At present, in the application field, the problems of tiny cracks in stone cultural relics has not been well addressed, and it is difficult to break through repairing methods. Therefore, we must guarantee the reliability and rationality of our project in every part of our design and try to fuse all parts of our project to make it reflect our project as a whole. On this page, we show some facts about Proof of Concept based on the project lab work, implementation plan, its robust modeling results, and some voices of support from industry experts.
Our whole project aims to explore novel and reliable methods for cultural relics restoration.
Lab work Confirmed at the molecular level that the engineered bacteria expressed and functioned according to our design. At the same time, we also saw the desired engineered bacterial metabolites under SEM and TEM. The modeling part, from a holistic perspective, illustrates the systematicity and integrity of the four modules of our project. This proves the application possibility of our engineered bacteria in the real comprehensive environment. Moreover, our implementation explains in detail how to use our products and the great advantages of “case by case” and customization for our project, which lays a foundation for strengthening the intersection of bio-mineralization and cultural heritage restoration in the future. In the experts' opinions and suggestions, they pointed out that natural mineralization products do exist on some cultural relics, which provides empirical evidence for our project design.
In the calcium carbonate production module, the engineered bacteria were cultured in a medium containing calcium ions for 4 days, and it was clearly observed that the medium changed to white turbid liquid. We obtained the carbonate precipitate by filtration and saw its crystalline form under SEM(Fig1-1,Fig1-2). In the Biological Scaffold Module, the experiments verified that the isolated EutM displayed large crystalline arrays with obvious hexameric organization and symmetry through transmission electron microscopy (Fig1-3).
Fig 1-1 A: Filtered and dried sediment products. B:Crystal morphology of carbonate precipitate under scanning electron microscope. C:EutM hexamers (red line indicates ordered crystalline lattice) under transmission electron microscope.
Fig 1-2 Crystal morphology of carbonate precipitate under scanning electron microscope.
Fig 1-3 EutM hexamers (red line indicates ordered crystalline lattice) under transmission electron microscope.
In order to test the feasibility of the combination between biological scaffold and calcium carbonate precipitation as a whole, we designed a co-culture experiment. The Bacillus subtilis with CA, EutM, Hag-SpyTag588 were cultured in 50 ml LB medium at 25 oC, 220 rpm, pH 8.0 for 4 days. At the first day and the third day 500μl 1M CaCl2 was added into the medium.
After the induction, we utilized the microscope to preliminary detect the cultures. Then we utilized the fluorescence microscope to detect the cultures treated with SYTO dyed to verify that there are bacteria in the briquette of the cultures. We also settled a control group which contained only calcium carbonate.
Fig 1-4 A: The culture medium after natural settlement for 10min.The yellow sediment and small partials of calcium carbonate can be clearly observed. B: The cultures were observed at 400* microscope. The small entity indicated by the arrow might be the complex of biological scaffold and calcium carbonate.
Fig 1-5 The image of the confocal microscope(FLUOVIEW FV3000). The bacterium were stained to indicate.The arrow noted the sediment.
Result: We observed that in the coculture group, the sediment possessed green fluorescent, while the control group did not. So we concluded that the sediment was the complex of bacterium and calcium carbonate.
Then we managed to test the yield of the complex. We sampled 800μl cultures into the 1.5 ml EP tube and the method showed below was adapted for the determination of the yield of the whole complex and the calcium carbonate precipitation respectively.
Step 1: The EP tubes were weighed(M1) before separating the bacteria solution to obtain M1 and we sampled 0.8ml cultural solution into the 1.5 ml ep tube. And we set five parallel experiments and numbered them 1-5.
Step 2: We collected the complex at 12000 rpm 2min. After the supernatant was discarded and the precipitation was fully dried, the EP tubes containing the precipitations were weighed(M2)
Step 3: We used a high concentration of hydrochloric acid to dissolve the precipitate. After there were no obvious bubbles, we utilized the pH dipstick to test and confirm that the pH was less than 7.
Step 4: After waiting for full reaction, we added triploid volume of 75% ethanol to dissolve salt and facilitate precipitation of protein. Then, we collected the precipitation at 12000rpm for 2min. After the supernatant was discarded and the precipitation was fully dried, the EP tubes containing the precipitations were weighed(M3).
Step 5: Calculate the yield according to the following formula.
A1=(M2-M1 ) / V * 100%
A2=(M3-M2) / V * 100%
Result: The results showed a high variation rate, but we could still know that the precipitation was a mixture of bacteria and mineralized products. So we can draw a conclusion that the bacteria and calcium carbonate precipitates could be cemented together
We combine microscopic molecular dynamics with macroscopic flow-curing behaviour to simulate and predict the entire process by which the restoration fluid completes its work.
We discretis the hydrodynamic behaviour and incorporate stochastic flow processes, using a cellular automaton to illustrate the hypoxia condition that can be achieved with our restoration fluid; we simulate the workflow of the quorum sensing module using a hybrid model; molecular dynamics simulations based on a system of differential equations illustrate the generation of cures; the solidification process is validated by the implementation of a cellular automaton to verify the feasibility of our idea. The exact process can be found in our modelling section.
In order to achieve good use effect, we developed the implementation for Story Light, including most of the using points that need to be taken into account. From the production transportation and storage to the correct and effective method of application, we have tried to give our guidelines for how to use it. Besides, we mention the great advantages of our project are the “case by case” and customization, which is suitable for the restoration of precious cultural relics.
To have a full understanding to our user manual, please turn to the Implementation page.
In order to provide empirical support for the biological mineralization repair of stone cultural relics, we visited Professor Zhang Bingjian, who serves as the director of China Cultural Relic Protection Technology Association and the expert group leader of China Stone Association Nursing Professional Committee(Fig2-1). At present, Zhang's Lab undertakes projects of restoring Leshan Giant Buddha and monitoring the preservation of stone relics in Lingyin Temple every year. To support our iGEM project, Zhang's Lab has provided us with some stone models for testing the mechanical strength of calcium carbonate fillers (Fig 2-1) and promised to assist us in putting our project into practice! With their professional help, our projects will certainly serve the real world in the future.
Fig 2-1The left: The teammates were listening carefully to Mr. Zhang's introduction to
the research and scientific progress of stone cultural relics restoration.
The right: Models provided by
Zhang's Lab. The top row is models of synthetic siltstone. The bottom row is models of natural limestone.
[1] ZHANG Bing-jian, LU Huan-ming, YIN Hai-yan, SHEN Zhong-yue. The Crude Conservation Film on Historic Store. Journal of Hangzhou Institute of Electronic Engineerin,2000(03):32-34.
