We provide a full set of BioBrick standard-compatible parts to be used for urease-promoted biomineralization in E. coli or B. subtilis. A B. subtilis–compatible expression plasmid was generated (BBa_K4417000) by mutating pCT5-bac 2.0 (created by Claudia Schmidt-Dannert's lab; Addgene: #119872). We generated 4 composite parts and cloned them into this backbone under an inducible cumate promoter: (1) urease genes ureABC (TU1, BBa_K4417012), containing three urease subunits for the breakdown of urea into ammonia and CO2; (2) ureEFG (TU2, BBa_K4417013) containing urease accessory genes, responsible for the activation of the ureABC proenzyme, transport and assembly of the nickel II center; (3) ureABCEFG (BBa_K4417018), the entire natural operon sequence from Sporosarcina pasteurii under the cumate-inducible promoter. Finally, two separate transcriptional units for ureABC and ureEFG were combined for strong induction of both transcriptional units (ureABC and ureEFG) individually. All parts contain the restriction enzyme NdeI to change the cumate promoter to a different promotor if desired.
pCT5c (Part:BBa_K4417000) : Type IIS compatible plasmid.
SDM1_Fw (Part:BBa_K4417001): SDM primer for BsaI site 1.
SDM1_Rv (Part:BBa_K4417002): SDM primer for BsaI site 1.
SDM2_Fw (Part:BBa_K4417003): SDM primer for BsaI site 2.
SDM1_Rv (Part:BBa_K4417004): SDM primer for BsaI site 2.
SDM3_Fw (Part:BBa_K4417005): SDM primer for BsaI site 3.
SDM3_Rv (Part:BBa_K4417006): SDM primer for BsaI site 3.
CuO (Part:BBa_K4417007): Cumate inducible system.
TU1 (Part:BBa_K4417012): urease genes ureABC.
TU2 (Part:BBa_K4417013): urease genes ureEFG.
Urease native operon sequence (Part:BBa_K4417013): native urease operon sequence from Sporosarcina pasteurii.