A modular toolkit for environmental , , and enables complex metabolic manipulation.

Soil-dwelling Actinomycetes are a diverse and ubiquitous component of the global microbiome but largely lack genetic tools comparable to those available in model species such as or , posing a fundamental barrier to their characterization and utilization as hosts for biotechnology. To address this, we have developed a modular plasmid assembly framework, along with a series of genetic control elements for the previously genetically intractable Gram-positive environmental isolate C208, and demonstrate conserved functionality in 11 additional environmental isolates of , , and . This toolkit encompasses five Mycobacteriale origins of replication, five broad-host-range antibiotic resistance markers, transcriptional and translational control elements, fluorescent reporters, a tetracycline-inducible system, and a counter-selectable marker.

Interrogating the Function of Bicistronic Translational Control Elements to Improve Consistency of Gene Expression.

Context independent gene expression is required for genetic circuits to maintain consistent and predicable behavior. Previous efforts to develop context independent translation have leveraged the helicase activity of translating ribosomes via bicistronic design translational control elements (BCDs) located within an efficiently translated leader peptide. We have developed a series of bicistronic translational control elements with strengths that span several orders of magnitude, maintain consistent expression levels across diverse sequence contexts, and are agnostic to common ligation sequences used in modular cloning systems.