The ProUSER2.0 Toolbox: Genetic Parts and Highly Customizable Plasmids for Synthetic Biology in Bacillus subtilis

Versatile DNA assembly
standards and compatible, well-characterized
part libraries are essential tools for creating effective designs
in synthetic biology. However, to date, vector standards for Gram-positive
hosts have limited flexibility. As a result, users often revert to
PCR-based methods for building the desired genetic constructs. These
methods are inherently prone to introducing mutations, which is problematic
considering vector backbone parts are often left unsequenced in cloning
workflows. To circumvent this, we present the ProUSER2.0 toolbox:
a standardized vector platform for building both integrative and replicative
shuttle vectors forBacillus subtilis. The ProUSER2.0 vectors consist of a ProUSER cassette for easy and
efficient insertion of cargo sequences and six exchangeable modules.
Furthermore, the standard is semicompatible with several previously
developed standards, allowing the user to utilize the parts developed
for these. To provide parts for the toolbox, seven novel integration
sites and six promoters were thoroughly characterized in B. subtilis. Finally, the capacity of the ProUSER2.0
system was demonstrated through the construction of signal peptide
libraries for two industrially relevant proteins. Altogether, the
ProUSER2.0 toolbox is a powerful and flexible framework for use in B. subtilis.