We aim to overexpress urease and carbonic anhydrase enzymes, the genes of which are naturally present in Sporosarcina pasteurii and Bacillus halodurans TSLV1, respectively.

We designed a complete urease operon for Bacillus subtilis 1A253 (RM125) using a mutated shuttle vector pCT5-bac 2.0 (Ordered from Addgene plasmid # 119872 from Claudia Smidt-Dannert's group). We removed the BsaI restriction sites in pCT5-bac 2.0 by site directed mutagenesis to make the vector Type IIS compatible. Sporosarcina pasteurii ureABC genes were synthesised as gBlocks by IDT based on our design, amplified by PCR (Polymerase Chain Reaction), and have cloned into our mutated backbone using restriction enzyme digests with BamHI/SacI and Gibson assembly. Accessory urease gene constructs (Sporosarcina pasteurii ureEFG) were subcloned via golden gate assembly to generate plasmids that contain two transcriptional units in tandem under the control of their own promoters and ribosome binding sites. All plasmids that we generate contained SapI sites flanking transcriptional units to comply with type IIS standards. The plasmids were first expressed in commercially available E. coli DH5-alpha with ampicillin resistance and tested using its inducible cumate promoter and urease assay. Successful transformation with this new BioBrick will be detected by the lack of colour change due to the removal of sfGFP genes in the original backbone. Diagnostic digests were performed, and agarose gel electrophoresis was run to analyse correct assembly. Gel extraction of DNA was used to obtain gene fragments/plasmids of known sizes from agarose gels. Plasmids were subsequently sequence verified. Urease assay, SDS-PAGE, and biocementation assay were performed for different constructs to validate our design.

Overall, we designed 4 constructs (Fig. 1) coding for ureABC, ureEFG, or the full urease operon using SnapGene. The basic parts were synthesized as gBlocks by IDT and cloned into pCT5c Type IIS compatible plasmid using restriction digest and gibson assembly. The four constructs were cloned using restriction digest and golden gate assembly.

Figure 1: Cloning strategy for urease constructs

All four constructs are compatible with Type IIS Standard, in order to facilitate sharing of the constructs among the scientific community. There are no forbidden restriction sites within the biobricks. All plasmid backbones contained an inducible cumate promoter, a strong RBS, and an rrnB T1 terminator. Construct TU1 and TU2 are urease structural and accessory proteins independently. Construct master plasmid is created as an intermediate plasmid for cloning the urease full operon. The nature urease operon (construct ureABCEFG) is cloned by parts 1 and 4 in Figure 1. Construct TU1 with TU2 is cloned using parts 2 and 6 in Figure 1. The final plasmid with multiple transcriptional units. This way we can compare the wide type of operon performance with the performance of ureABC and ureEFG each from a different transcriptional unit. We can see if our modular approach works too.

A more graphical detailed description of the 4 constructs can be found below (Fig. 2). Here we explain the role of each of the individual components.

Figure 2: The four constructs in Type IIS Standard

Construct TU1 (BBa_K4417012): CuO-RBS-ureABC-rrnB T1 Terminator. This enzyme contains three urease subunits and is able to catalyze the breakdown of urea into ammonia and CO2. UreA, ureB, and ureC have α, ß, and γ active sites, respectively, which work in coordination with structural change to break down urea.

Construct TU2 (BBa_K4417013): CuO-RBS-ureEFG-rrnB T1 Terminator. This composite part is the ureEFG gene from Sporosarcina pasteurii. ureEFG are urease (accessory) proteins, responsible for the activation of the ureABC proenzyme. ureEFG is responsible for the transport and assembly of the nickel II center.

Construct ureABCEFG native urease operon (BBa_K4417018): CuO-RBS-ureABC-ureEFG-rrnB T1 Terminator. This part retained the native urease operon sequence from Sporosarcina pasteurii under a novel cumate inducible promoter.

Construct MTU (BBa_K4417019): CuO-RBS-ureABC-rrnB T1 terminator-CuO-RBS-ureEFG-rrnB T1 Terminator. This part contains both ureABC and ureEFG genes with their own cumate inducible promoter, RBS, and terminator. NdeI sites are included to change promoters for different transcriptional units.