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.

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.

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.

Modular cloning systems that rely on type IIS enzymes for DNA assembly have many advantages for complex pathway engineering. 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 users 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.

Changing environmental conditions greatly affect the accumulation of many proteins; therefore, the analysis of alterations in the proteome is essential to understand the plant response to abiotic stress. Proteomics provides a platform for the identification and quantification of plant proteins responsive to cold stress and taking part in cold acclimation. Here, we describe the preparation of proteins for LC-MS measurement to monitor the changes of protein patterns during cold treatment in Arabidopsis thaliana. In our protocol, proteins are precipitated using TCA/acetone, quantified with 2D Quant Kit and digested with trypsin using a filter-based method and analyzed using an LC-MS approach. The acquired results can be further applied for label-free protein quantification.

The complexity of the obligate symbiotic interaction of arbuscular mycorrhizal (AM) fungi and their host roots requires sophisticated molecular methods. In particular, to capture the dynamic of the interaction, cell-specific methods for gene expression analysis are required. In situ hybridization is a technique that allows to determine the location of transcript accumulation within tissues, being of special interest for these fungi that cannot be genetically modified. The method requires proper fixation and embedding methods as well as specific probes for the hybridization allowing detection of specific transcripts. In this chapter, we present a method to prepare roots, which have established a symbiosis with an arbuscular mycorrhizal fungus for the detection of fungal transcripts. This includes chemical fixation, subsequent embedding in a suitable medium, sectioning and pretreatment of sections, the hybridization procedure itself, as well as the immunological detection of RNA-RNA hybrids.

Availability of efficient DNA assembly methods is a basic requirement for synthetic biology. A variety of modular cloning systems have been developed, based on Golden Gate cloning for DNA assembly, to enable users to assemble multigene constructs from libraries of standard parts using a series of successive one-pot assembly reactions. Standard parts contain the DNA sequence coding for a genetic element of interest such as a promoter, coding sequence or terminator. Standard parts for the modular cloning system MoClo must be flanked by two BsaI restriction sites 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 basic parts. This protocol requires the following steps (1) defining the type of basic part that needs to be cloned, (2) designing primers for amplification, (3) performing PCR amplification, (4) cloning of the fragments using Golden Gate cloning, and finally (5) sequencing of the part. For large basic parts, it is preferable to first clone subparts as intermediate level −1 constructs. These subparts are sequenced individually and are then further assembled to make the final level 0 module.