Modular cloning systems that rely on type IIS enzymes for DNA assembly have many advantages for construct engineering for biological research and synthetic biology. These systems are simple to use, efficient, and allow users to assemble multigene constructs by performing a series of one-pot assembly steps, starting from libraries of cloned and sequenced parts. The efficiency of these systems also facilitates the generation of libraries of construct variants. We describe here a protocol for assembly of multigene constructs using the modular cloning system MoClo. Making constructs using the MoClo system requires to first define the structure of the final construct to identify all basic parts and vectors required for the construction strategy. The assembly strategy is then defined following a set of standard rules. Multigene constructs are then assembled using a series of one-pot assembly steps with the set of identified parts and vectors.

Efficient DNA assembly methods are an essential prerequisite in the field of synthetic biology. Modular cloning systems, which rely on Golden Gate cloning for DNA assembly, are designed to facilitate assembly of multigene constructs from libraries of standard parts through a series of streamlined one-pot assembly reactions. Standard parts consist of the DNA sequence of a genetic element of interest such as a promoter, coding sequence, or terminator, cloned in a plasmid vector. Standard parts for the modular cloning system MoClo, also called level 0 modules, must be flanked by two BsaI restriction sites in opposite orientations and should not contain internal sequences for two type IIS restriction sites, BsaI and BpiI, and optionally for a third type IIS enzyme, BsmBI. We provide here a detailed protocol for cloning of level 0 modules. This protocol requires the following steps: (1) defining the type of part that needs to be cloned, (2) designing primers for amplification, (3) performing polymerase chain reaction (PCR) amplification, (4) cloning of the fragments using Golden Gate cloning, and finally (5) sequencing of the part. For large standard parts, it is preferable to first clone sub-parts as intermediate level-1 constructs. These sub-parts are sequenced individually and are then further assembled to make the final level 0 module.

Scientific workflows facilitate the automation of data analysis tasks by integrating various software and tools executed in a particular order. To enable transparency and reusability in workflows, it is essential to implement the FAIR principles. Here, we describe our experiences implementing the FAIR principles for metabolomics workflows using the Metabolome Annotation Workflow (MAW) as a case study. MAW is specified using the Common Workflow Language (CWL), allowing for the subsequent execution of the workflow on different workflow engines. MAW is registered using a CWL description on WorkflowHub. During the submission process on WorkflowHub, a CWL description is used for packaging MAW using the Workflow RO-Crate profile, which includes metadata in Bioschemas. Researchers can use this narrative discussion as a guideline to commence using FAIR practices for their bioinformatics or cheminformatics workflows while incorporating necessary amendments specific to their research area.

Dalbergia melanoxylon Guill. & Perr (Fabaceae) is widely utilized in the traditional medicine of East Africa, showing effects against a variety of ailments including microbial infections. Phytochemical investigation of the root bark led to the isolation of six previously undescribed prenylated isoflavanones together with eight known secondary metabolites comprising isoflavanoids, neoflavones and an alkyl hydroxylcinnamate. Structures were elucidated based on HR-ESI-MS, 1- and 2-D NMR and ECD spectra. The crude extract and the isolated compounds of D. melanoxylon were tested for their antibacterial, antifungal, anthelmintic and cytotoxic properties, applying established model organisms non-pathogenic to humans. The crude extract exhibited significant antibacterial activity against Gram-positive Bacillus subtilis (97% inhibition at 50 μg/mL) and antifungal activity against the phytopathogens Phytophthora infestans, Botrytis cinerea and Septoria tritici (96, 89 and 73% at 125 μg/mL, respectively). Among the pure compounds tested, kenusanone H and (3R)-tomentosanol B exhibited, in a panel of partially human pathogenic bacteria and fungi, promising antibacterial activity against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium showing MIC values between 0.8 and 6.2 μg/mL. The observed biological effects support the traditional use of D. melanoxylon and warrant detailed investigations of its prenylated isoflavanones as antibacterial lead compounds.

Over the past decades, Colombia has suffered complex social problems related to illicit crops, including forced displacement, violence, and environmental damage, among other consequences for vulnerable populations. Considerable effort has been made in the regulation of illicit crops, predominantly Cannabis sativa, leading to advances such as the legalization of medical cannabis and its derivatives, the improvement of crops, and leaving an open window to the development of scientific knowledge to explore alternative uses. It is estimated that C. sativa can produce approximately 750 specialized secondary metabolites. Some of the most relevant due to their anticancer properties, besides cannabinoids, are monoterpenes, sesquiterpenoids, triterpenoids, essential oils, flavonoids, and phenolic compounds. However, despite the increase in scientific research on the subject, it is necessary to study the primary and secondary metabolism of the plant and to identify key pathways that explore its great metabolic potential. For this purpose, a genome-scale metabolic reconstruction of C. sativa is described and contextualized using LC-QTOF-MS metabolic data obtained from the leaf extract from plants grown in the region of Pesca-Boyaca, Colombia under greenhouse conditions at the Clever Leaves facility. A compartmentalized model with 2101 reactions and 1314 metabolites highlights pathways associated with fatty acid biosynthesis, steroids, and amino acids, along with the metabolism of purine, pyrimidine, glucose, starch, and sucrose. Key metabolites were identified through metabolomic data, such as neurine, cannabisativine, cannflavin A, palmitoleic acid, cannabinoids, geranylhydroquinone, and steroids. They were analyzed and integrated into the reconstruction, and their potential applications are discussed. Cytotoxicity assays revealed high anticancer activity against gastric adenocarcinoma (AGS), melanoma cells (A375), and lung carcinoma cells (A549), combined with negligible impact against healthy human skin cells.

Signaling proteins trigger a sequence of molecular switches in the cell, which permit development, growth, and rapid adaptation to changing environmental conditions. SCF-type E3 ubiquitin ligases recognize signaling proteins prompting changes in their fate, one of these being ubiquitylation followed by degradation by the proteasome. SCFs together with their ubiquitylation targets (substrates) often serve as phytohormone receptors, responding and/or assembling in response to fluctuating intracellular hormone concentrations. Tracing and understanding phytohormone perception and SCF-mediated ubiquitylation of proteins could provide powerful clues on the molecular mechanisms utilized for plant adaptation. Here, we describe an adaptable in vitro system that uses recombinant proteins and enables the study of hormone-triggered SCF-substrate interaction and the dynamics of protein ubiquitylation. This system can serve to predict the requirements for protein recognition and to understand how phytohormone levels have the power to control protein fate.

Liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics experiments have become increasingly popular because of the wide range of metabolites that can be analyzed and the possibility to measure novel compounds. LC-MS instrumentation and analysis conditions can differ substantially among laboratories and experiments, thus resulting in non-standardized datasets demanding customized annotation workflows. We present an ecosystem of R packages, centered around the MetaboCoreUtils, MetaboAnnotation and CompoundDb packages that together provide a modular infrastructure for the annotation of untargeted metabolomics data. Initial annotation can be performed based on MS1 properties such as m/z and retention times, followed by an MS2-based annotation in which experimental fragment spectra are compared against a reference library. Such reference databases can be created and managed with the CompoundDb package. The ecosystem supports data from a variety of formats, including, but not limited to, MSP, MGF, mzML, mzXML, netCDF as well as MassBank text files and SQL databases. Through its highly customizable functionality, the presented infrastructure allows to build reproducible annotation workflows tailored for and adapted to most untargeted LC-MS-based datasets. All core functionality, which supports base R data types, is exported, also facilitating its re-use in other R packages. Finally, all packages are thoroughly unit-tested and documented and are available on GitHub and through Bioconductor.

Multicomponent reactions (MCRs) are recently expanding the plethora of solid-phase protocols for the synthesis and derivatization of peptides. Herein, we describe a solid-phase-compatible strategy based on MCRs as a powerful strategy for peptide cyclization and ligation . We illustrate, using Gramicidin S as a model peptide, how the execution of on-resin Ugi reactions enables the simultaneous backbone N-functionalization and cyclization, which are important types of derivatizations in peptide-based drug development or for incorporation of conjugation handles, or labels.

Protein expression in plants by agroinfiltration and subsequent purification is increasingly used for the biochemical characterization of plant proteins. In this chapter we describe the purification of secreted, His-tagged proteases from the apoplast of agroinfiltrated Nicotiana benthamiana using immobilized metal affinity chromatography (IMAC). We show quality checks for the purified protease and discuss potential problems and ways to circumvent them. As a proof of concept, we produce and purify tomato immune protease Pip1 and demonstrate that the protein is active after purification.

Jasmonates are essential engineers of plant defense responses against many pests, including herbivorous insects. Herbivory induces the production of jasmonic acid (JA) and its bioactive conjugate jasmonoyl-l-isoleucine (JA-Ile), which then triggers a large transcriptional reprogramming to promote plant acclimation. The contribution of the JA pathway, including its components and regulators, to defense responses against insect herbivory can be evaluated by conducting bioassays with a wide range of host plants and insect pests. Here, we describe a detailed and reproducible protocol for testing feeding behavior of the generalist herbivore Spodoptera littoralis on the model plant Arabidopsis thaliana and hence infer the contribution of JA-mediated plant defense responses to a chewing insect.