón
Keyword(s):  
Rational Design ◽  
Selection Process ◽  
Logic Gates ◽  
Genetic Circuit ◽  
Evolutionary Engineering ◽  
Genetic Circuits ◽  
E Coli ◽  
Search And Selection ◽  
Selection Of

Success in synthetic biology depends on the efficient construction of robust genetic circuitry. However, even the direct engineering of the simplest genetic elements (switches, logic gates) is a challenge and involves intense lab work. As the complexity of biological circuits grows, it becomes more complicated and less fruitful to rely on the rational design paradigm, because it demands many time-consuming trial-and-error cycles. One of the reasons is the context-dependent behavior of small assembly parts (like BioBricks), which in a complex environment often interact in an unpredictable way. Therefore, the idea of evolutionary engineering (artificial directed in vivo evolution) based on screening and selection of randomized combinatorial genetic circuit libraries became popular. In this article we build on the so-called dual selection technique. We propose a plasmid-based framework using toxin-antitoxin pairs together with the relaxase conjugative protein, enabling an efficient autonomous in vivo evolutionary selection of simple Boolean circuits in bacteria (E. coli was chosen for demonstration). Unlike previously reported protocols, both on and off selection steps can run simultaneously in various cells in the same environment without human intervention; and good circuits not only survive the selection process but are also horizontally transferred by conjugation to the neighbor cells to accelerate the convergence rate of the selection process. Our directed evolution strategy combines a new dual selection method with fluorescence-based screening to increase the robustness of the technique against mutations. As there are more orthogonal toxin-antitoxin pairs in E. coli, the approach is likely to be scalable to more complex functions. In silico experiments based on empirical data confirm the high search and selection capability of the protocol.

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