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

Goosens, VJ; Walker, KT; Aragon, SM; Singh, A; Senthivel, VR; Dekker, L; Caro-Astorga, J; ... Ellis, T; + view all (2021) Komagataeibacter Tool Kit (KTK): A Modular Cloning System for Multigene Constructs and Programmed Protein Secretion from Cellulose Producing Bacteria. ACS Synthetic Biology , 10 (12) pp. 3422-3434. 10.1021/acssynbio.1c00358. Green open access

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Abstract

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.

Type: Article
Title: Komagataeibacter Tool Kit (KTK): A Modular Cloning System for Multigene Constructs and Programmed Protein Secretion from Cellulose Producing Bacteria
Location: United States
Open access status: An open access version is available from UCL Discovery
DOI: 10.1021/acssynbio.1c00358
Publisher version: https://doi.org/10.1021/acssynbio.1c00358
Language: English
Additional information: This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions.
Keywords: Cellulose, Cloning, Molecular, Genetic Engineering, Plasmids, Synthetic Biology
UCL classification: UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Cell and Developmental Biology
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
URI: https://discovery.ucl.ac.uk/id/eprint/10146599
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