An official website of the United States government
The ability to precisely engineerAgrobacteriumstrains is crucial for advancing their utility in plant biotechnology. We recently implemented the CRISPR RNA-guided transposase system, INTEGRATE, as an efficient tool for genetic modification inAgrobacterium. Despite its promise, the practical application of INTEGRATE inAgrobacteriumstrain engineering remains underexplored. Here, we present a standardized and optimized workflow that enables researchers to harness INTEGRATE for targeted genome modifications. By addressing common challenges, such as crRNA design, transformation efficiency, and vector eviction, this protocol expands the genetic toolkit available forAgrobacterium, facilitating both functional genomics and strain development for plant transformation. As a demonstration, we domesticatedAgrobacterium rhizogenesK599 strain by deleting the 15-kb T-DNA region from its root-inducing plasmid pRi2659 and inactivating a thymidylate synthase gene to render the strain auxotrophic for thymidine. The protocol provides detailed guidance for each step, including target site selection, crRNA spacer cloning,Agrobacteriumtransformation, screening for targeted insertion and Cre/loxP-mediated deletion, and vector removal. This resource will empower new users to perform efficient and reproducible genome engineering inAgrobacteriumusing the INTEGRATE system, paving the way for broader adoption and innovation in plant biotechnology.
more »
« less
An extensible vector toolkit and parts library for advanced engineering of plant genomes
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
- PAR ID:
- 10401108
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- The Plant Genome
- Volume:
- 16
- Issue:
- 2
- ISSN:
- 1940-3372
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
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
-
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
-
Sustainably enhancing crop production is a global necessity to meet the escalating demand for staple crops while sustainably managing their associated carbon/nitrogen inputs. Leveraging plant-associated microbiomes is a promising avenue for addressing this demand. However, studying these communities and engineering them for sustainable enhancement of crop production have remained a challenge due to limited genetic tools and methods. In this work, we detail the development of the Maize Root Microbiome ToolKit (MRMTK), a rapid Modular Cloning (MoClo) toolkit that only takes 2.5 h to generate desired constructs (5400 potential plasmids) that replicate and express heterologous genes in Enterobacter ludwigii strain AA4 (Elu), Pseudomonas putida strain AA7 (Ppu), Herbaspirillum robiniae strain AA6 (Hro), Stenotrophomonas maltophilia strain AA1 (Sma), and Brucella pituitosa strain AA2 (Bpi), which comprise a model maize root synthetic community (SynCom). In addition to these genetic tools, we describe a highly efficient transformation protocol (107–109 transformants/μg of DNA) 1 for each of these strains. Utilizing this highly efficient transformation protocol, we identified endogenous Expression Sequences (ES; promoter and ribosomal binding sites) for each strain via genomic promoter trapping. Overall, MRMTK is a scalable and adaptable platform that expands the genetic engineering toolbox while providing a standardized, high-efficiency transformation method across a diverse group of root commensals. These results unlock the ability to elucidate and engineer plant–microbe interactions promoting plant growth for each of the 5 bacterial strains in this study.more » « less
