The Geobacillus Plasmid Set: A Modular Toolkit for Thermophile Engineering

Geobacillus thermoglucosidasius is a Gram-positive thermophile of industrial interest that exhibits rapid growth and can utilize a variety of plant-derived feedstocks. It is an attractive chassis organism for high temperature biotechnology and synthetic biology applications but is currently limited by a lack of available genetic tools. Here we describe a set of modular shuttle vectors, including a promoter library and reporter proteins. The compact plasmids are composed of interchangeable modules for molecular cloning in Escherichia coli and stable propagation in G. thermoglucosidasius and other Geobacillus species. Modules include two origins of replication, two selectable markers and three reporter proteins for characterization of gene expression. For fine-tuning heterologous expression from these plasmids, we include a characterized promoter library and test ribosome binding site design. Together, these gene expression tools and a standardized plasmid set can facilitate modularity and part exchange to make Geobacillus a thermophile chassis for synthetic biology.