Synthetic biology is inherently engineering-oriented. By incorporating engineering principles into the study of life sciences, synthetic biology has established standardized, modular, and systematic research approaches. In our project, we have designed three distinct modules, which are subsequently integrated into a chassis and subjected to efficacy testing. This process follows a cycle of design, construction, testing, and learning, involving the expression of urease through UreABC, utilization of the low-temperature inducible promoter (pCspA), and incorporation of the galU gene. Through multiple iterations, we have successfully constructed this engineered microorganism.

Design

We utilized the ureA, ureB, and ureC genes derived from Bacillus subtilis to express urease and assess its expression level in CaCO3 formation. The principle and components of this process are illustrated in Figure 1.

Figure 1 Schematic diagram of UreABC.

Build

UreABC was cloned into the pET23b vector, as shown in Figure 2. Simultaneously, the growth status of the UreABC recombinant strains, the production of CaCO3, and the dependence on nickel ions were tested.

Figure 2  Design of UreABC.

Test

The UreABC engineered strains were cultured in large scale, and crude enzyme extracts were collected to determine protein concentration. Finally, the activity of UreABC was measured. As shown in Figure 3a, the urease activity of pT7-UreABC reached 0.4 mg/ml. Meanwhile, the strains were inoculated in urea-CaCl2 medium supplemented with NiCl2 and antibiotics. All samples were pre-washed with hydrochloric acid. The concentration of CaCO3 was then determined using a titration method. As shown in Figure 3b, pT7-UreABC generated more calcium carbonate precipitation. In addition, we studied the dependence of UreABC activity on Ni2+ (Figure 3c). The recombinant strains were cultured in LB medium supplemented with ampicillin and different concentrations of NiCl2. Bacteria were then collected, and urease activity was measured. The results showed that UreABC activity depended on the concentration of Ni2+, but concentrations higher than 5 mM exhibited cell toxicity. Introduction of the urease gene through pT7-UreABC resulted in a significant increase in urease activity. Therefore, more calcium carbonate precipitation was observed. Additionally, it was found that an appropriate concentration of nickel ions could enhance urease activity. However, nickel ions at concentrations higher than 5 mM exhibited toxic effects on bacterial cells.

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

Learn

Through the above experiments, it was found that the pT7-UreABC engineered strain produced more calcium carbonate precipitation compared to the wild type, and an appropriate concentration of nickel ions could enhance urease activity.However,this experiment was conducted at 37℃, but in reality, the temperature will be lower than 37℃. Consequently, enzyme activity and gene expression levels are likely to decrease. Therefore, it is necessary to design a cold-inducible promoter to enhance gene expression at lower temperatures.

Design

From Pcold E. coli, we discovered a cold-inducible promoter that allows expression at low temperatures. Therefore, we have decided to introduce pCspA and couple it with mRFP downstream of the promoter for detection purposes.

Build

The CspA promoter was extracted from Pcold E. coli and inserted upstream of the urease gene, as shown in Figure 4. It was then introduced into E. coli Rosetta. In the pSB1A3 vector, mRFP was cloned downstream of the cold-inducible promoter cspA and transformed into E. coli Rosetta. The transformed cells were inoculated into LB media and cultured. After a certain period, the bacterial liquid concentration and mRFP fluorescence intensity were measured using an microplate reader. The fluorescence intensity expressed per unit of bacterial cell was calculated.

Figure 4 Design of Cold-inducible promoterp(pCspA)

Test

As shown in Figure 5a, the growth of urease at 25 ° C and 37 ° C respectively. We can see that OD600 at 37 ° C (about 1.7) is significantly higher than OD600 at 25 ° C (about 0.9). The absorbance and fluorescence intensity of mRFP were measured using an microplate reader. Finally, the relative fluorescence unit (RFU) was obtained by dividing the fluorescence intensity of row mRFP by OD600. As shown in Figure 5b and5c, the fluorescence intensity of mRFP gene expression coupled at different temperatures showed that the fluorescence of mRFP at 25 degrees (100A.U.) was higher than that of mRFP at 37 degrees (50A.U.). It can be seen that after the introduction of CspA promoter, the expression  levels were increased under low temperature conditions.

Figure 5  The test of Cold-inducible promoter 

Learn

The research findings suggest that the CspA promoter can enhance the expression of the target gene urease. However, pure calcium carbonate is extremely fragile and easily breakable, so it requires the introduction of specific genes to increase the expression level of extracellular polysaccharides. This is done in order to enhance the adhesion between bricks and overall strength during practical applications.

Design

 Clone GalU into the pET28a vector and transform it into E.coli Rosetta.

Build

Inoculate the engineered bacteria into LB medium and induce the expression of UDP glucose pyrophosphorylase (GalU) by adding IPTG chemical inducer. Harvest the bacterial culture and measure the extracellular polysaccharide (EPS) yield using the anthrone-sulfuric acid method and compare it with the EPS yield of the wild-type strain.

Figure 6 Design of galU

Test

Figure 7 Comparison of EPS production data between the wild-type control bacterial group and the pT7-galU engineered bacterial group.

As shown in Figure 7 above, it can be seen that, compared to the wild-type strain, the EPS yield of the engineered strain induced by IPTG is approximately twice as high.

Learn

The results of this study provide clear evidence that the expression of the galU gene effectively enhances EPS production in E. coli. Moreover, the successful application of an EPS coating has significantly improved the resilience and strength of the restored cultural relics, thereby minimizing the potential for secondary damage to these precious artifacts.

Our ureaABC has undergone three rounds of iterative improvements, ultimately resulting in the development of an engineered strain that is capable of highly efficient expression and production of calcium carbonate precipitation at low temperatures. Furthermore, this strain exhibits enhanced viscosity and toughness. UreABC holds potential for applications in agriculture, environmental conservation, biotechnology, and medicine.