At present, the restoration of ancient murals is a key issue in the field of cultural relic protection. For the restoration of murals, we need to comprehensively consider the stability of pigments, whether the restoration process will cause secondary damage to the murals, and the degree of restoration of the murals. The main way of mural restoration is artificial, the main problems are: 1. Time-consuming, repair time can not be shortened 2. Manual repair is prone to errors. 3. Fewer professionals and longer training time. Therefore, we focuse on a photoinduced biofilm based mineralization technology and the expression of fusion proteins to develop a product for biotechnological restoration of murals. In the aspect of dyeing, we plan to use blue light sensor p70-VVD-VVD-T7R-179 to control the synthesis of CsgA-Mfp3S-pep and amilCP biological proteins, and recolor the mural with peeling pigment. We plan to use these methods to implement a system that avoids these problems. First, our system will automatically and continuously mineralize the frescoes, which saves time and improves efficiency. Second, our system uses Escherichia coli to make precise repairs from a microscopic perspective, which improves our accuracy and avoids errors that often occur with manual repairs. Third, using our system to work, only need to draw the required pattern on the computer, and then our light control system will emit blue light, control E. coli repair, thus reducing the requirements for professional and skilled personnel. Not only that, we replaced artificial beating and harsh chemical pigments with mild mineralized coating and chromoprotein to prevent secondary damage to the mural. This technology mainly uses the platform of E. coli biofilm induced by blue light photocontrol. The transformed bacteria can sense the expression of fusion protein initiated by external blue light, and the resulting biofilm can be further mineralized to form composite materials. Since the biofilm is inherently viscous, the resulting composite will adhere to the substrate.
Fig.1 Process and micrograph of blue light regulation[1]
Accurate patterned living composite materials are obtained by projecting different blue patterns According to the viscosity characteristics of the biofilm and blue for the spatial distribution of accurate control, use the sticky biofilm fixed Polystyrene PS (Polystyrene, abbreviation) ball filler has realized the fixed-point fill the cracks, and through sedimentary hydroxyapatite mineralization of crack filling, further to complete the crack repair and improve the mechanical strength.
To prove our blue light system viable ,we decided to design a set of controlled experiments.Firstly, a plasmid containing the photoreceptor system gene was synthesized and transferred into dh-5 α receptor state together with amilCP-CsgA-Mfp3S-pep plasmid. In the first experiment, the bacteria would be poured evenly on two plates: one completely wrapped in tin foil to block light, and the other wrapped in a light-absorbing cloth with a specific pattern cut out. In the second experiment, the bacteria were inoculated at 1:100 in two fresh LB tubes, one of which was completely wrapped in tin foil and shielded from light. After two days, the results were as follows
Fig.2(Left) Light culture (Right) Sheltered culture
After 12000rpm centrifugation for 1min, while the left was shielded from light and the right was exposed to the light,it was obvious that the expression of amilCP-CsgA-Mfp3S-pep in experiment 1 was regulated by blue light. In experiment 2, the bacteria exposed to light showed obvious blue color. Two experimental results show that the photosensitive system is feasible
For ATC-induced biofilm formation, seed cultures (strains) were inoculated from frozen glycerol stock and grown on LB medium with 34 μg mL-1 chloramphenicol. Seed cultures were oscillated at 220 RPM in 14 mL tubes at 37°C for 12 h. Cells collected from the above seed cultures by centrifugation were suspended in M63 medium. After the OD 600 was adjusted to 1, cells were added into M63 medium in a ratio of 1:100. The medium was supplemented with 1 mM MgSO4, 0.2% (wt/vol) glucose (hereinafter referred to as glucose supplemental M63), 34 μg ml-1 chloramphenicol, and 250 ng mL-1 aTc inducer. The mixture was then used for experimental culture. All liquid experimental cultures for biofilm growth are placed in an incubator and cultured without shaking at 30°C.
For blue light induced biofilm patterns general conditions for biofilm growth in liquid culture are applied as described above. Recombinant strains were used to inoculate cultures. Biofilms were grown on petri dishes containing M63, supplemented with 500 μg mL-1 benzyl, an antibiotic. All liquid experimental cultures for biofilm growth were placed in an incubator and cultured without oscillation at 30°C. Blue light is emitted from a commercial projector (Aodian) to induce biofilm formation. The projector was attached to the petri dish and projected a preset blue light pattern prepared as a Microsoft PowerPoint
After the photoreceptor-CsgA-Mfp3S-pep strain was cultured on a petri dish (diameter, 150 mm) under patterned blue light, the attached biofilm matrix was first gently rinsed 3 times with ddH2O. Subsequently, 30 mL 0.1% (WT/VOL) CV (Sigma) aqueous solution was added and the biofilms were stained at room temperature for 15 min. The sample is then immersed in water and rinsed until no visible blue dye is observed in the washing solution. Staining samples were imaged using commercial digital cameras.
Fig.3 Crystal violet stain sample
A plate of petri dish with IPTG and blank control petri dish with amilCP bacteria were taken for crystal violet staining, and the staining results are shown in the figure below CV staining. After the photoreceptor-CsgA-Mfp3S-pep strain was cultured on a petri dish (diameter, 150 mm) under patterned blue light, the attached biofilm matrix was first gently rinsed 3 times with ddH2O. Subsequently, 30 mL 0.1% (WT/VOL) CV (Sigma) aqueous solution was added and the biofilms were stained at room temperature for 15 min. The sample is then immersed in water and rinsed until no visible blue dye is observed in the washing solution. Staining samples were imaged using commercial digital cameras.
Prepare 1.5×SBF solution according to the method previously reported. The prepared SBF contained 0.1M NaCl, 4.2 mM NaHCO3, 3 mM KCl, 1 mM K2HPO4·3H2O, 1.5 mM MgCl2 ·6H2O, 2.6 mM CaCl2, 0.5 mM Na2SO4 and 0.1M Tris, adjust the pH value of solution to 7.4 with 1 M HCl. Biofilm samples were immersed in 1.5×SBF in an incubator and cultured at 37°C to mineralize HA crystals. For time-dependent mineralization experiments, 1.5×SBF was removed after 1, 3, 5, or 7 days of incubation. Otherwise, change the SBF solution every 2 days until the mineralization process was completed after 7 days of incubation.
The samples on day 0 and 7 were sent to scanning electron microscopy for detection, and the detection results are as follows
Fig.4 Sem images show the surface morphology of biofilms (i.e., unmineralized). Scale: 10um (left)3μm (right)
Fig.5 Scanning electron microscopy (SEM) image shows the surface morphology of the mineralized composite (mineralized for 7 days). Scale: 10um (left)3μm (right)
The photosensitive system-T7RNAP plasmid and amilCP-CsgA-Mfp3S-pep recombinant plasmid were transferred into Dh-5 α sensing state. The medium was wrapped with black photographic cloth and specific patterns were cut out. After three days of induction culture under blue LED lamp, crystal violet staining was used to confirm the formation of biofilms and mineralization was carried out together with blank control containing amilCP bacteria. The mineralization results were as follows
Fig.6 Result picture
The left plate was a mineralized petri dish containing photosensitive system-T7RNAP plasmid and amilCP-CsgA-Mfp3S-pep bacteria Mineralized petri dishes containing amilCP bacteria were shown on the right Compared with petri dishes containing amilCP, obvious mineralization of specific shape biofilms could be observed. The results proved the success of amilCP-CsgA-Mfp3S-pep combined with the blue light photosensitive system, that is, the success of our experiment and the feasibility of our project.
In fact, our system is still deficient in multiple color controls, and we can only control the production of a single pigment with blue light. To address this question, we turned to the papers of Fernandez-Rodriguez J, Moser F, Song M et al.[2]We envision a genetic coding system that enables E. coli to discriminate between red, green, and blue (RGB) light and respond by altering gene expression. We use this system to make "color murals" on bacterial culture plates. We consider adding red and green sensors (probably based on phytochrome) and connecting them to a not gate to accomplish this. In each not gate, the input promoter drives off the expression of the repressor (CI or PhlF, respectively) of the output promoter.These circuits are connected Overlowering repressor expression to reduce toxicity and mutating CI output promoters in response to appropriately colored light offers the possibility of achieving more complex signal processing. After signal processing, red, green and blue signals induced different promoters. These initiators are linked to the representation of the actuator through a resource allocation system. In the same way, drawing on the experience of the paper, we designed the RGB system to be composed of 14 cistrons divided into four plasmids. This requires altering ribo-binding sites (RBSs) to balance expression and altering plasmid sources to avoid homologous recombination caused by reusing parts. In our system, only one color can be mineralized using amilCP to produce, but since the resulting pigment amilCP depends on their structure, we have gained some enlightenment: Through different enzymes producing colored pigments, different colored compounds will be expressed, and the RGB color strains will be used to make the products of each enzyme reaction rapidly form insoluble precipitate, so that the mural can be permanently colored.
Fig.7 Color photography by Escherichia coli. Colored images (insets) were projected onto plates of bacteria containing the RGB system.
We can achieve more accurate mineralization by changing the genes and adding light control systems, using projection equipment to make repair more precise and efficient.
[1]Wang Y, An B, Xue B, et al. Living materials fabricated via gradient mineralization of light-inducible biofilms[J]. Nature Chemical Biology, 2021, 17(3): 351-359. [2]Fernandez-Rodriguez J, Moser F, Song M, et al. Engineering RGB color vision into Escherichia coli[J]. Nature chemical biology, 2017, 13(7): 706-708.
