Throughout China's history, clay tiles have been widely used in the construction of roofs for
buildings, such as the Hakka earthen buildings. Clay tiles are a type of construction material made from clay and are
used for roofing. Over time, these clay tiles deteriorate due to physical, chemical, and biological weathering
processes. Our project aims to address and protect against this phenomenon.

Physical weathering involves erosion from rainfall, wet-dry cycles, and the freezing and thawing of water. Chemical
weathering encompasses processes such as the dissolution and migration of salts, hydration, oxidation, reduction, and
the carbonation of water with O2, CO2, and SO2.

Eroded Clay Tiles

Decaying Ancient Building

Biology Weathering

1.1Existing Solutions and Their Pros and Cons

1.1.1Traditional Methods

(1) Replacement of New Tiles: In cases of significant damage, this method involves creating
entirely new tiles using materials that match or closely resemble the original building materials. These new tiles are
then used to replace the damaged ones.

(2) Patching and Repair of Old Tiles: For minor damage, the process begins with brushing away debris from the damaged
tiles. Subsequently, a mortar similar to the original is applied into the cracks using a trowel to fill them.

Process of Repairing Roof

1.1.2Pros and Cons of Traditional Methods

(1) Replacement of New Tiles:
Pros: Simple and straightforward process.
Cons: Low cost-effectiveness due to the need to replace entire tiles; original building materials are lost, leading to
the loss of traditional craftsmanship; public preference for retaining traditional old tiles.

(2) Patching and Repair of Old Tiles:
Pros: Relatively lower cost compared to replacing new tiles; repaired tiles retain their original characteristics.
Cons: During the filling and repair process, accidents may irreversibly damage cultural artifacts; requires more
manpower and effort; the lifespan of repaired tiles is reduced, necessitating frequent follow-up repairs.

1.2. Introduction of MICP Technology

Microbially Induced Calcium Carbonate Precipitation (MICP) is an efficient biomineralization
technique. It involves the use of specific types of bacteria, typically calcifying bacteria, under specific
environmental conditions to catalyze a chemical reaction between calcium ions (Ca2+) and carbonate ions (CO3²⁻),
resulting in the precipitation of calcium carbonate (CaCO3). This precipitation can fill cracks and improve the strength
and stability of materials.

In recent years, various studies have reported successful implementations of MICP technology in a wide range of
potential applications, such as soil improvement, foundation reinforcement, concrete repair, preservation of historical
buildings, and stone conservation.

Diagrams of MICP Technology


MICP technology reduces the demand for traditional stone and concrete, enhancing the durability and impermeability of
materials and reducing maintenance costs. It also improves soil quality in ecological restoration projects. That
contributes to carbon emission reduction, promotes the development of sustainable construction and infrastructure
projects.

The widespread application of MICP technology demonstrates the potential of microorganisms to innovate in the fields of
material science and soil engineering, enhancing material performance and sustainability.

Diagrams of MICP Technology

 We aim to utilize microbial-induced carbonate precipitation technology as an alternative to
the traditional manual restoration process for historical building roofs. Our goal is to minimize the high costs and
potential irreversible damage to cultural heritage artifacts associated with manual restoration. While ensuring that the
historical appearance is preserved, we seek to enhance the strength and resilience of the roof tiles, thereby extending
their lifespan and effectively protecting the historical building's roof.

Tile Repair Diagram

3.1chassis microorganism

 We chose E. coli Rosetta as the host organism for our project. This strain of E. coli is
known for its ability to express large quantities of foreign proteins with high stability, making it well-suited for
protein expression and purification purposes.

Picture of E. coli Rosetta

3.2The Whole System Consists of Three Subsystems

3.2.1.System one: Calcium Carbonate Precipitation Systerm

 We selected the urease genes ureA, B, and C from Bacillus subtilis for our project.
Engineered bacteria will express urease, which breaks down externally supplied urea into ammonia and carbon dioxide.
These two products will rapidly increase the pH and the concentration of carbon dioxide in the cellular
microenvironment, creating the alkaline conditions necessary to induce calcium carbonate precipitation. Additionally,
since microbial cell surfaces typically carry numerous negatively charged functional groups, they will adsorb positively
charged Ca2+ ions artificially added to the solution. When Ca2+ encounters high concentrations of carbon dioxide, it
reacts to form calcium carbonate, which precipitates on the cell surface.

 The principle of repairing damaged tile cracks using a calcium carbonate precipitation
system

3.2.2.System two: Low-Temperature Inducible Promoter System

3.2.2.1.Field Obstacle The ideal survival temperature for Escherichia coli (E. coli) is 37°C, but
achieving this ideal temperature in the actual environment can be challenging. Therefore, we need a system that ensures
its robust survival and efficient expression at low temperatures

3.2.2.2 Working Principle We selected the CspA promoter from Pcold Escherichia coli. By inserting this
promoter upstream of the target gene in a plasmid, it will expresses a significant amount of CspA protein under cold
conditions.
Under low-temperature conditions, the CspA cold-shock protein will be activated and form a complex with the
low-temperature inducible promoter. This promoter complex recruits RNA polymerase II to the target gene's promoter
region. Once RNA polymerase binds to the promoter, it moves along the DNA template strand, synthesizing new mRNA
molecules. Thus, through the activation of the low-temperature inducible promoter, transcription factors can enhance the
binding of RNA polymerase to the gene promoter region, thereby increasing the transcription process of the target
gene.[1]

Principle Diagram of RNA Polymerase Binding to Low-Temperature Inducible Promoter Under Cold Conditions.

Indeed, this will result in an increased production of mRNA for controlling urease synthesis, ultimately leading to the
synthesis of more target proteins. This accomplishes the goal of achieving better expression of the target gene at lower
temperatures.


###3.2.3.System three:Tile Resilience Enhancement System

3.2.3.1 Increased Production of Biopolymer Reaction SubstratesThe galU gene encodes for UDP glucose
pyrophosphorylase, which, when transcribed and translated, forms GalU. Normally, the reaction of UDP glucose to produce
UDP glucose pyrophosphate is inefficient in the regular cytoplasm. Therefore, we need to utilize GalU to catalyze and
enhance this reaction, resulting in the production of more UDP glucose pyrophosphate.

3.2.3.2 Working Principle The expression of the galU gene will increase the production of
glucose-1-phosphate uridylyltransferase, promoting the reaction:
UDP-glucose + pyrophosphate → UDP-glucose pyrophosphate + inorganic phosphate.
As a result, the substrate UDP-glucose pyrophosphate, which is used in the subsequent EPS generation, is increased.
Inside the cell, under the catalysis of various enzymes, the reaction substrates will combine with each other, and
pyrophosphate (PPi) will undergo thermal decomposition to provide energy. After a series of reactions, UDP-glucose
pyrophosphate will be converted into polysaccharides (EPS), and the final EPS product will coat clay tiles and calcium
carbonate, thereby enhancing the strength and resilience of the repaired tiles.

Schematic Diagram Illustrating the Indirect Increase in EPS Production by the galU Gene

Overall Gene Circuit Diagram

Our approach involves using microbial-induced carbonate precipitation technology to protect cultural artifacts.

Firstly, we insert a low-temperature inducible promoter to ensure efficient expression of the two target genes by our
microorganisms in lower-temperature environments. The ureABC genes are combined to form a complete urease gene, which
expresses urease to break down urea in the environment, leading to the subsequent formation of calcium carbonate
precipitation. To enhance the durability of the restored tiles, we introduce the galU gene. The expression of GalU
enzyme generated by this gene indirectly increases extracellular polysaccharide (EPS) production, thereby strengthening
the adhesion strength and toughness between bricks and tiles in the repaired ancient building roofs.

5.1Target Audience

 The target audience for our designed product comprises organizations involved in historic
building preservation and individuals working in the field of historic building restoration. We have engaged in
discussions with architectural experts and historic building preservation specialists, reaching a consensus that our
product possesses the capability to restore historic building roofs. Additionally, we are in communication with historic
building preservation authorities and plan to collaborate with them in the future to gradually introduce our product for
the maintenance of historic structures.

5.2Production Process

We combine several crucial ingredients, including powdered calcium chloride (providing
calcium ions), powdered nickel chloride (expediting reaction rates), powdered cellulose, and powdered urea.
Simultaneously, we mass-cultivate our engineered bacteria in the factory for subsequent production of our product. After
cultivation, the bacterial strains are freeze-dried for easy transportation and storage. All products are individually
packaged and stored in boxes.

5.3.Storage and Activation of Our Product

The freeze-dried bacterial strains exhibit remarkable stability and can maintain 70%-80% activity even after two years,
allowing for long-term storage.

To activate the strains, pour the freeze-dried powder into a container filled with warm water (optimal at 37°C) and add
glucose water to provide energy, promoting bacterial activation and higher activity levels.

5.4.Instruction of Our Product

After activating the bacterial strains, combine them with other materials to form a
paste-like fluid. Apply this mixture to the surface of the crevices that require repair. The product will penetrate the
deepest parts of the cracks due to capillary action generated by the tile joints and begin generating calcium carbonate
precipitates to repair the gaps.

5.5.Ensuring Biosafety

To ensure that microorganisms are not released from the coating, we consider Extracellular
Polymeric Substances (EPS) to enhance the adhesion between calcium carbonate and the microbial coating. EPS can provide
adhesive properties for microorganisms to adhere to calcium carbonate particles, promoting microbial settlement and
growth on the tile surface. While cells produce calcium carbonate, they also produce Extracellular Polymeric Substances
(EPS). EPS possesses strong adhesive properties and, along with cellulose, traps the engineered bacteria. Subsequently,
the microorganisms' self-generated calcium carbonate can confine themselves within the narrow crevices of the cultural
heritage objects, ensuring that transgenic microorganisms and their genes do not leak, thereby meeting biosafety
requirements.

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