The continual demand for specialized molecular cloning techniques that suit a broad range of applications has driven the development of many different cloning strategies. One method that has gained significant traction is Golden Gate assembly, which achieves hierarchical assembly of DNA parts by utilizing Type IIS restriction enzymes to produce user-specified sticky ends on cut DNA fragments. This technique has been modularized and standardized, and includes different subfamilies of methods, the most widely adopted of which are the MoClo and Golden Braid standards. Moreover, specialized toolboxes tailored to specific applications or organisms are also available. Still, the quantity and range of assembly methods can constitute a barrier to adoption for new users, and even experienced scientists might find it difficult to discern which tools are best suited toward their goals. In this review, we provide a beginner-friendly guide to Golden Gate assembly, compare the different available standards, and detail the specific features and quirks of commonly used toolboxes. We also provide an update on the state-of-the-art in Golden Gate technology, discussing recent advances and challenges to inform existing users and promote standard practices.

Encapsulins are protein nanocompartments that house various cargo enzymes, including a family of decameric ferritin-like proteins. Here, we study a recombinant Haliangium ochraceum encapsulin:encapsulated ferritin complex using cryo–electron microscopy and hydrogen/deuterium exchange mass spectrometry to gain insight into the structural relationship between the encapsulin shell and its protein cargo. An asymmetric single-particle reconstruction reveals four encapsulated ferritin decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the protein cargo and the icosahedral encapsulin shell. The encapsulated ferritin decamers are offset from the interior face of the encapsulin shell. Using hydrogen/deuterium exchange mass spectrometry, we observed the dynamic behavior of the major fivefold pore in the encapsulin shell and show the pore opening via the movement of the encapsulin A-domain. These data will accelerate efforts to engineer the encapsulation of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications.

Encapsulins (Enc) are protein nanocompartments which house various cargo enzymes, including a family of decameric ferritin-like proteins. Previously, we elucidated the structure and activity of these ferritin-like proteins (EncFtn) and demonstrated that they must be encapsulated in a nanocompartment for iron storage. Here, we study a recombinant Haliangium ochraceum Enc:EncFtn complex using electron cryo-microscopy (Cryo-EM) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) to gain insight into the structural relationship between Enc and EncFtn. An asymmetric single particle reconstruction reveals four EncFtn decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the EncFtn cargo and the icosahedral encapsulin shell. The EncFtn decamers are offset from the interior face of the encapsulin shell and are resolved at a much lower overall resolution in the final reconstruction. This flexibility, and the fixed number of EncFtn decamers sequestered within, implies that the loading of the encapsulin nanocompartment is limited by the steric effect of both engaged and free encapsulin localization sequences. Using a combination of focused refinements and HDX-MS, we observed dynamic behavior of the major five-fold pore, and show the pore opening via the movement of the encapsulin A-domain. These data can accelerate efforts to engineer the sequestration of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications.

Synthetic biology aims to improve the development of biological systems and increase their reproducibility through the use of engineering principles, such as standardisation and modularisation. It is important that these systems can be represented and shared in a standard way to ensure they are easily understood, reproduced, and utilised by other researchers. The Synthetic Biology Open Language (SBOL) is a data standard for sharing biological designs and information about their implementation and characterisation. Thus far, this standard has been used to represent designs in homogeneous systems, where the same design is implemented in every cell. In recent years there has been increasing interest in multicellular systems, where biological designs are split across multiple cells to optimise the system behaviour and function. Here 1 .