Our technology involves a total of three systems. System I is used to repair clay bricks. System II can cope with lower temperatures in the real environment. System 3 increases the toughness and strength of the repaired brick.

Figure 1 Diagram and schematic representation of the genetic elements for constructing an engineered strain.

We amplified and expressed urease gene (UreABC) from Bacillus subtilis, and then utilized UreABC to promote the production of CaCO3. We used this microorganism to produce calcium ions, which formed a MICP (Microbially Induced Calcium Carbonate Precipitation) coating applied to the surface of clay bricks, in order to repair and enhance their durability.

UreABC was cloned into pET23b vector, the bacterium was suspended, the crude enzyme solution was collected, the protein concentration was determined and finally the activity of UreABC was determined. As shown in Fig. 2a, the urease activity of pT7-UreABC will reach 0.4 mg/ml. meanwhile, the cells of the test strain were inoculated into the Urea-cacl2 medium, and the recombinant strain was added to the in the culture medium of the recombinant strain with the addition of NiCl2 and antibiotics. All samples were pre-washed with HCl. The experimental group produced more calcium carbonate.​​​​​​​​​​​​​​​​

​Figure 2 depicts the growth status of UreABC genetically engineered bacteria, the production of CaCO3, and its dependence on nickel ions.

Figure 3  Ribosome Cold Adaptation Model【1】
In pSB1A3 vector, mRFP was cloned into the downstream of cold-induced promoter cspA, and after verification, it was transformed into E.coli Rosetta and inoculated into LB medium.As shown in Figure a, the engineering bacteria was grown at 25°C and 37°C, respectively. We can see that the OD600 at 37°C (~1.7) is significantly higher than that at 25°C  (~0.9). The absorbance and fluorescence intensity of mRFP were measured using a microplate readerr. Finally, the fluorescence intensity of row mRFP was divided by OD600 to obtain relative fluorescence units (R F U). As shown in Fig. bc, the fluorescence intensity of coupled mRFP gene expression at different temperatures indicated that the fluorescence intensity of the coupled mRFP gene expression at 25°C (100 A.U.) was higher than that of the coupled mRFP gene expression at 25°C (100 A.U.). ) the fluorescence of mRFP at 25°C (100 A.U.) is higher than that of urease at 37°C (50 A.U.). ) fluorescence of urease at 37°C (50 A.U.). Insertion of the cspa promoter upstream of the urease expression target gene did improve urease expression and bacterial adaptation to low temperatures, and facilitated ESP production in subsequent manipulations.

​Figure 4  Test of pCspA.

​Figure 5  Schematic diagram of the principle of GalU

To address the fragility of calcium carbonate, we explored the formation of bioshells via EPS coatings to improve the elasticity and adhesive properties of repaired areas. We employed UDP-glucose pyrophosphorylase to catalyse the generation of additional EPS substrates to facilitate the UDP-glucose pyrophosphate production reaction, thereby increasing glucose yield. 

The engineered bacteria were inoculated into the medium and cultured, followed by induction, as shown in Figure 6. It can be seen that the wild-type extracellular polysaccharide yield was 46 mM, whereas the engineered strain induced with 0.5 mM extracellular polysaccharide had an extracellular polysaccharide yield of 105 mm. Finally, the anthrone-sulfuric acid method was used to determine the EPS  yield of EPS. The results of this study suggest that the introduction of galU gene can enhance the production of crust in E. coli. In conclusion, the formation of biological crusts involves the initial adhesion of microorganisms, crust production and fixation, as well as microbial interactions and ecological functions. The galu gene increases extracellular polysaccharide expression, plays an important catalytic role in the EPS extracellular polysaccharide production reaction, triggers and accelerates the production of EPS substrates and UDP-glucose pyrophosphate, and promotes microbial adhesion, stabilisation, and symbiosis to form a complex and diverse ecosystem.

 Figure 6  Comparison of EPS production. 

Figure 7     Experimental picture of tiles

Figure 8     The impact force generated when weights fall from different heights is used to determine the hardness of repairing tiles.

Figure 9   The repaired tiles can withstand 10 weights of 100 grams placed on them
The hardness of the repaired tiles was measured by the impact force generated by a heavy object dropped from different heights, as shown in  Figure 8. We found that the repaired tiles can withstand the maximum impact of a 300g heavy object at a height of 15 cm, and can withstand the damage of a 400g heavy object at a height of 15 cm height can withstand damage from a 400g weight. And in Fig. 9 we can see that if only a heavy object is placed on the repaired tile, it can withstand 10 100g weights placed on the tile. The practical results show that the repair is effective and can withstand the impact of 1000g weight or 300g weight at 15cm.

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.

[1]吴波. 大肠杆菌（E.coli）冷激蛋白CspA启动子区调控表达机制初探[D].东北师范大学,2007.