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

Abstract

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.

From empirical to data-driven host selection: a broad-host-range expression platform to facilitate chassis screening

“…Previously green, yellow and cyan fluorescent proteins have been engineered for improved thermostability 53 . However, considering the high autofluorescence of bacteria, particularly Bacilli , using a fluorescent protein in the red range (561 nm excitation) can allow higher resolution 22 . mRuby2 was previously shown to be a suitable fluorescent protein when a low signal-to-noise ratio of the system is crucial, and so was used for this study 22 .…”

“…As there is a limited number of resistance markers that can be used in thermophiles, we provide pProUSER63I2G to allow broad-host-range expression amongst mesophiles. When B. subtilis is selected as desired production host, pProUSER63C1G can be used to simply create more stable strains through integration into the genome at the previously reported, favorable glmS integration site 22 . Considering that the TetR-P tet * system is in itself an advantageous expression system in thermophiles, we further provide pProUSER94H0G and pProUSER94J0G.…”

“…The modified TetR-P tet regulator and promoter sequence was ordered from IDT Technologies as Gblock, and designed using the pProUSER13G2F sequence as template 22 , with specific point mutations introduced to remove the identified and undesired restriction sites.…”

“…To our knowledge, all studies using variants of the tetracycline inducible system only show application within one bacterial phylum. Here, the sequence as previously proposed in C. difficile 28 and thoroughly characterized in B. subtilis 22 , was used as starting sequence.…”

“…To function in B. subtilis, the tet regulatory elements were inserted and modified in the strong xylose promoter (Pxyl) 20,21 . This variant was previously shown to be silent, titratable and homogenous in B. subtilis, as well as silent in an E. coli DH5α cloning strain, critical to allowing easy cloning of desired target genes 22 . Different variants of this promoter have also been shown to function in Actinomyces oris 23 , Staphylococcus species [24][25][26] , Bacillus licheniformis 27 , Clostridium difficile 28,29 , Chlamydia trachosomatis 30 , and Acetobacterium woodii 31 , however systematic analysis of whether one variant can function across hosts is lacking.…”