scholarly journals Start-Stop Assembly: a functionally scarless DNA assembly system optimised for metabolic engineering

2018 ◽  
Author(s):  
George M. Taylor ◽  
Paweł M. Mordaka ◽  
John T. Heap
Keyword(s):  
Metabolic Engineering ◽  
Design Space ◽  
Combinatorial Libraries ◽  
Dna Assembly ◽  
Destination Vector ◽  
E Coli ◽  
Assembly Method ◽  
Wide Range ◽  
Combinatorial Assembly ◽  
Sequence Boundaries

ABSTRACTDNA assembly allows individual DNA constructs or designed mixtures to be assembled quickly and reliably. Most methods are either: (i) Modular, easily scalable and suitable for combinatorial assembly, but leave undesirable ‘scar’ sequences; or (ii) bespoke (non-modular), scarless but less suitable for construction of combinatorial libraries. Both have limitations for metabolic engineering. To overcome this trade-off we devised Start-Stop Assembly, a multi-part, modular DNA assembly method which is both functionally scarless and suitable for combinatorial assembly. Crucially, 3 bp overhangs corresponding to start and stop codons are used to assemble coding sequences into expression units, avoiding scars at sensitive coding sequence boundaries. Building on this concept, a complete DNA assembly framework was designed and implemented, allowing assembly of up to 15 genes from up to 60 parts (or mixtures); monocistronic, operon-based or hybrid configurations; and a new streamlined assembly hierarchy minimising the number of vectors. Only one destination vector is required per organism, reflecting our optimisation of the system for metabolic engineering in diverse organisms. Metabolic engineering using Start-Stop Assembly was demonstrated by combinatorial assembly of carotenoid pathways inE. coliresulting in a wide range of carotenoid production and colony size phenotypes indicating the intended exploration of design space.GRAPHICAL ABSTRACT

scholarly journals Direct preparation of Cas9 ribonucleoprotein from E. coli for PCR-free seamless DNA assembly

2018 ◽  
Author(s):  
Wenqiang Li ◽  
Shuntang Li ◽  
Jie Qiao ◽  
Fei Wang ◽  
Yang Liu ◽  
...  
Keyword(s):  
Large Scale ◽  
Genome Engineering ◽  
Restriction Enzymes ◽  
General Strategy ◽  
Dna Assembly ◽  
E Coli ◽  
Assembly Method ◽  
Direct Preparation

AbstractCRISPR-Cas9 is a versatile and powerful genome engineering tool. Recently, Cas9 ribonucleoprotein (RNP) complexes have been used as promising biological tools with plenty of in vivo and in vitro applications, but there are by far no efficient methods to produce Cas9 RNP at large scale and low cost. Here, we describe a simple and effective approach for direct preparation of Cas9 RNP from E. coli by co-expressing Cas9 and target specific single guided RNAs. The purified RNP showed in vivo genome editing ability, as well as in vitro endonuclease activity that combines with an unexpected superior stability to enable routine uses in molecular cloning instead of restriction enzymes. We further develop a RNP-based PCR-free method termed Cas-Brick in a one-step or cyclic way for seamless assembly of multiple DNA fragments with high fidelity up to 99%. Altogether, our findings provide a general strategy to prepare Cas9 RNP and supply a convenient and cost-effective DNA assembly method as an invaluable addition to synthetic biological toolboxes.

scholarly journals Combinatorial Assembly and Optimisation of Designer Cellulosomes - A Galactomannan Case Study

2021 ◽  
Author(s):  
Julie Vanderstraeten ◽  
Maria João Maurício da Fonseca ◽  
Philippe De Groote ◽  
Dennis Grimon ◽  
Hans Gerstmans ◽  
...  
Keyword(s):  
Dna Assembly ◽  
Assembly Method ◽  
Rapid Construction ◽  
Starting Point ◽  
Combinatorial Assembly ◽  
Designer Cellulosome ◽  
Enzyme Complexes ◽  
Enzyme Ratio ◽  
Combinatorial Arrangements

Abstract Background: Designer cellulosomes are self-assembled chimeric enzyme complexes that can be used to improve lignocellulosic biomass degradation. They are composed of a synthetic multimodular backbone protein, termed the scaffoldin, and a range of different chimeric docking enzymes that degrade polysaccharides. Over the years, several functional designer cellulosomes have been constructed. Since many parameters influence the efficiency of these multi-enzyme complexes, there is a need to optimise designer cellulosome architecture by testing combinatorial arrangements of docking enzyme and scaffoldin variants. However, the modular cloning procedures are tedious and cumbersome. Results: VersaTile is a combinatorial DNA assembly method, allowing the rapid construction and thus comparison of a range of modular proteins. Here, we present the extension of the VersaTile platform to facilitate the construction of designer cellulosomes. We have constructed a tile repository, composed of dockerins, cohesins, linkers, tags and enzymatically active modules. The developed toolbox allows us to efficiently create and optimise designer cellulosomes at an unprecedented speed. As a proof of concept, a trivalent designer cellulosome able to degrade the specific hemicellulose substrate, galactomannan, was constructed and optimised. The main factors influencing cellulosome efficiency were found to be the selected dockerins and linkers and the docking enzyme ratio on the scaffoldin. The optimised designer cellulosome was able to hydrolyse the galactomannan polysaccharide and release mannose and galactose monomers. Conclusion: We have eliminated one of the main technical hurdles in the designer cellulosome field and anticipate the VersaTile platform to be a starting point in the development of more elaborate multi-enzyme complexes.

scholarly journals Automation assisted anaerobic phenotyping for metabolic engineering

2021 ◽  
Author(s):  
Kaushik Raj ◽  
Naveen Venayak ◽  
Patrick Diep ◽  
Sai Akhil Golla ◽  
Alexander F. Yakunin ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
High Throughput ◽  
Large Scale ◽  
Small Scale ◽  
Chemical Production ◽  
E Coli ◽  
Strain Design ◽  
Phenotypic Screen ◽  
Wide Range ◽  
Scale Down

Microorganisms can be metabolically engineered to produce a wide range of commercially important chemicals. Advancements in computational strategies for strain design and synthetic biological techniques to construct the designed strains have facilitated the generation of large libraries of potential candidates for chemical production. Consequently, there is a need for a high-throughput, laboratory scale methods to characterize and screen these candidates to select strains for further investigation in large scale fermentation processes. Several small-scale fermentation techniques, in conjunction with laboratory automation have enhanced the throughput of enzyme and strain phenotyping experiments. However, such high throughput experimentation typically entails large operational costs and generate massive amounts of laboratory plastic waste. In this work, we develop an eco-friendly automation workflow that effectively calibrates and decontaminates fixed-tip liquid handling systems to reduce tip waste. We also investigate inexpensive methods to establish anaerobic conditions in microplates for high-throughput anaerobic phenotyping. To validate our phenotyping platform, we assess its performance in two case studies - an anaerobic enzyme screen, and a microbial phenotypic screen. We used our automation platform to investigate conditions under which several strains of E. coli exhibit the same phenotypes in 0.5 L bioreactors and in our scaled-down fermentation platform. Further, we propose the use of dimensionality reduction through t-distributed stochastic neighbours embedding, in conjunction with our phenotyping platform to serve as an effective scale-down model for bioreactor phenotypes. By integrating an in-house data-analysis pipeline, we were able to accelerate the 'test' phase of the design-build-test-learn cycle of metabolic engineering.

scholarly journals A VersaTile-driven platform for rapid hit-to-lead development of engineered lysins

2020 ◽  
Vol 6 (23) ◽  
pp. eaaz1136 ◽  
Author(s):  
H. Gerstmans ◽  
D. Grimon ◽  
D. Gutiérrez ◽  
C. Lood ◽  
A. Rodríguez ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Bacterial Pathogen ◽  
Combinatorial Libraries ◽  
Resistant Bacteria ◽  
Dna Assembly ◽  
Burn Wound ◽  
Development Platform ◽  
High Antibacterial Activity ◽  
Wound Model ◽  
Assembly Method

Health care authorities are calling for new antibacterial therapies to cope with the global emergence of antibiotic-resistant bacteria. Bacteriophage-encoded lysins are a unique class of antibacterials with promising (pre)clinical progress. Custom engineering of lysins allows for the creation of variants against potentially any bacterial pathogen. We here present a high-throughput hit-to-lead development platform for engineered lysins. The platform is driven by VersaTile, a new DNA assembly method for the rapid construction of combinatorial libraries of engineered lysins. We constructed approximately 10,000 lysin variants. Using an iterative screening procedure, we identified a lead variant with high antibacterial activity against Acinetobacter baumannii in human serum and an ex vivo pig burn wound model. This generic platform could offer new opportunities to populate the preclinical pipeline with engineered lysins for diverse (therapeutic) applications.

Small Molecule Permeation Across Membrane Channels: Chemical Modification to Quantify Transport Across OmpF

2018 ◽  
Author(s):  
Jiajun Wang ◽  
Jayesh Arun Bafna ◽  
Satya Prathyusha Bhamidimarri ◽  
Mathias Winterhalter
Keyword(s):  
Dwell Time ◽  
Covalent Binding ◽  
Channel Structure ◽  
Ion Current ◽  
E Coli ◽  
Current Fluctuation ◽  
Wide Range ◽  
Outer Membrane Channel ◽  
Biological Channels ◽  
Constriction Region

Biological channels facilitate the exchange of small molecules across membranes, but surprisingly there is a lack of general tools for the identification and quantification of transport (i.e., translocation and binding). Analyzing the ion current fluctuation of a typical channel with its constriction region in the middle does not allow a direct conclusion on successful transport. For this, we created an additional barrier acting as a molecular counter at the exit of the channel. To identify permeation, we mainly read the molecule residence time in the channel lumen as the indicator whether the molecule reached the exit of the channel. As an example, here we use the well-studied porin, OmpF, an outer membrane channel from <i>E. coli</i>. Inspection of the channel structure suggests that aspartic acid at position 181 is located below the constriction region (CR) and we subsequently mutated this residue to cysteine, where else cysteine free and functionalized it by covalent binding with 2-sulfonatoethyl methanethiosulfonate (MTSES) or the larger glutathione (GLT) blockers. Using the dwell time as the signal for transport, we found that both mono-arginine and tri-arginine permeation process is prolonged by 20% and 50% respectively through OmpF<sub>E181C</sub>MTSES, while the larger sized blocker modification OmpF<sub>E181C</sub>GLT drastically decreased the permeation of mono-arginine by 9-fold and even blocked the pathway of the tri-arginine. In case of the hepta-arginine as substrate, both chemical modifications led to an identical ‘blocked’ pattern observed by the dwell time of ion current fluctuation of the OmpF<sub>wt</sub>. As an instance for antibiotic permeation, we analyzed norfloxacin, a fluoroquinolone antimicrobial agent. The modulation of the interaction dwell time suggests possible successful permeation of norfloxacin across OmpF<sub>wt</sub>. This approach may discriminate blockages from translocation events for a wide range of substrates. A potential application could be screening for scaffolds to improve the permeability of antibiotics.

An Improved Membrane Filtration Method for Enumeration of Faecal Coliforms and E. Coli by a Fluorogenic β-Glucuronidase Assay

1993 ◽  
Vol 27 (3-4) ◽  
pp. 267-270 ◽  
Author(s):  
M. T. Augoustinos ◽  
N. A. Grabow ◽  
B. Genthe ◽  
R. Kfir
Keyword(s):  
Escherichia Coli ◽  
Water Samples ◽  
Membrane Filtration ◽  
Faecal Coliforms ◽  
Environmental Waters ◽  
Filtration Method ◽  
E Coli ◽  
Wide Range ◽  
Pure Cultures ◽  
Glucuronidase Assay

A fluorogenic β-glucuronidase assay comprising membrane filtration followed by selective enumeration on m-FC agar at 44.5°C and further confirmation using tlie 4-metliylumbelliferyl-β-D-glucuronide (MUG) containing medium was evaluated for the detection of Escherichia coli in water. A total of 200 typical blue and non-typical blue colonies were isolated from sea and fresh water samples using initial selective enumeration on m-FC agar. Pure cultures of the selected colonies were further tested using the MUG assay and identified using the API 20E method. Of the colonies tested which were shown to be positive using the MUG assay 99.4% were Escherichia coli. The results of this study indicate the combination of the m-FC method followed by the MUG assay to be highly efficient for the selection and confirmation of E. coli from a wide range of environmental waters.

Design of Synthetic Quorum Sensing Achieving Induction Timing-Independent Signal Stabilization for Dynamic Metabolic Engineering of E. coli

2021 ◽  
Author(s):  
Yuki Soma ◽  
Masatomo Takahashi ◽  
Yuri Fujiwara ◽  
Tamaki Shinohara ◽  
Yoshihiro Izumi ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Quorum Sensing ◽  
E Coli

scholarly journals Metabolic engineering in woody plants: challenges, advances, and opportunities

aBIOTECH ◽  
2021 ◽  
Author(s):  
Shu Yu ◽  
Cody S. Bekkering ◽  
Li Tian
Keyword(s):  
Renewable Energy ◽  
Metabolic Engineering ◽  
Ecosystem Services ◽  
Plant Transformation ◽  
Woody Plants ◽  
Woody Plant ◽  
High Biomass ◽  
Generation Times ◽  
Wide Range ◽  
Biochemical Diversity

AbstractWoody plant species represent an invaluable reserve of biochemical diversity to which metabolic engineering can be applied to satisfy the need for commodity and specialty chemicals, pharmaceuticals, and renewable energy. Woody plants are particularly promising for this application due to their low input needs, high biomass, and immeasurable ecosystem services. However, existing challenges have hindered their widespread adoption in metabolic engineering efforts, such as long generation times, large and highly heterozygous genomes, and difficulties in transformation and regeneration. Recent advances in omics approaches, systems biology modeling, and plant transformation and regeneration methods provide effective approaches in overcoming these outstanding challenges. Promises brought by developments in this space are steadily opening the door to widespread metabolic engineering of woody plants to meet the global need for a wide range of sustainably sourced chemicals and materials.

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.

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