An official website of the United States government
Abstract Plant biotechnology is rife with new advances in transformation and genome engineering techniques. A common requirement for delivery and coordinated expression in plant cells, however, places the design and assembly of transformation constructs at a crucial juncture as desired reagent suites grow more complex. Modular cloning principles have simplified some aspects of vector design, yet many important components remain unavailable or poorly adapted for rapid implementation in biotechnology research. Here, we describe a universal Golden Gate cloning toolkit for vector construction. The toolkit chassis is compatible with the widely accepted Phytobrick standard for genetic parts, and supports assembly of arbitrarily complex T‐DNAs through improved capacity, positional flexibility, and extensibility in comparison to extant kits. We also provision a substantial library of newly adapted Phytobricks, including regulatory elements for monocot and dicot gene expression, and coding sequences for genes of interest such as reporters, developmental regulators, and site‐specific recombinases. Finally, we use a series of dual‐luciferase assays to measure contributions to expression from promoters, terminators, and from cross‐cassette interactions attributable to enhancer elements in certain promoters. Taken together, these publicly available cloning resources can greatly accelerate the testing and deployment of new tools for plant engineering.
more »
« less
This content will become publicly available on January 1, 2026
BEVA2.0: modular assembly of golden gate-compatible vectors with expanded utility for genetic engineering
This expansion for the modular vector assembly platform BEVA (Bacterial Expression Vector Archive) introduces 11 new BEVA parts including two new cloning site variants, two new antibiotic resistance modules, three new origins of replication, and four new accessary modules. As a result, the modular system is now doubled in size and expanded in its capacity to produce diverse replicating plasmids. Furthermore, it is now amenable to genetic engineering methods involving genome-manipulation of target strains through deletions or integrations. In addition to introducing the new modules, we provide several BEVA-derived Golden Gate cloning plasmids that are used to validate parts and that may be useful for genetic engineering of proteobacteria and other bacteria. We also introduce new parts to allow compatibility with the CIDAR MoClo parts libraries.
more »
« less
- Award ID(s):
- 2243818
- PAR ID:
- 10633783
- Publisher / Repository:
- Canadian Journal of Microbiology
- Date Published:
- Journal Name:
- Canadian Journal of Microbiology
- Volume:
- 71
- ISSN:
- 0008-4166
- Page Range / eLocation ID:
- 1 to 13
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Mienda, Bashir Sajo (Ed.)Engineered plasmids have been workhorses of recombinant DNA technology for nearly half a century. Plasmids are used to clone DNA sequences encoding new genetic parts and to reprogram cells by combining these parts in new ways. Historically, many genetic parts on plasmids were copied and reused without routinely checking their DNA sequences. With the widespread use of high-throughput DNA sequencing technologies, we now know that plasmids often contain variants of common genetic parts that differ slightly from their canonical sequences. Because the exact provenance of a genetic part on a particular plasmid is usually unknown, it is difficult to determine whether these differences arose due to mutations during plasmid construction and propagation or due to intentional editing by researchers. In either case, it is important to understand how the sequence changes alter the properties of the genetic part. We analyzed the sequences of over 50,000 engineered plasmids using depositor metadata and a metric inspired by the natural language processing field. We detected 217 uncatalogued genetic part variants that were especially widespread or were likely the result of convergent evolution or engineering. Several of these uncatalogued variants are known mutants of plasmid origins of replication or antibiotic resistance genes that are missing from current annotation databases. However, most are uncharacterized, and 3/5 of the plasmids we analyzed contained at least one of the uncatalogued variants. Our results include a list of genetic parts to prioritize for refining engineered plasmid annotation pipelines, highlight widespread variants of parts that warrant further investigation to see whether they have altered characteristics, and suggest cases where unintentional evolution of plasmid parts may be affecting the reliability and reproducibility of science.more » « less
-
Abstract Modular cloning systems streamline laboratory workflows by consolidating genetic ‘parts’ into reusable and modular collections, enabling researchers to fast-track strain construction. The GoldenBraid 2.0 modular cloning system utilizes the cutting property of type IIS restriction enzymes to create defined genetic ‘grammars’, which facilitate the reuse of standardized genetic parts and assembly of genetic parts in the right order. Here, we present a GoldenBraid 2.0 toolkit of genetic parts designed to accelerate cloning in the model bacterium Escherichia coli. This toolkit features 478 pre-made parts for gene expression and protein tagging as well as strains to expedite cloning and strain construction, enabling researchers to quickly generate functional plasmid-borne or chromosome-integrated expression constructs. In addition, we provide a complete laboratory manual with overviews of common reagent recipes, E. coli protocols, and community resources to promote toolkit utilization. By streamlining the assembly process, this resource will reduce the financial and temporal burdens of cloning and strain building in many laboratory settings.more » « less
-
Ruby, Edward G. (Ed.)ABSTRACT A conspicuous roadblock to studying marine bacteria for fundamental research and biotechnology is a lack of modular synthetic biology tools for their genetic manipulation. Here, we applied, and generated new parts for, a modular plasmid toolkit to study marine bacteria in the context of symbioses and host-microbe interactions. To demonstrate the utility of this plasmid system, we genetically manipulated the marine bacteriumPseudoalteromonas luteoviolacea, which stimulates the metamorphosis of the model tubeworm,Hydroides elegans. Using these tools, we quantified constitutive and native promoter expression, developed reporter strains that enable the imaging of host-bacteria interactions, and used CRISPR interference (CRISPRi) to knock down a secondary metabolite and a host-associated gene. We demonstrate the broader utility of this modular system for testing the genetic tractability of marine bacteria that are known to be associated with diverse host-microbe symbioses. These efforts resulted in the successful conjugation of 12 marine strains from the Alphaproteobacteria and Gammaproteobacteria classes. Altogether, the present study demonstrates how synthetic biology strategies enable the investigation of marine microbes and marine host-microbe symbioses with potential implications for environmental restoration and biotechnology. IMPORTANCEMarine Proteobacteria are attractive targets for genetic engineering due to their ability to produce a diversity of bioactive metabolites and their involvement in host-microbe symbioses. Modular cloning toolkits have become a standard for engineering model microbes, such asEscherichia coli, because they enable innumerable mix-and-match DNA assembly and engineering options. However, such modular tools have not yet been applied to most marine bacterial species. In this work, we adapt a modular plasmid toolkit for use in a set of 12 marine bacteria from the Gammaproteobacteria and Alphaproteobacteria classes. We demonstrate the utility of this genetic toolkit by engineering a marinePseudoalteromonasbacterium to study their association with its host animalHydroides elegans. This work provides a proof of concept that modular genetic tools can be applied to diverse marine bacteria to address basic science questions and for biotechnology innovations.more » « less
-
Robots are often built from standardized assemblies, (e.g. arms, legs, or fingers), but each robot must be trained from scratch to control all the actuators of all the parts together. In this paper we demonstrate a new approach that takes a single robot and its controller as input and produces a set of modular controllers for each of these assemblies such that when a new robot is built from the same parts, its control can be quickly learned by reusing the modular controllers. We achieve this with a framework called MeMo which learns (Me)aningful, (Mo)dular controllers. Specifically, we propose a novel modularity objective to learn an appropriate division of labor among the modules. We demonstrate that this objective can be optimized simultaneously with standard behavior cloning loss via noise injection. We benchmark our framework in locomotion and grasping environments on simple to complex robot morphology transfer. We also show that the modules help in task transfer. On both structure and task transfer, MeMo achieves improved training efficiency to graph neural network and Transformer baselines.more » « less
