Overview

We envision our project to be a reliable solution for cultural relics protection workers. We want to be able to provide new methods for repairing tiny cracks in stone artifacts that are not yet covered by a solution. To achieve this goal, we have designed a total of four modules.

1. Calcium Carbonate Production module: We designed the filler material using Microbial Induced Calcite Precipitation(MICP). We introduce the carbonic anhydrase to enhance the ability of bacteria to produce HCO₃-（CO₃^2-), and the Amorphous calcium carbonate binding protein(ACCBP) to accelerate the precipitation rate of calcium carbonate.

2. Biological scaffold Module: We design a biological scaffold system based on EutM, Spycatcher-SpyTag, and the flagella of Bacillus subtilis to provide the attaching surface for sediments, achieving better structure and strength of mineralization products.

3. Quorum Sensing Module: We take advantage of oxygen-limitation induced promotor and use subtilisin as sensing molecular. The former enables our chassis to get into the depth of the cracks and express the material then, while the latter could also work as an antibiotic to inhibit the growth of contaminated bacteria.

4. Biosafety Module: We introduce a sucrose-induced promoter PsacB and the conservative toxin protein MazE/F to achieve regulating the death of cell, which makes sure the safety of our project

We select Bacillus subtilis as the chassis organism. Bacillus subtilis  is an industrially used GRAS (Generally Recognized as Safe) bacteria known to have excellent protein secretion capabilities. Importantly, it remains highly resistant to adversity and allows it to survive extreme conditions. Therefore, it can be well suited for the application of restoration of relics. Of course, we also use mathematical modeling and hardware design to make our project more applicable to the real world.

See our model and hardware here!

Fig 1 Schematic design of the project

Module 1:Calcium Carbonate Production

The reinforcement of HCO₃^- production

This module focuses on the production of calcium carbonate. We introduce carbonic anhydrase (CA) to accelerate the precipitation of CaCO₃, and amorphous calcium carbonate binding protein(ACCBP) to regulate the formation of ACC.

Although the wild Bacillus subtilis can complete the biological mineralization process spontaneously, the mineralization efficiency is low.Therefore, we design a carbonic anhydrase enhancement module to accelerate its mineralization process. For biological mineralization, urease and carbonic anhydrase are two commonly used catalysts to accelerate mineralization.

Fig 2 Comparison plots of the two methods

However, when urease is used, the by-product NH₃ is produced and escapes, and NH₃ is considered a polluting gas that contributes to global warming. Considering that, we choose the green enzyme for biomineralization:carbonic anhydrase.

We transfer the gene of carbonic anhydrase (CA) into our bacteria to speed up the microbial-mediated mineralization process. The CA is derived from Bacillus halodurans TSLV1, which encodes a zinc-containing enzyme α-carbonic anhydrase(BhCA).It can efficiently catalyze the hydration of CO₂ and rapidly generate HCO₃^- and H⁺(I).

H₂O+CO₂↔HCO₃^-+H⁺（I）

HCO₃^- will diffuse out of the cell along the concentration gradient. At this time, we provide Ca₂⁺ in the extracellular medium, and bicarbonate can combine with Ca₂⁺ to form CaCO₃ precipitation. CaCO₃ deposited in the tiny cracks of the cultural relics will play a certain role of fixation, adhesion and support.

The BhCA naturally produced by Bacillus halodurans is a homomeric enzyme. BhCA is stable at pH6.0-11.0 and has good thermal stability [1] , indicating that it can adapt to the complex environment of the surface of stone relics.

The acceleration of ACC fromation

When CaCO₃ are produced, they will automatically aggregate and form prenucleation clusters, which further form amorphous calcium carbonate (ACC) through certain pathways. ACC will transform into minerals of different crystal forms [2] . In this process, organisms mainly regulate the biological mineralization process by regulating ACC formation and transformation.

To promote the production of mineralization products, namely ACC, we introduced amorphous calcium calcium carbonate binding protein (ACCBP) derived from Streptodera trachelostropha and enabled the engineered bacteria to express and secrete. While the solution possesses a low concentration of magnesium ions, ACCBP can promote the formation of ACC and the conversion of ACC to calcite. So this design can enable engineered Bacillus subtilis to acquire the ability to convert amorphous calcium carbonate into calcite.

Fig 3 Schematic design of calcium carbonate production module

Module 2:Biological Scaffold

In order to make the engineered mineralized products produced have better morphological structure and mechanical strength, we utilized two sets of engineered proteins: Ethanolamine utilizing bacterial micro compartments protein EutM , the Spytag-Spycatcher system based on the protein of Streptococcus pyogenes, and the flagella of Bacillus subtilis.

EutM is a self-assembling protein that when heterologously expressed (e.g. using Bacillus subtilis and E. coli) can form a two-dimensional planar structure [3] .These scaffold proteins are highly amenable to engineering and tolerating N- and C-terminal fusions. In the SpyCatcher-SpyTag system, the SpyCatcher can specially bind the SpyTag and catalyze the formation of an isopeptide bond between SpyCatcher and SpyTag. We design a C-terminal SpyCatcher domain for the covalent linkage of SpyTag-modified proteins to scaffolds [4] . Besides, Bacillus subtilis can be engineered to display SpyTags on the flagella for the cross-linking of EutM scaffolds.

While we first need to optimize secretion by Bacillus subtilis of our EutM-SpyCatcher. EutM-SpyCatcher are not normally secreted. In addition, EutM-SpyCatcher rapidly self- assembles both in vivo and in vitro into large structures [5][6]. So we choose the secretion signal sequence of the SacB gene of Bacillus subtilis which is ligated at its N-terminus to ensure the efficiency of protein secretion.

We therefore rationalized the Bacillus subtilis that can be engineered to secrete EutM-SpyCatcher building blocks and attach itself covalent via sapeptide bond formation by the SpyTag-SpyCatcher system to the formed scaffolds to become both a structural biological material [7].

Fig 4 Schematic design of biological scaffold module

Module 3:Quorum Sensing

Biomineralization requires a suitable starting signal, and in order to achieve deep repair of tiny cracks in stone cultural relics and ensure the structural strength of restored stone cultural relics. Therefore, we designed the quorum sensing module. In this part we draw inspiration from iGEM2010 Team Newcastle(BBa_K302021, BBa_K302018). The module consists of three parts, namely: subtilisin production module, signal module and immune module.

Fig 5 Schematic design of quorum sensing module

In the first subtilisin production part, The subtilisin production module is mediated by a hypoxic promoter and functions through a gene cluster composed of SpaBCTS. The population of engineered Bacillus subtilis keeps growing in the cracks, consuming oxygen, and at the same time, the engineered bacteria penetrate deep into the tiny cracks, and the environment lacks oxygen, thus realizing hypoxic conditions, and the subtilisin production module is activated [8].

The SpaS gene expresses the subtilisin precursor peptide, which is converted into subtilisin through the correction of the expression products of SpaB and SpaC genes, and then the subtilisin is transported out of the cell by the expression product of SpaT. Subtilisin, as a signaling molecule of downstream gene circuits, will play an important role in the initiation of biomineralization. Subtilin is also an antibiotic, so it can also remove bacteria from deep cracks.

Secondly, the immunity part consists of the spaIFEG gene cluster under a constitutive promoter, Pveg. SpaI is a lipoprotein that will bind to and interacts with subtilin at the cell surface. SpaFEG forms an ABC transporter homologue that will expel subtilin from the cell membrane into the extra cellular matrix,preventing bacteria from being poisoned.

The final signal module part consists of the constitutive promoter SpaR/K and the gene cluster SpaRK. When subtilisin interacts with the expression products of SpaK and SpaR, it can act as a phosphorylation regulator to initiate the expression of the native SpaS promoter. Through our design, the signaling module will be inserted into the front end of the carbonic anhydrase production module and mineralization and reinforcement module, and the SpaS promoter will initiate downstream expression, thereby realizing the subtilisin-mediated biomineralization system [9].

Module 4:Biosafety

In this Module,we introduce a sucrose-induced promoter PsacB and a conservative toxin protein MazE/F to achieve regulating the death of cell, which makes sure the safety of our project.

Maz E/F sucrose induced kill switch

In our design, we use the toxin-antitoxin system documented in the iGEM Parts (BBa_K2292006) for our design this time. Since the system is mainly expressed in E. coli, we use the sucrose-inducible promoter from Bacillus subtilis to adapt our chassis and modify it.

With the increase of culture time, the concentration of sucrose in the medium will decrease, the activity of sucrose promoter will decrease, and MazE cannot be continuously expressed. Based on its instability, its extracellular concentration will decline faster than MazF, resulting in MazF playing its toxic role and eventually leading to cell death.

Sucrose induced promoter PsacB

The sucrose promoter PsacB is the third commonly used promoter system in Bacillus subtilis. The expression activity of Bacillus subtilis endogenous promoter in the presence of sucrose is 100 times higher than that in the absence of sucrose, which is used to regulate the expression of downstream MazE/F module [10].

We improve this part and you can find more details on Safety Page.

Fig 6 Schematic design of biosafety module