Latest revision as of 13:50, 12 October 2023

ureABC

Description

This part codes for the ureABC operon from Sporosarcina pasteurii, enabling the expression of the three subunits of urease enzymes.

Figure 1: ureABC operon from Sporosarcina pasteurii

Usage and Biology

• Urease (EC 3.5.1.5) is an enzyme that catalyses urea hydrolysis, forming carbon dioxide and ammonia. In biomineralization, carbonic anhydrase acts as a hydrator, catalysing the reaction of carbon dioxide (CO₂) with water into carbonic acid, which spontaneously decays into carbonate ions.
• ureABC are urease structural genes.
• Most bacterial urease has a trimer structure (ureABC)₃ of two small (ureA and ureB) and one large (ureC) subunit.

Figure 2: Urease assembly and maturation process (Veaudor T, 2019). Urease catalytic subunits (ureA, ureB, and ureC) will assemble automatically and start carbamylation, resulting in inactive apo-urease ure(ABC)3. With the presence of accessory protein, Ni will bind with apo-urease ure(ABC)₃ turning into active holo-urease.

Reference

1. Veaudor T, Cassier-Chauvat C, Chauvat F. Genomics of Urea Transport and Catabolism in Cyanobacteria: Biotechnological Implications. Front Microbiol. 2019 Sep 4;10:2052. doi: 10.3389/fmicb.2019.02052. PMID: 31551986; PMCID: PMC6737895.

Thinker-Guangdong 2023

Description

Taking into account our intention to utilize synthetic biology techniques to develop a strain capable of precipitating calcium carbonate with calcium ions, suitable for coating clay tiles for repair purposes, we consider that calcium ions are more easily deposited in alkaline environments. NH3 can create such an environment. Therefore, we aim to express a substance capable of decomposing substrates to generate NH3. To achieve this, we have made the decision to overexpress the urease gene sourced from Bacillus subtilis, as it has the ability to decompose urea and produce NH3.

Usage and Biology

We express the UreABC gene by utilizing the pCspA promoter and the ribosome binding site B0034. The pET23b vector will be employed, and the engineered plasmid will be introduced into E.coli Rosetta for efficient expression.

Figure 1 Design of the UreABC gene encoding urease.

Characterization

Figure 2 Gel electrophoresis of the UreABC gene encoding urease.

Figure 3 Growth status of engineered bacterial strain with UreABC gene, CaCO3 production, and dependency on nickel ions.

UreABC was cloned into pET23b vector, the bacteria were suspended, the crude enzyme extract was collected, the protein concentration was determined, and finally the activity of UreABC was measured. As shown in Figure a, urease activity of pT7-UreABC will reach 0.4mg/ml. At the same time, the cells of the test strain were inoculated into urea-CACL2 medium, and the medium of the recombinant strain was supplemented with NiCl2 and antibiotics. All samples were pre-washed with HCl. Then the CaCO3 concentration was measured by titration. As shown in Figure b, pT7-UreABC produces more calcium carbonate precipitation. We also investigated the UreABC active site Ni2+ dependence (Figure c) of urease in LB medium supplemented with ampicillin and different concentrations of NiCl2, then collected bacteria, and finally measured urease activity. The results showed that UreABC activity was Ni2+ concentration-dependent, but greater than 5 mM resulted in cytotoxicity. After the urease gene of pT7-UreABC was added, the expression of urease activity was significantly improved, and more calcium carbonate precipitation was generated. It was also found that the urease activity could be improved with appropriate concentration of nickel ions, but if the concentration of nickel ions was higher than 5mM, it would have a toxic effect on bacteria.

Potential application directions

UreABC is an enzyme complex that catalyzes the breakdown of urea into carbon dioxide and ammonia. It has the potential to be used in various applications such as urea fertilizer production, urea pollution control, bioenergy production, and as a fundamental component in biosensors for detecting urea or related substances. By measuring the fluctuations in UreABC activity, it is possible to rapidly, sensitively, and accurately detect urea concentration. Overall, UreABC holds great promise for applications in agriculture, environmental conservation, biotechnology, and medicine.

Sequence and Features