BacilloFlex: A modular DNA assembly toolkit for<i>Bacillus subtilis</i>synthetic biology

Wicke, Niels; Radford, David; Verrone, Valeria; Wipat, Anil; French, Christopher E.

doi:10.1101/185108

Cited by 7 publications

(6 citation statements)

References 37 publications

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“…were published for important model organisms such as E. coli, 22,23 S. cerevisiae, 24 and B. subtilis. 25 Here we describe the design, construction, and evaluation of a novel Golden Gate-based toolbox, termed the Marburg Collection, optimized for working in V. natriegens. Through a detailed, quantitative characterization of the genetic parts in this new chassis organism, this toolbox will provide the growing V. natriegens community with the resources to work efficiently with this unique bacterium.…”

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confidence: 99%

The Marburg Collection: A Golden Gate DNA Assembly Framework for Synthetic Biology Applications in Vibrio natriegens

et al. 2021

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Vibrio natriegens is known as the world's fastest growing organism with a doubling time of less than 10 min. This incredible growth speed empowers V. natriegens as a chassis for synthetic and molecular biology, potentially replacing E. coli in many applications. While first genetic parts have been built and tested for V. natriegens, a comprehensive toolkit containing wellcharacterized and standardized parts did not exist. To close this gap, we created the Marburg Collectiona highly flexible Golden Gate cloning toolbox optimized for the emerging chassis organism V. natriegens, containing 191 genetic parts. The Marburg Collection overcomes the paradigm of plasmid constructionintegrating inserts into a backboneby enabling the de novo assembly of plasmids from basic genetic parts. This allows users to select the plasmid replication origin and resistance part independently, which is highly advantageous when limited knowledge about the behavior of those parts in the target organism is available. Additional design highlights of the Marburg Collection are novel connector parts, which facilitate modular circuit assembly and, optionally, the inversion of individual transcription units to reduce transcriptional crosstalk in multigene constructs. To quantitatively characterize the genetic parts contained in the Marburg Collection in V. natriegens, we developed a reliable microplate reader measurement workflow for reporter experiments and overcame organism-specific challenges. We think the Marburg Collection with its thoroughly characterized parts will provide a valuable resource for the growing V. natriegens community.

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confidence: 99%

The Marburg Collection: A Golden Gate DNA Assembly Framework for Synthetic Biology Applications in Vibrio natriegens

et al. 2021

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“…The resulting transformants are selected on media lacking the target nutrient [86,87]. Libraries of modular plasmids combining antibiotic resistance markers, origins of replication, multiple cloning sites (MCS), and other genetic parts have been developed for different classes of organisms, and they are very useful for the rapid testing of genetic parts [88][89][90].…”

Section: Maintaining and Selecting For Dnamentioning

confidence: 99%

Approaches to genetic tool development for rapid domestication of non-model microorganisms

Riley

Guss

2021

Biotechnol Biofuels

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Non-model microorganisms often possess complex phenotypes that could be important for the future of biofuel and chemical production. They have received significant interest the last several years, but advancement is still slow due to the lack of a robust genetic toolbox in most organisms. Typically, “domestication” of a new non-model microorganism has been done on an ad hoc basis, and historically, it can take years to develop transformation and basic genetic tools. Here, we review the barriers and solutions to rapid development of genetic transformation tools in new hosts, with a major focus on Restriction-Modification systems, which are a well-known and significant barrier to efficient transformation. We further explore the tools and approaches used for efficient gene deletion, DNA insertion, and heterologous gene expression. Finally, more advanced and high-throughput tools are now being developed in diverse non-model microbes, paving the way for rapid and multiplexed genome engineering for biotechnology.

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“…The modular cloning (MoClo) system (21), for instance, allows the rapid assembly of multi-gene constructs in a hierarchical manner, and several toolboxes with basic genetic parts (promoters, ribosome binding sites, terminators, etc.) were published for important model organisms such as E. coli (22,23), S. cerevisiae (24) and B. subtilis (25).…”

Section: Introductionmentioning

confidence: 99%

The Marburg Collection: A Golden Gate DNA Assembly Framework for Synthetic Biology Applications inVibrio natriegens

Stukenberg

Hensel

Hoff

et al. 2021

Preprint

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Vibrio natriegens is known as the world's fastest growing organism with a doubling time of less than 10 minutes. This incredible growth speed empowers V. natriegens as a chassis for synthetic and molecular biology, potentially replacing E. coli in many applications. While first genetic parts have been built and tested for V. natriegens, a comprehensive toolkit containing well-characterized and standardized parts did not exist. To close this gap, we created the Marburg Collection - a highly flexible Golden Gate Assembly-based cloning toolbox optimized for the emerging chassis organism V. natriegens. The Marburg Collection overcomes the paradigm of plasmid construction - integrating inserts into a backbone - by enabling the de novo assembly of plasmids from basic genetic parts. This allows users to select the plasmid replication origin and resistance part independently, which is highly advantageous when limited knowledge about the behavior of those parts in the target organism is available. Additional design highlights of the Marburg Collection are novel connector parts, which facilitate modular circuit assembly and, optionally, the inversion of individual transcription units to reduce transcriptional crosstalk in multigene constructs. To quantitatively characterize the genetic parts contained in the Marburg Collection in V. natriegens, we developed a reliable microplate reader measurement workflow for reporter experiments and overcame organism-specific challenges. We think the Marburg Collection with its thoroughly characterized parts will provide a valuable resource for the growing V. natriegens community.

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BacilloFlex: A modular DNA assembly toolkit forBacillus subtilissynthetic biology

Cited by 7 publications

References 37 publications

The Marburg Collection: A Golden Gate DNA Assembly Framework for Synthetic Biology Applications in Vibrio natriegens

The Marburg Collection: A Golden Gate DNA Assembly Framework for Synthetic Biology Applications in Vibrio natriegens

Approaches to genetic tool development for rapid domestication of non-model microorganisms

The Marburg Collection: A Golden Gate DNA Assembly Framework for Synthetic Biology Applications inVibrio natriegens

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