DNA assembly standards: Setting the low-level programming code for plant biotechnology

Vázquez‐Vilar, Marta; Orzáez, Diego; Patron, Nicola J.

doi:10.1016/j.plantsci.2018.02.024

Cited by 23 publications

(15 citation statements)

References 74 publications

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“…[10][11][12] Several DNA assembly methods are used across the synthetic biology community, among which the most used method is isothermal or Gibson assembly (after Daniel Gibson who developed the technique). [13][14][15] In a Gibson assembly reaction, T5 exonuclease, thermostable high-fidelity DNA polymerase, and Taq DNA ligase are combined in a cocktail with overlapping fragments of double-stranded DNA. Exonuclease chews back from the 5¢ end to create 3¢ overhangs, allowing the single-stranded homologous ends to anneal.…”

Section: Introductionmentioning

confidence: 99%

Single Plasmid-Based, Upgradable, and Backward-Compatible Adenoviral Vector Systems

Liu¹,

Zhang

et al. 2019

Human Gene Therapy

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Existing adenoviral vector systems have two drawbacks. It is labor-intensive and time-consuming to load a transgene in these systems, and transgene-harboring vectors are dead ends: they cannot be reused to construct a vector carrying another transgene or achieving new characteristics. To conquer these shortcomings, single plasmid-based adenoviral vector systems were constructed where a unique PmeI site was located at the position for insertion of the exogenous gene. The polymerase chain reaction (PCR) amplified transgene could be cloned into PmeI-linearized starting plasmids using one step of Gibson assembly to generate target adenoviral plasmids, which were then ready for virus rescue. This procedure was termed restriction assembly. To expand the application of these systems, two ClaI sites were created upstream and downstream of the fiber gene to generate an upgraded starting plasmid pKAd5f11pABR-EPG. The modified fiber gene, amplified by overlap extension PCR, could be used to substitute the original fiber in pKAd5f11pABR-EPG to generate an adenoviral plasmid with a new fiber by restriction assembly. On the other hand, pKAd5f11pABR-EPG was also a starting adenoviral plasmid for expressing other transgenes.In conclusion, easy-to-use and upgradable adenoviral vector systems are introduced here, which offer extensive versatility and can serve as a basic platform and functional component library for the synthetic biology of adenoviral vectors.

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Section: Introductionmentioning

confidence: 99%

Single Plasmid-Based, Upgradable, and Backward-Compatible Adenoviral Vector Systems

Liu¹,

Zhang

et al. 2019

Human Gene Therapy

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“…In the first place, biosynthetic platforms for the rational design, construction, and quantitative characterization of a bigger number of variants of genetic parts need to be established. Toward this goal, adequate vectors and high-throughput DNA assembly methods are already in place (Patron, 2014;Vazquez-Vilar et al, 2018). However, experimental approaches to quantitatively and functionally describe synthetic modules, as well as hand-in-hand work with mathematical modelers to improve predictability and reliability, still lag behind.…”

Section: Discussion and Perspectivesmentioning

confidence: 99%

Synthetic Switches and Regulatory Circuits in Plants

2019

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Synthetic biology is an established but ever-growing interdisciplinary field of research currently revolutionizing biomedicine studies and the biotech industry. The engineering of synthetic circuitry in bacterial, yeast, and animal systems prompted considerable advances for the understanding and manipulation of genetic and metabolic networks; however, their implementation in the plant field lags behind. Here, we review theoretical-experimental approaches to the engineering of synthetic chemical-and light-regulated (optogenetic) switches for the targeted interrogation and control of cellular processes, including existing applications in the plant field. We highlight the strategies for the modular assembly of genetic parts into synthetic circuits of different complexity, ranging from Boolean logic gates and oscillatory devices up to semi-and fully synthetic open-and closed-loop molecular and cellular circuits. Finally, we explore potential applications of these approaches for the engineering of novel functionalities in plants, including understanding complex signaling networks, improving crop productivity, and the production of biopharmaceuticals.

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“…Further, it has ability to stack multiple synthetic operons. Major limitation in this context is size of the transgene as engineering metabolic pathways require engineering of the multiple genes involved in that particular pathway [42][43]. The identification of intercistronic expression elements (minimum sequence elements involved in the proper processing of polycistronic transcript into monocistronic) has helped to devise workable synthetic operons for the expression of multiple proteins involved in the biosynthesis of vitamin E [44], artemisinic acid [45], carotenoids [46], and dhurrin [47] or other metabolic pathways [48].…”

Section: Role Of Synthetic Biology In Engineering Plastid Metabolic Pmentioning

confidence: 99%

Technical Advances in Chloroplast Biotechnology

Khan¹,

Mustafa²,

Joyia³

2019

Transgenic Crops - Emerging Trends and Future Perspectives

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Chloroplasts are highly organized cellular organelles after master organelle nucleus. They not only play a central role in photosynthesis but are also involved in several crucial cellular activities. Advancements in molecular biology and transgenic technology have further groomed importance of the organelle, and they are the most ideal ones for the expression of transgene. No doubt, limitations are there, but still research is advancing to resolve those. Certain valuable traits have been engineered for improved agronomic performance of crop plants. Industrial enzymes and therapeutic proteins have been expressed using plastid transformation system. Synthetic biology has been explored to play a key role in engineering metabolic pathways. Further, producing dsRNA in a plant's chloroplast rather than in its cellular cytoplasm is more effective way to address desired traits. In this chapter, we highlight technological advancements in chloroplast biotechnology and its implication to develop biosafe engineered plants.

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DNA assembly standards: Setting the low-level programming code for plant biotechnology

Cited by 23 publications

References 74 publications

Single Plasmid-Based, Upgradable, and Backward-Compatible Adenoviral Vector Systems

Single Plasmid-Based, Upgradable, and Backward-Compatible Adenoviral Vector Systems

Synthetic Switches and Regulatory Circuits in Plants

Technical Advances in Chloroplast Biotechnology

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