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.

scholarly journals BacilloFlex: A modular DNA assembly toolkit forBacillus subtilissynthetic biology

2017 ◽  
Author(s):  
Niels Wicke ◽  
David Radford ◽  
Valeria Verrone ◽  
Anil Wipat ◽  
Christopher E. French
Keyword(s):  
Synthetic Biology ◽  
Cellular Differentiation ◽  
Genetic Manipulation ◽  
Surface Display ◽  
Dna Assembly ◽  
Golden Gate ◽  
E Coli ◽  
Production Platform ◽  
Bacterial Model ◽  
Golden Gate Cloning

AbstractBacillus subtilisis a valuable industrial production platform for proteins, a bacterial model for cellular differentiation and its endospores have been proposed as a vehicle for vaccine delivery. As suchB. subtilisis a major synthetic biology chassis but, unlikeEscherichia coli, lacks a standardized toolbox for genetic manipulation. EcoFlex is a versatile modular DNA assembly toolkit forE. colisynthetic biology based on Golden Gate cloning. Here we introduce BacilloFlex, an extension of the EcoFlex assembly standard toB. subtilis. Transcription units flanked by sequences homologous to loci in theB. subtilisgenome were rapidly assembled by the EcoFlex standard and subsequently chromosomally integrated. At present, BacilloFlex includes a range of multi-functionalB. subtilisspecific parts with applications including metabolic engineering, biosensors and spore surface display. We hope this work will form the foundation of a widely adopted cloning standard forB. subtilisfacilitating collaboration and the sharing of parts.

Consensus architecture of promoters and transcription units in Escherichia coli: design principles for synthetic biology

2017 ◽  
Vol 13 (4) ◽  
pp. 665-676 ◽  
Author(s):  
Cynthia Rangel-Chavez ◽  
Edgardo Galan-Vasquez ◽  
Agustino Martinez-Antonio
Keyword(s):  
Escherichia Coli ◽  
Synthetic Biology ◽  
Design Principles ◽  
Genetic Circuits ◽  
E Coli ◽  
Transcriptional Units

Consensus of the architecture and composition of the elements that form transcriptional units inE. coliand comparison with synthetic genetic circuits.

scholarly journals Golden Gate Assembly of Aerobic and Anaerobic Microbial Bioreporters

2021 ◽  
Author(s):  
Aaron J. Hinz ◽  
Benjamin Stenzler ◽  
Alexandre J. Poulain
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Synthetic Biology ◽  
Reporter Gene ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Fluorescent Microscopy ◽  
Genetic Circuit ◽  
Regulatory Sequences ◽  
Golden Gate

Microbial bioreporters provide direct insight into cellular processes by producing a quantifiable signal dictated by reporter gene expression. The core of a bioreporter is a genetic circuit in which a reporter gene (or operon) is fused to promoter and regulatory sequences that govern its expression. In this study, we develop a system for constructing novel Escherichia coli bioreporters based on Golden Gate assembly, a synthetic biology approach for the rapid and seamless fusion of DNA fragments. Gene circuits are generated by fusing promoter and reporter sequences encoding yellow fluorescent protein, mCherry, bacterial luciferase, and an anaerobically active flavin-based fluorescent protein. We address a barrier to the implementation of Golden Gate assembly by designing a series of compatible destination vectors that can accommodate the assemblies. We validate the approach by measuring the activity of constitutive bioreporters and mercury and arsenic biosensors in quantitative exposure assays. We also demonstrate anaerobic quantification of mercury and arsenic in biosensors that produce flavin-based fluorescent protein, highlighting the expanding range of redox conditions that can be examined by microbial bioreporters. IMPORTANCE Microbial bioreporters are versatile genetic tools with wide-ranging applications, particularly in the field of environmental toxicology. For example, biosensors that produce a signal output in the presence of a specific analyte offer less costly alternatives to analytical methods for the detection of environmental toxins such as mercury and arsenic. Biosensors of specific toxins can also be used to test hypotheses regarding mechanisms of uptake, toxicity, and biotransformation. In this study, we develop an assembly platform that uses a synthetic biology technique to streamline construction of novel Escherichia coli bioreporters that produce fluorescent or luminescent signals either constitutively or in response to mercury and arsenic exposure. Beyond the synthesis of novel biosensors, our assembly platform can be adapted for numerous applications, including labelling bacteria for fluorescent microscopy, developing gene expression systems, and modifying bacterial genomes.

scholarly journals Refactoring of a synthetic raspberry ketone pathway with EcoFlex

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Simon J. Moore ◽  
Yonek B. Hleba ◽  
Sarah Bischoff ◽  
David Bell ◽  
Karen M. Polizzi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
Combinatorial Libraries ◽  
Value Added ◽  
Microtiter Plate ◽  
Fine Tuning ◽  
Golden Gate ◽  
E Coli ◽  
Fine Chemical ◽  
Raspberry Ketone

Abstract Background  A key focus of synthetic biology is to develop microbial or cell-free based biobased routes to value-added chemicals such as fragrances. Originally, we developed the EcoFlex system, a Golden Gate toolkit, to study genes/pathways flexibly using Escherichia coli heterologous expression. In this current work, we sought to use EcoFlex to optimise a synthetic raspberry ketone biosynthetic pathway. Raspberry ketone is a high-value (~ £20,000 kg−1) fine chemical farmed from raspberry (Rubeus rubrum) fruit. Results  By applying a synthetic biology led design-build-test-learn cycle approach, we refactor the raspberry ketone pathway from a low level of productivity (0.2 mg/L), to achieve a 65-fold (12.9 mg/L) improvement in production. We perform this optimisation at the prototype level (using microtiter plate cultures) with E. coli DH10β, as a routine cloning host. The use of E. coli DH10β facilitates the Golden Gate cloning process for the screening of combinatorial libraries. In addition, we also newly establish a novel colour-based phenotypic screen to identify productive clones quickly from solid/liquid culture. Conclusions  Our findings provide a stable raspberry ketone pathway that relies upon a natural feedstock (L-tyrosine) and uses only constitutive promoters to control gene expression. In conclusion we demonstrate the capability of EcoFlex for fine-tuning a model fine chemical pathway and provide a range of newly characterised promoter tools gene expression in E. coli.

scholarly journals Leveraging modern DNA assembly techniques for rapid, markerless genome modification

2016 ◽  
Vol 1 (1) ◽  
Author(s):  
Ilya B Tikh ◽  
James C Samuelson
Keyword(s):  
High Efficiency ◽  
Point Mutations ◽  
Host Cells ◽  
Dna Assembly ◽  
E Coli ◽  
Recombination Efficiency ◽  
Genome Modification ◽  
Counter Selection ◽  
K 12 ◽  
Polymerase Chain Reaction Primers

Abstract The ability to alter the genomic material of a prokaryotic cell is necessary for experiments designed to define the biology of the organism. In addition, the production of biomolecules may be significantly improved by application of engineered prokaryotic host cells. Furthermore, in the age of synthetic biology, speed and efficiency are key factors when choosing a method for genome alteration. To address these needs, we have developed a method for modification of the Escherichia coli genome named FAST-GE for Fast Assembly-mediated Scarless Targeted Genome Editing. Traditional cloning steps such as plasmid transformation, propagation and isolation were eliminated. Instead, we developed a DNA assembly-based approach for generating scarless strain modifications, which may include point mutations, deletions and gene replacements, within 48 h after the receipt of polymerase chain reaction primers. The protocol uses established, but optimized, genome modification components such as I-SceI endonuclease to improve recombination efficiency and SacB as a counter-selection mechanism. All DNA-encoded components are assembled into a single allele-exchange vector named pDEL. We were able to rapidly modify the genomes of both E. coli B and K-12 strains with high efficiency. In principle, the method may be applied to other prokaryotic organisms capable of circular dsDNA uptake and homologous recombination.

scholarly journals Correction to “The Marburg Collection: A Golden Gate DNA Assembly Framework for Synthetic Biology Applications in Vibrio natriegens”

2021 ◽  
Author(s):  
Daniel Stukenberg ◽  
Tobias Hensel ◽  
Josef Hoff ◽  
Benjamin Daniel ◽  
René Inckemann ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Dna Assembly ◽  
Golden Gate

Biological Circuit Components

2014 ◽  
Author(s):  
Richard M. Murray
Keyword(s):  
Synthetic Biology ◽  
Bacterial Chemotaxis ◽  
Dynamical Model ◽  
Genetic Circuit ◽  
Toggle Switch ◽  
Simple Circuit ◽  
Natural Systems ◽  
E Coli ◽  
Feedforward Loop ◽  
Circuit Components

This chapter describes some simple circuit components that have been constructed in E. coli cells using the technology of synthetic biology and then considers a more complicated circuit that already appears in natural systems to implement adaptation. It first analyzes the negatively autoregulated gene fabricated in E. coli bacteria, before turning to the toggle switch, which is composed of two genes that mutually repress each other. The chapter next illustrates a dynamical model of a “repressilator”—an oscillatory genetic circuit consisting of three repressors arranged in a ring fashion. The activator–repressor clock is then considered, alongside an incoherent feedforward loop (IFFL). Finally, the chapter examines bacterial chemotaxis, which E. coli use to move in the direction of increasing nutrients.

scholarly journals Interfacing gene circuits with microelectronics through engineered population dynamics

2020 ◽  
Vol 6 (21) ◽  
pp. eaaz8344 ◽  
Author(s):  
M. Omar Din ◽  
Aida Martin ◽  
Ivan Razinkov ◽  
Nicholas Csicsery ◽  
Jeff Hasty
Keyword(s):  
Population Dynamics ◽  
Synthetic Biology ◽  
Integrated Circuits ◽  
Population Control ◽  
Genetic Circuits ◽  
Bacterial Colony ◽  
Bacterial Response ◽  
Impedance Profile ◽  
Changes Over Time ◽  
Engineered Bacteria

While there has been impressive progress connecting bacterial behavior with electrodes, an attractive observation to facilitate advances in synthetic biology is that the growth of a bacterial colony can be determined from impedance changes over time. Here, we interface synthetic biology with microelectronics through engineered population dynamics that regulate the accumulation of charged metabolites. We demonstrate electrical detection of the bacterial response to heavy metals via a population control circuit. We then implement this approach to a synchronized genetic oscillator where we obtain an oscillatory impedance profile from engineered bacteria. We lastly miniaturize an array of electrodes to form “bacterial integrated circuits” and demonstrate its applicability as an interface with genetic circuits. This approach paves the way for new advances in synthetic biology, analytical chemistry, and microelectronic technologies.

scholarly journals The occurrence of antibiotic-resistant bacteria, including Escherichia coli, in municipal wastewater and river water

2019 ◽  
Vol 100 ◽  
pp. 00061 ◽  
Author(s):  
Adriana Osińska ◽  
Ewa Korzeniewska ◽  
Monika Harnisz ◽  
Sebastian Niestępski ◽  
Piotr Jachimowicz
Keyword(s):  
Escherichia Coli ◽  
Wastewater Treatment ◽  
River Water ◽  
Municipal Wastewater ◽  
High Efficiency ◽  
Treated Wastewater ◽  
Resistant Bacteria ◽  
Antibiotic Resistant Bacteria ◽  
Antibiotic Resistant ◽  
E Coli

Wastewater treatment plants (WWTPs) are major reservoirs of antibiotic-resistant bacteria (ARB) which are transported to the natural environment with discharged effluents. Samples of untreated wastewater (UWW) and treated wastewater (TWW) from four municipal WWTPs and samples of river water collected upstream (URW) and downstream (DRW) from the effluent discharge point were analyzed in the study. The total counts of bacteria resistant to β-lactams and tetracyclines and the counts of antibiotic-resistant Escherichia coli were determined. Antibiotic-resistant bacteria, including antibiotic-resistant E. coli, were removed with up to 99.9% efficiency in the evaluated WWTPs. Despite the above, ARB counts in TWW samples were high at up to 1.25x105 CFU/mL in winter and 1.25x103 CFU/mL in summer. Antibiotic-resistant bacteria were also abundant (up to 103 CFU/ml) in URW and DRW samples collected in winter and summer. In both UWW and TWW samples, the counts of ARB and antibiotic-resistant E. coli were at least one order of magnitude lower in summer than in winter. The study revealed that despite the high efficiency of bacterial removal in the wastewater treatment processes, considerable amounts of ARB are released into the environment with TWW and that the percentage of ARB in total bacterial counts increases after wastewater treatment.

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.

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