A User's Guide to Golden Gate Cloning Methods and Standards.

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.

Copper microenvironments in the human body define patterns of copper adaptation in pathogenic bacteria.

Copper is an essential micronutrient for most organisms that is required as a cofactor for crucial copper-dependent enzymes encoded by both prokaryotes and eukaryotes. Evidence accumulated over several decades has shown that copper plays important roles in the function of the mammalian immune system. Copper accumulates at sites of infection, including the gastrointestinal and respiratory tracts and in blood and urine, and its antibacterial toxicity is directly leveraged by phagocytic cells to kill pathogens. Copper-deficient animals are more susceptible to infection, whereas those fed copper-rich diets are more resistant. As a result, copper resistance genes are important virulence factors for bacterial pathogens, enabling them to detoxify the copper insult while maintaining copper supply to their essential cuproenzymes. Here, we describe the accumulated evidence for the varied roles of copper in the mammalian response to infections, demonstrating that this metal has numerous direct and indirect effects on immune function. We further illustrate the multifaceted response of pathogenic bacteria to the elevated copper concentrations that they experience when invading the host, describing both conserved and species-specific adaptations to copper toxicity. Together, these observations demonstrate the roles of copper at the host-pathogen interface and illustrate why bacterial copper detoxification systems can be viable targets for the future development of novel antibiotic drug development programs.

Synthetic biology approaches to copper remediation: bioleaching, accumulation and recycling.

One of the current aims of synthetic biology is the development of novel microorganisms that can mine economically important elements from the environment or remediate toxic waste compounds. Copper, in particular, is a high-priority target for bioremediation owing to its extensive use in the food, metal and electronic industries and its resulting common presence as an environmental pollutant. Even though microbe-aided copper biomining is a mature technology, its application to waste treatment and remediation of contaminated sites still requires further research and development. Crucially, any engineered copper-remediating chassis must survive in copper-rich environments and adapt to copper toxicity; they also require bespoke adaptations to specifically extract copper and safely accumulate it as a human-recoverable deposit to enable biorecycling. Here, we review current strategies in copper bioremediation, biomining and biorecycling, as well as strategies that extant bacteria use to enhance copper tolerance, accumulation and mineralization in the native environment. By describing the existing toolbox of copper homeostasis proteins from naturally occurring bacteria, we show how these modular systems can be exploited through synthetic biology to enhance the properties of engineered microbes for biotechnological copper recovery applications.

Copper tolerance in bacteria requires the activation of multiple accessory pathways.

Copper is a required micronutrient for bacteria and an essential cofactor for redox-active cuproenzymes. Yet, excess copper is extremely toxic, and is exploited as a bacteriocide in medical and biotechnological applications and also by the mammalian immune system. To evade copper toxicity, bacteria not only control intracellular copper homeostasis, but they must also repair the damage caused by excess copper. In this review, we summarize the bacterial cell-wide response to copper toxicity in Enterobacteria. Tapping into the abundant research data on two key organisms, Escherichia coli and Salmonella enterica, we show that copper resistance requires both the direct copper homeostatic response and also the indirect accessory pathways that deal with copper-induced damage. Since patterns of copper response are conserved through the Proteobacteria, we propose a cell-wide view of copper detoxification and copper tolerance that can be used to identify novel targets for copper-based antibacterial therapeutics.