1. Construction and Validation of the Dual-Plasmid Expression System
To validate the core functionality of "engineered bacteria creating patterns," the first step is to construct a dual-plasmid expression system and confirm its effectiveness. We designed a complete workflow:
1.1 Gene and Vector Preparation
The target sequences for the optogenetic system (genes for photosensitive chromophores and inducer proteins) and the pigment synthesis pathway (enzyme genes from tryptophan to indigo and its derivatives), along with their vectors, were synthesized/obtained.
1.2 Plasmid Construction
Plasmid 1 (pigment synthesis vector) and Plasmid 2 (optogenetic system vector) were constructed separately via homologous recombination or restriction enzyme digestion and ligation.
1.3 Chassis Transformation and Validation
The two plasmids were co-transformed into E. coli. Methods such as colony PCR and sequencing were used to verify the successful construction and stable transfer of the plasmids, ensuring the proper presence of the dual-plasmid system in the chassis.
2. Validation of Functional Modules
The project core consists of the pigment synthesis module, the optogenetic system module, and the hydrogel immobilization module. The validation strategy for each module is as follows:
2.1 Pigment Synthesis Module
Using tryptophan and its brominated/nitrated derivatives as precursors, indigo (blue), 6,6'-dibromoindigo (purple), and 4,4'-dinitroindigo (green) were synthesized. The synthesized indigo was analyzed by absorbance measurement and HPLC.
2.2 Optogenetic System Module
Inducible Promoter Response Efficiency: A reporter gene (e.g., GFP) was placed downstream of the light-inducible promoters. The expression level of the reporter gene (quantified by fluorescence intensity) was measured under "light/dark" conditions and with "different light wavelengths/durations" to validate the promoter's ability to transmit and respond to light signals.
2.3 Hydrogel Immobilization and In-Situ Staining Module
Hydrogel immobilization is key to achieving in-situ staining and controlling the spatial distribution of patterns. It was validated as follows:
2.3.1 Hydrogel Immobilization Efficacy
The embedding efficiency, cell viability (via live cell counting), and the stability of cell morphology and distribution within the hydrogel under long-term culture (observed visually) were assessed.
2.3.2 In-Situ Staining Effect
Light-controlled pigment synthesis experiments were conducted within the hydrogel.
3. Practical Application and Extended Validation
Ultimately, we expect the engineered bacteria to be capable of creating patterns and possess application potential. Therefore, the following validations are planned:
Color matching for Van Gogh's "Irises" through modeling analysis: Perform color analysis on the original painting "Irises" by Van Gogh; simultaneously, measure the parameters of the three pigments synthesized by the engineered bacteria to verify their match with the target colors in the original artwork.
Future work could explore the scalability of pattern creation: for instance, designing patterns of varying complexity, from simple geometric shapes to intricate petal structures, to validate the capability of the "light control strategy" and "hydrogel system" in supporting complex patterns.
Additionally, the staining efficiency and pattern stability of the bacteria-hydrogel system after "multiple light cycles" and "long-term storage at 4°C" will be tested to assess the sustainability for practical applications.