Biofoundries have enabled the ability to automate the construction of genetic constructs using computer-aided design. In this study, we have developed the methodology required to abstract and automate the construction of yeast-compatible designs. We demonstrate the use of our in-house software tool, AMOS, to coordinate with design software, JMP, and robotic liquid handling platforms to successfully manage the construction of a library of 88 yeast expression plasmids. In this proof-of-principle study, we used three fluorescent genes as proxy for three enzyme coding sequences. Our platform has been designed to quickly iterate around a design cycle of four protein coding sequences per plasmid, with larger numbers possible with multiplexed genome integrations in Saccharomyces cerevisiae. This work highlights how developing scalable new biotechnology applications requires a close integration between software development, liquid handling robotics, and protocol development.

Plant-colonizing fungi secrete a cocktail of effector proteins during colonization. After secretion, some of these effectors are delivered into plant cells to directly dampen the plant immune system or redirect host processes benefitting fungal growth. Other effectors function in the apoplastic space either as released proteins modulating the activity of plant enzymes associated with plant defense or as proteins bound to the fungal cell wall. For such fungal cell wall-bound effectors, we know particularly little about their molecular function. In this review, we describe effectors that are associated with the fungal cell wall and discuss how they contribute to colonization.

Fluorogenic protein tagging systems have been less developed for prokaryotes than for eukaryotic cell systems. Here, we extend the concept of noncovalent fluorogenic protein tags in bacteria by introducing transcription factor-based tags, namely, LmrR and RamR, for probe binding and fluorescence readout under aerobic and anaerobic conditions. We developed two chemogenetic protein tags that impart fluorogenicity and a longer fluorescence lifetime to reversibly bound organic fluorophores, hence the name Chemogenetic Tags with Probe Exchange (CTPEs). We present an extensive characterization of 30 fluorophores reversibly interacting with the two different CTPEs and conclude that aromatic planar structures bind with high specificity to the hydrophobic pockets of these tags. The reversible binding of organic fluorophores to the CTPEs and the superior photophysical properties of organic fluorophores enable long-term fluorescence microscopy of living bacterial cells. Our protein tags provide a general tool for investigating (sub)cellular protein localization and dynamics, protein–protein interactions, and prolonged live-cell microscopy, even under oxygen-free conditions.

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