Overview
To enable engineered bacteria to complete patterns, we constructed a dual-plasmid expression system in E. coli: one plasmid carries the light-control system, while another controls pigment synthesis. We aimed to regulate both the type and location of pigment production via light induction. Additionally, bacterial cells were immobilized using hydrogel to achieve in situ dyeing.
Chassis Organism Selection
For this project, E. coli was selected as the chassis organism due to its well-characterized genetics, rapid growth, scalability, and extensive use in synthetic biology. Previous studies have successfully employed E. coli for pigment synthesis and compatibility with various photosensitive proteins, providing important guidance for our experiments.
During plasmid construction, E. coli DH5α was used. This strain lacks recombinase (recA), ensuring plasmid and foreign DNA stability. For pigment production and light-control validation, E. coli BL21(DE3) was employed. Its protease deficiency reduces recombinant protein degradation, allowing longer retention of photosensitive proteins and pigment synthesis enzymes.
In future applications, we plan to introduce genetic components into amino acid auxotrophic E. coli strains to enhance biocontainment.
Pigment Selection
Based on color analysis of Van Gogh’s Irises, we selected blue, purple, and green as target colors. Literature review indicated that tryptophan can serve as a precursor for various colored indigo derivatives. Considering synthesis difficulty, cost, and dyeing effect, we chose indigo, 6,6'-dibromoindigo, and 4,4'-dinitroindigo as target products, exhibiting blue, purple, and green colors, respectively. Using tryptophan as a common precursor simplifies cultivation and reduces fermentation costs. Moreover, tryptophan and its derivatives (e.g., violacein and indigo) possess unique bioactivities such as antibacterial, antioxidant, and antitumor properties due to their indole side chains.
Indigo, also known as indigo blue, appears as a deep blue powder with a maximum absorption wavelength (λmax) of ~660 nm (in THF) and is nearly insoluble in water. Traditionally extracted from Indigofera tinctoria leaves, conventional methods often leave carcinogenic aniline or N-methylaniline residues, posing sustainability concerns. Over the past decade, tryptophan-based indigo synthesis via the indole pathway has become significant in biological pigment production. The synthesis from tryptophan to indigo typically involves two steps: conversion of tryptophan to indole, followed by oxidation of indole to cis-dihydroxyindole, which spontaneously dimerizes into indigo.
6,6'-dibromoindigo, also known as Tyrian purple, features a bromine group attached at the C6 position of the indole structure. Its maximum absorption wavelength is red-shifted compared to indigo, resulting in a purple-black to deep violet powder. Traditionally extracted from gland secretions of Murex mollusks via light-induced oxidation, its complex production once symbolized royalty in ancient Rome. Currently, 6,6'-dibromoindigo is mainly chemically synthesized, but synthetic biology offers great potential. Microbial pathways typically involve bromination of tryptophan by halogenases, followed by oxidation and dimerization via tryptophanase and monooxygenase to form 6,6'-dibromoindigo.
4,4'-dinitroindigo, another indigo derivative, has a maximum absorption wavelength below 500 nm and appears green. Research on its biological synthesis remains limited, but it shows promise in textile dyeing and antimicrobial applications. This project proposes a potential synthesis pathway to support future studies.
Pigments Synthesis Pathway
Light-Controlled Pigment Synthesis
Two plasmids were introduced into E. coli: one for light response and one for pigment synthesis.
The light-control system consists of:
(1) Photosensory chromophore: responds to specific light wavelengths, undergoes conformational changes upon illumination, and modulates the activity of associated proteins.
(2) Inducer protein: relays light signals within the cell, enabling binding to specific inducible promoters under light stimulation. Pigment synthesis enzymes are placed under these promoters for regulated expression.
Module 1: Blue Pigment Expression
Blue Pigment Synthesis[1]
Indigo synthesis from tryptophan involves two steps: oxidation of tryptophan to indole, followed by conversion of indole to indigo.
We overexpressed E. coli tryptophanase (EcTnaA) to convert tryptophan to indole, producing pyruvate and ammonia—a classic pathway from the shikimate route. Among several routes from indole to indigo, we selected the hydroxylation pathway for its simplicity, minimal external requirements, and fewer intermediates. We introduced a flavin-dependent monooxygenase (MaFMO) from Methylophaga aminisulfidivorans.
Under NADPH and oxygen, MaFMO oxidizes indole to 3-hydroxyindole, which tautomerizes to 3-indoxyl. Two 3-indoxyl molecules then spontaneously dimerize, releasing water and forming indigo.
Blue Light Control[2]
We introduced a Per-Arnt-Sim (PAS) sensing system to enable bacterial response to blue light.
The PAS domain binds flavin mononucleotide (FMN) and is referred to as a light-oxygen-voltage (LOV) domain. The light-inactivated histidine kinase YF1 is a fusion of the blue light-sensing domain from Bacillus subtilis YtvA and the histidine kinase domain from Bradyrhizobium japonicumFixL. Its LOV domain binds FMN; blue light irradiation inactivates its phosphorylation function.
To achieve light-induced expression, we incorporated the repressor PhlF to construct an inverter circuit. In darkness, YF1 phosphorylates the response regulator FixJ. FixJ-P activates the pfixK2 promoter, expressing PhlF, which binds pPhlF to suppress downstream pigment synthesis genes.
Under 470 nm blue light, YF1 undergoes conformational changes, losing phosphorylation activity. FixJ-P levels decrease, halting PhlF expression and derepressing indigo synthesis enzymes EcTnaA and MaFMO.
Module 2: Purple Pigment Expression
Purple Pigment Synthesis[3]
To synthesize halogenated indigo derivatives, current strategies involve regioselective tryptophan halogenation followed by oxidation via tryptophanase and monooxygenase.
We used Trp 6-halogenase SttH from Streptomyces toxytricini, which specifically halogenates the C6 position of indole. To enhance bromination activity and supply the cofactor FADH₂, we fused E. coli flavin reductase Fre to the N-terminus of SttH via an L3 rigid linker peptide for efficient production of bromotryptophan.
Studies show that bromotryptophan can be oxidized by EcTnaA and converted to bromoindigo by MaFMO. We designed a two-cell system: first, the fusion halogenase SttH-L3-Fre was expressed in E. coli ΔtnaA supplemented with tryptophan and KBr to produce 6-bromotryptophan; second, 6-bromotryptophan was introduced into cells overexpressing EcTnaA and MaFMO for 6,6'-dibromoindigo production.
Red Light Control[4]
We introduced the NETMAP system, based on diguanylate cyclase PadC and the transcriptional activator MrkH responsive to c-di-GMP, for red light response.
Biliverdin (BV) was used as the chromophore. Heme oxygenase gene bphOwas introduced to convert heme to BV. BV binding to PadC4 enables red light activation. To reduce background noise, we introduced E. coli enzyme YhjH with constitutive DGC activity to lower c-di-GMP levels in the dark.
In darkness, insufficient c-di-GMP prevents MrkH from activating the PmrkA promoter driving halogenase expression.
Under 710 nm red light, PadC4 becomes active, producing c-di-GMP from GTP. Increased c-di-GMP promotes MrkH binding to PmrkA, initiating SttH-L3-Fre expression and subsequent bromoindigo production.
Module 3: Green Pigment Expression
Green Pigment Synthesis[5],[6]
Synthesis of nitroindigo involves nitration of tryptophan followed by oxidation via tryptophanase and monooxygenase.
Cytochrome P450 TxtE from Streptomyces scabiesselectively nitrates aromatic compounds. Using O₂, NO, NADPH, and tryptophan, TxtE nitrates the C4 position of tryptophan to produce 4-nitrotryptophan. To supply NO, we introduced nitric oxide synthase TxtD, which generates NO from arginine.
To support electron transfer for P450 catalysis, we introduced a class I electron transfer system using codon-optimized ferredoxin (Fdx) and ferredoxin reductase (Fdr) from Mycobacteriumsp. HXN-1500.
A two-cell system was designed: nitrated 4-nitrotryptophan was introduced into cells overexpressing EcTnaA and MaFMO for oxidation to 4,4'-dinitroindigo.
Green Light Control[7]
We introduced the green light-sensing system from SynechocystisPCC 6803 for green light response.
To heterologously produce phycocyanobilin (PCB) in E. coli, we introduced heme oxygenase (ho1) and ferredoxin reductase (pcyA) to convert heme to PCB. CcaS, a cyanobacterial sensor histidine kinase with a PCB-binding GAF domain, senses green light. The response regulator CcaR controls downstream gene expression.
In darkness, CcaS remains in a green light-absorbing state, unable to activate the PcpcG2 promoter.
Under 520 nm green light, CcaS switches to a red light-absorbing state, phosphorylating CcaR. Phosphorylated CcaR binds PcpcG2, activating TxtE and TxtD expression for nitroindigo synthesis.
Hydrogel Immobilization and Dyeing Design
To achieve precise in situ dyeing without bacterial diffusion, we encapsulated engineered bacteria in biocompatible sodium alginate hydrogel.
Sodium alginate rapidly forms hydrogel with calcium ions, offering good mechanical properties and biocompatibility. To control reaction speed and improve pore uniformity/hydration, we prepared a suspension of sodium alginate with lightweight CaCO₃, mixed with bacterial culture, nutrients, and glucono-δ-lactone (GDL). The suspension was sprayed onto fabric via a nozzle, applied 2–3 times until fully soaked, and solidified into gel within 3–5 minutes. We aimed for pore sizes between 100–500 nm to restrict bacterial movement while allowing pigment diffusion for dyeing. This design supports potential industrial application.
Painting Procedure
Preperation
The hydrogel material is supplemented with corresponding nutrients and the substrate amino acid, making it suitable for the survival of Escherichia coli and pigment production. Three strains of Escherichia coli are introduced into this system. The engineered bacteria are uniformly dispersed and immobilized within the hydrogel material to facilitate the synthesis of three pigment.
Red/Green Light Induced
In coordination with a hardware-based photoexcitation printer, the hydrogel is first exposed to patterned illumination using 520 nm green light and 710 nm red light. This step induces a site-specific tryptophan substitution reaction in the irradiated regions.
Tryptophan Derivatization
In areas exposed to green light, 4-nitrotryptophan is generated, while red light illumination leads to the formation of 6-bromotryptophan. These modified tryptophan derivatives serve as substrates for subsequent reactions and remain immobilized within the hydrogel.
Pigment Synthesis
Following the region-specific substitution, 470 nm blue light is projected onto all areas designated for dye production. Unmodified tryptophan is converted into the blue pigment indigo via the action of EctnaA and MaFMO enzymes. The nitrated 4-nitrotryptophan is transformed into the green pigment 4,4'-dinitroindigo, while the brominated 6-bromotryptophan yields the violet pigment 6,6'-dibromoindigo.
Diffusion & Painting
The pigments produced by Escherichia coli diffuse through the hydrogel and fixed onto the fabric, thereby completing the process of microbial painting. Through this approach, we can create a living artwork, iRIS, generated by living processes.
Safety
To prevent bacterial escape, we plan to use amino acid auxotrophic strains in future applications outside the lab, ensuring cell death upon leaving the hydrogel. For enhanced physical containment, we encapsulated the alginate hydrogel in a tougher shell with smaller pores, combining polyacrylamide (elastic polymer network) and alginate (energy-dissipating network via ion crosslinking). This shell is tough, permeable to small molecules, but restrictive to bacteria. In preliminary experiments, without the shell, live bacteria were detected in surrounding media after 24h of shaking; with the shell, no escape was observed. This physical containment complements biochemical strategies for near-zero escape rates.
Heating and UV sterilization modules were incorporated into the hardware for post-dyeing sterilization of hydrogel, fabric, and equipment.