Joint Universal Modular Plasmids (JUMP): A flexible and comprehensive platform for synthetic biology

Valenzuela-Ortega, Marcos; French, Christopher E.

doi:10.1101/799585

Cited by 7 publications

(3 citation statements)

References 33 publications

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“…Here, we adapted a dual reporter tool for the CyanoGate MoClo Assembly system that provides a normalized quantification of terminator efficiency within and between species. The pDUOTK1-L1 vector is compatible with several available libraries and thus facilitates easy adoption and sharing of parts with the community ( Andreou and Nakayama, 2018 ; Lai et al, 2018 ; Valenzuela-Ortega and French, 2019 ; Vasudevan et al, 2019 ), and is accessible to any lab currently using Golden Gate cloning. The robustness of our system was validated by comparing results in E. coli against previously published data ( Chen et al, 2013 ).…”

Section: Discussionmentioning

confidence: 99%

Evaluation and Comparison of the Efficiency of Transcription Terminators in Different Cyanobacterial Species

Gale

McCormick

2021

Front. Microbiol.

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Cyanobacteria utilize sunlight to convert carbon dioxide into a wide variety of secondary metabolites and show great potential for green biotechnology applications. Although cyanobacterial synthetic biology is less mature than for other heterotrophic model organisms, there are now a range of molecular tools available to modulate and control gene expression. One area of gene regulation that still lags behind other model organisms is the modulation of gene transcription, particularly transcription termination. A vast number of intrinsic transcription terminators are now available in heterotrophs, but only a small number have been investigated in cyanobacteria. As artificial gene expression systems become larger and more complex, with short stretches of DNA harboring strong promoters and multiple gene expression cassettes, the need to stop transcription efficiently and insulate downstream regions from unwanted interference is becoming more important. In this study, we adapted a dual reporter tool for use with the CyanoGate MoClo Assembly system that can quantify and compare the efficiency of terminator sequences within and between different species. We characterized 34 intrinsic terminators in Escherichia coli, Synechocystis sp. PCC 6803, and Synechococcus elongatus UTEX 2973 and observed significant differences in termination efficiencies. However, we also identified five terminators with termination efficiencies of >96% in all three species, indicating that some terminators can behave consistently in both heterotrophic species and cyanobacteria.

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Section: Discussionmentioning

confidence: 99%

Evaluation and Comparison of the Efficiency of Transcription Terminators in Different Cyanobacterial Species

Gale

McCormick

2021

Front. Microbiol.

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“…Modular GG-based toolkits have revolutionised molecular and synthetic biology in other organisms including in yeasts, mammalian cells, plants, and grampositive and gram-negative bacteria (Andreou and Nakayama, 2018;Engler et al, 2014;Hernanz-Koers et al, 2018;Iverson et al, 2016;Lee et al, 2015;Martella et al, 2017;Moore et al, 2016;Weber et al, 2011;Wicke et al, 2017). Toolkits with both broad or specific host-ranges, have been developed and are transforming their relevant fields (Chiasson et al, 2019;Geddes et al, 2019;Valenzuela-Ortega and French, 2019;Vasudevan et al, 2019;Wu et al, 2018). These cloning systems allow for rapid assembly of DNA constructs from modular genetic parts to build multigene systems.…”

Section: Introductionmentioning

confidence: 99%

Komagataeibacter tool kit (KTK): a modular cloning system for multigene constructs and programmed protein secretion from cellulose producing bacteria

Goosens

Walker

Aragon

et al. 2021

Preprint

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Bacteria proficient at producing cellulose are an attractive synthetic biology host for the emerging field of Engineered Living Materials (ELMs). Species from the Komagataeibacter genus produce high yields of pure cellulose materials in a short time with minimal resources, and pioneering work has shown that genetic engineering in these strains is possible and can be used to modify the material and its production. To accelerate synthetic biology progress in these bacteria, we introduce here the Komagataeibacter tool kit (KTK), a standardised modular cloning system based on Golden Gate DNA assembly that allows DNA parts to be combined to build complex multigene constructs expressed in bacteria from plasmids. Working in Komagataeibacter rhaeticus, we describe basic parts for this system, including promoters, fusion tags and reporter proteins, before showcasing how the assembly system enables more complex designs. Specifically, we use KTK cloning to reformat the Escherichia coli curli amyloid fibre system for functional expression in K. rhaeticus, and go on to modify it as a system for programming protein secretion from the cellulose producing bacteria. With this toolkit, we aim to accelerate modular synthetic biology in these bacteria, and enable more rapid progress in the emerging ELMs community.

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“…As the initial RE site is removed in this process, the ligation is cemented and no recutting of the same site can occur. These cloning systems allow for rapid assembly of DNA constructs from modular genetic parts to build multigene systems, and as such several toolkits with both broad or specific host-ranges have been developed for synthetic biology and are transforming their relevant fields. − …”

mentioning

confidence: 99%

Komagataeibacter Tool Kit (KTK): A Modular Cloning System for Multigene Constructs and Programmed Protein Secretion from Cellulose Producing Bacteria

et al. 2021

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Bacteria proficient at producing cellulose are an attractive synthetic biology host for the emerging field of Engineered Living Materials (ELMs). Species from the Komagataeibacter genus produce high yields of pure cellulose materials in a short time with minimal resources, and pioneering work has shown that genetic engineering in these strains is possible and can be used to modify the material and its production. To accelerate synthetic biology progress in these bacteria, we introduce here the Komagataeibacter tool kit (KTK), a standardized modular cloning system based on Golden Gate DNA assembly that allows DNA parts to be combined to build complex multigene constructs expressed in bacteria from plasmids. Working in Komagataeibacter rhaeticus, we describe basic parts for this system, including promoters, fusion tags, and reporter proteins, before showcasing how the assembly system enables more complex designs. Specifically, we use KTK cloning to reformat the Escherichia coli curli amyloid fiber system for functional expression in K. rhaeticus, and go on to modify it as a system for programming protein secretion from the cellulose producing bacteria. With this toolkit, we aim to accelerate modular synthetic biology in these bacteria, and enable more rapid progress in the emerging ELMs community.

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Joint Universal Modular Plasmids (JUMP): A flexible and comprehensive platform for synthetic biology

Cited by 7 publications

References 33 publications

Evaluation and Comparison of the Efficiency of Transcription Terminators in Different Cyanobacterial Species

Evaluation and Comparison of the Efficiency of Transcription Terminators in Different Cyanobacterial Species

Komagataeibacter tool kit (KTK): a modular cloning system for multigene constructs and programmed protein secretion from cellulose producing bacteria

Komagataeibacter Tool Kit (KTK): A Modular Cloning System for Multigene Constructs and Programmed Protein Secretion from Cellulose Producing Bacteria

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