One step DNA assembly for combinatorial metabolic engineering

2014 ◽  
Vol 23 ◽  
pp. 70-77 ◽  
Author(s):  
Pieter Coussement ◽  
Jo Maertens ◽  
Joeri Beauprez ◽  
Wouter Van Bellegem ◽  
Marjan De Mey
Keyword(s):  
Metabolic Engineering ◽  
Dna Assembly ◽  
One Step

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

2018 ◽  
Vol 47 (3) ◽  
pp. e17-e17 ◽  
Author(s):  
George M Taylor ◽  
Paweł M Mordaka ◽  
John T Heap
Keyword(s):  
Metabolic Engineering ◽  
Assembly System ◽  
Dna Assembly

scholarly journals Customized one-step preparation of sgRNA transcription templates via overlapping PCR Using short primers and its application in vitro and in vivo gene editing

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Zheng Hu ◽  
Li Wang ◽  
Zhaoying Shi ◽  
Jing Jiang ◽  
Xiangning Li ◽  
...  
Keyword(s):  
Dna Assembly ◽  
Dna Templates ◽  
Core Fragment ◽  
Cas9 Nuclease ◽  
Overlap Extension ◽  
One Step ◽  
Dna Fragment ◽  
Polymerase Chain

Abstract Overlap extension polymerase chain reaction (PCR) is a powerful technology for DNA assembly. Based on this technology, we synthesized DNA templates, which were transcribed into sgRNA in vitro, and further detected their efficiency of purified sgRNAs with Cas9 nuclease. The sgRNAs synthesized by this approach can effectively cleave the DNA fragments of interest in vitro and in vivo. Compared with the conventional method for generating sgRNA, it does not require construction of recombinant plasmids and design of primers to amplify sgRNA core fragment. Only several short primers with overlapped sequences are needed to assemble a DNA fragment as the template of sgRNA. This modified and simplified method is highly applicable and less time-consuming.

scholarly journals Challenges and opportunities of bioprocessing 5-aminolevulinic acid using genetic and metabolic engineering: a critical review

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Ying-Chen Yi ◽  
I-Tai Shih ◽  
Tzu-Hsuan Yu ◽  
Yen-Ju Lee ◽  
I-Son Ng
Keyword(s):  
Metabolic Engineering ◽  
Pyridoxal Phosphate ◽  
Aminolevulinic Acid ◽  
Tca Cycle ◽  
Industrial Applications ◽  
Trna Synthetase ◽  
The Us ◽  
Challenges And Opportunities ◽  
One Step ◽  
C5 Pathway

Abstract5-Aminolevulinic acid (5-ALA), a non-proteinogenic five-carbon amino acid, has received intensive attentions in medicine due to its approval by the US Food and Drug Administration (FDA) for cancer diagnosis and treatment as photodynamic therapy. As chemical synthesis of 5-ALA performed low yield, complicated processes, and high cost, biosynthesis of 5-ALA via C4 (also called Shemin pathway) and C5 pathway related to heme biosynthesis in microorganism equipped more advantages. In C4 pathway, 5-ALA is derived from condensation of succinyl-CoA and glycine by 5-aminolevulic acid synthase (ALAS) with pyridoxal phosphate (PLP) as co-factor in one-step biotransformation. The C5 pathway involves three enzymes comprising glutamyl-tRNA synthetase (GltX), glutamyl-tRNA reductase (HemA), and glutamate-1-semialdehyde aminotransferase (HemL) from α-ketoglutarate in TCA cycle to 5-ALA and heme. In this review, we describe the recent results of 5-ALA production from different genes and microorganisms via genetic and metabolic engineering approaches. The regulation of different chassis is fine-tuned by applying synthetic biology and boosts 5-ALA production eventually. The purification process, challenges, and opportunities of 5-ALA for industrial applications are also summarized.

scholarly journals Y la biología se convirtió en ingeniería: La adopción de estándares para sistemas vivos

2020 ◽  
Author(s):  
Victor De Lorenzo
Keyword(s):  
Synthetic Biology ◽  
Production Process ◽  
Industrial Production ◽  
Industrial Design ◽  
Dna Assembly ◽  
Fundamental Knowledge ◽  
Biological Objects ◽  
Activity Measurements ◽  
One Step ◽  
Design Construction

For decades, molecular biologists have been removing or inserting genes into all kinds of organisms with biotechnological intent or simply to generate fundamental knowledge. Synthetic biology (SynBio) goes one step further by incorporating conceptual frameworks from computing, electronics, and industrial design. This change makes it possible to conceive the creation of complex biological objects that were previously considered too difficult to assemble. To do this, the stages of any industrial production process must be adopted: design, construction of the components, assembly, and final manufacture. This objective requires standardisation of the physical and functional formats of the components involved, DNA assembly methods, activity measurements, and descriptive languages.

scholarly journals OSCA: A one-step construction of adenovirus system using the Gibson DNA Assembly technology

2021 ◽  
Author(s):  
Na Ni ◽  
Fang Deng ◽  
Fang He ◽  
Hao Wang ◽  
Deyao Shi ◽  
...  
Keyword(s):  
Dna Assembly ◽  
One Step ◽  
Assembly Technology

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 Method of preparing an equimolar DNA mixture for one-step DNA assembly of over 50 fragments

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Kenji Tsuge ◽  
Yukari Sato ◽  
Yuka Kobayashi ◽  
Maiko Gondo ◽  
Masako Hasebe ◽  
...  
Keyword(s):  
Dna Assembly ◽  
One Step ◽  
Dna Mixture
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