K. rhaeticus culture conditions and culturing approaches

Two culture media were used in this study to culture K. rhaeticus. HS-glucose media (2% glucose, 10 g l−1 yeast extract, 10 g l−1 peptone, 2.7 g l−1 Na2HPO4 and 1.3 g l−1 citric acid, pH 5.6–5.8) and coconut water media (coconut water (Vita Coco), 0.05% (vol/vol) acetic acid). Coconut water media was sterilized by filtration, except in situations where more than 1 l was required. In those situations, media supplements were sterilized separately and combined with coconut water, which had been opened and decanted out with aseptic technique, in the culturing container.

When K. rhaeticus was cultured on solid media, HS-glucose media was always used and supplemented with 1.5% agar. K. rhaeticus liquid cultures fell into the following two separate approaches: shaking cultures and stationary cultures. In shaking cultures, the media in use was supplemented with 2% (vol/vol) cellulase (Sigma-Aldrich, C2730) to allow for turbid growth without clumping. In stationary culture, where the goal is pellicle formation, media would be supplemented with 1% (vol/vol) ethanol to enhance pellicle production. In both approaches, where antibiotics were required for plasmid maintenance, media was supplemented with 340 μg ml−1 chloramphenicol or 200 μg ml−1 spectinomycin.

To facilitate consistency when inoculating multiple pellicles, K. rhaeticus cells would be grown in shaking growth conditions until turbid, normalized in OD600 across samples, pelleted by centrifugation and washed in the subsequent media to remove cellulase. The washed cells were used as a preculture and added, at a ratio of 1:25, into the culturing container and left in stationary conditions at 30 °C to form pellicles. In the case of forming large pellicles consistently (>25 cm2), a glycerol aliquot approach was used. The K. rhaeticus strain of interest would be grown, shaking at 30 °C in 100 ml of HS-glucose media until it reached an OD600 of ~0.6 to 1. At this point, the cells would be pelleted by centrifugation, washed in HS-glucose media, before being pelleted again and resuspended in 10 ml of HS-glucose media containing 25% glycerol. The resuspended cells would be separated into 1 ml aliquots and stored at −80 °C until use. When used, an aliquot would be thawed and added to the media in the final culturing container.

Molecular biology and strain construction

DNA parts and plasmids used in this study are listed in the supplementary materials. E. coli Turbo (NEB) cells were used for plasmid construction. The tyr1 DNA sequence was ordered from Twist Bioscience, with compatible 3′ and 5′ overhangs for entry into the KTK via Golden Gate Cloning. Constitutive tyrosinase constructs were built using the KTK. The procedures and protocols for working with the KTK are described in ref. 26. Plasmids containing the various versions of the Opto-T7RNAP system were kindly sent to us by A. Baumschlager and M. Khammash from ETH Zürich. Due to the presence of multiple KTK-incompatible restriction sites in the T7-Opto coding sequences, Gibson cloning was used to build both the pOpto-T7RNAP*(563-F2)-target plasmid and the five pOpto-T7RNAP suicide plasmids for genomic integration. The primers for Gibson cloning are listed in the supplementary materials.

K. rhaeticus electrocompetent cells were prepared as in ref. 24. K. rhaeticus cells were transformed using electroporation and selected for HS-glucose agar plates containing either 340 μg ml−1 chloramphenicol or 500 μg ml−1 spectinomycin, depending on the plasmid selection marker in use. Note, here a higher concentration of spectinomycin is used during normal culturing. Genetic constructs that were integrated into the chromosome of K. rhaeticus were done so by homologous recombination using a pUC19 suicide plasmid, as described in ref. 26.

Melanated pellicle production

Melanated pellicles were produced using a two-step approach. First, a tyr1 expression strain would be inoculated into a sterile culture container. Typically, 24-well deep well plates (Axygen) were used to make small pellicles. Each well contained 5 ml of growth media and was inoculated at a ratio of 1:25 with preculture. Growth media was enriched with 0.5 g l−1l-tyrosine and 10 μM CuSO4 to promote the highest eumelanin production. Once the pellicles had reached the desired thickness, they were collected, placed in a bath of sterile dH2O and washed for 1 min by gently shaking by hand. The washed pellicles are then passed into a bath of eumelanin development buffer. A large ratio of buffer to pellicle was used, that is, one pellicle in 25 ml of buffer in a 50-ml falcon tube; this was to prevent the overwhelming of the buffer by remaining acid in the pellicle. The pellicle would be allowed to produce eumelanin at >30 °C in shaking conditions over 24 h.

Large melanated pellicle production

To produce the melanated pellicle used to make the wallet, a 200 × 300 Eurobox container was sterilized and filled with 3 l of coconut water media supplemented with 0.5 g l−1l-tyrosine, 10 μM CuSO4 and 1% ethanol. The media was inoculated with a 1 ml K. rhaeticus ctyr1 glycerol aliquot and covered in a paper towel before being placed into a stationary incubator set to 30 °C. After 10 days of growth, the pellicle was collected, washed briefly in dH2O before being placed in a 300 × 400 mm Eurobox containing 2 l of concentrated eumelanin development buffer (10× PBS). The development container was then placed into a shaking incubator set to 45 °C and allowed to produce eumelanin over 2 days, at which point the cellulose had become completely black. The melanated cellulose was then washed again to remove excess eumelanin development buffer before being autoclaved. To make the material pliable after drying, the cellulose sheet was left in a 5% glycerol solution. This glycerol process may improve the strength of dried BC by maintaining some of the properties of wet BC, by preventing hornification55. The sample was then pressed to remove bulk water and air-dried for 24 h. This process typically leads to around a 98% reduction in mass due to the removal of water.

To produce the melanated pellicle used to make the shoe, a custom-shaped vessel, containing an apparatus that held a network of tightly strung yarn, was sterilized and filled with 2 l of coconut water media supplemented with 0.5 g l−1l-tyrosine, 10 μM CuSO4, 340 μg ml−1 chloramphenicol and 1% ethanol. The media was inoculated with a ~500 ml precultured K. rhaeticus ptyr1 pellicle. To accommodate the fed-batch procedure and unique vessel size necessary to incorporate the yarn apparatus, the culture was left to grow at room temperature in stationary conditions, until a thin pellicle had formed. At this point, fresh coconut water media supplemented with 0.5 g l−1l-tyrosine, 10 μM CuSO4, 340 μg ml−1 chloramphenicol and 1% ethanol was added, to raise the pellicle to just below the level of the tensed yarn. After a longer growth period of 2 weeks due to lower temperature, the media was drained and replaced with concentrated eumelanin development buffer (10× PBS). The full container was placed into a shaking incubator set to 30 rpm, and developed at 30 °C for 1 day, at which point the pellicle had become completely black. The vessel was then drained of eumelanin development buffer, replaced with 70% ethanol and left overnight to sterilize. The ethanol was replaced with a 5% glycerol solution before the melanated cellulose was removed from the apparatus and wrapped around a shoe-shaped mold (last) to air-dry at 45 °C for 24 h. Once air-dried, the shoe upper and last were placed onto a sole and photographed.

Eumelanin production assay

The eumelanin production assay uses a 384-square-well microtiter plate as a reaction plate. An OT-2 liquid handling robot (Opentrons) was used to prepare these reaction plates for the assay. Development buffer was placed into a deep well plate, from which 40 μl was transferred to each well in the reaction plate using an eight-channel 300 μl OT-2 Gen2 pipette. The reaction plate was kept at 4 °C to slow eumelanin production during plate preparation using the OT-2 thermo-module. Cells and supernatant potentially containing tyrosinase were placed into a 96-well plate. Cells were mixed in one round of aspiration using an eight-channel 20 μl OT-2 Gen2 pipette before 10 μl of cells were transferred into each well of the 384-well plate. Once full, the reaction plate was centrifuged for 10 s to draw liquid to the bottom of the wells before being sealed with a Breath-Easy sealing membrane. The reaction plate was placed into a plate reader and heated to 45 °C to accelerate eumelanin production and prevent potential cell growth from affecting optical density readings. To measure cell density in the reaction plate, an initial measurement at OD600 is taken, after which OD405 measurements are taken every 10 min for 12 h, while the plate is shaken at high speed.

Eumelanin production assay (supernatant)

K. rhaeticus ptyr1, K.rhaeticus tyr1, and wild-type K. rhaeticus starter cultures were grown in 3 ml of HS-glucose media, with 2% cellulase, 0.5 g l−1 tyrosine, 10 μM CuSO4 and, if appropriate, 340 μg ml−1 chloramphenicol, in shaking conditions for 24 h. The cultures were normalized for OD600 and inoculated into shaking flasks containing 25 ml of the same prior media for 48 h. At this point, the cells were pelleted by centrifugation and the supernatant was transferred to a separate container on ice. The supernatant was sterilized using a 0.2-μm filter and the pH was adjusted to pH 7 by 1 M NaOH titration. The cell pellets were resuspended in eumelanin development buffer and 10 μl of the resulting mixture was placed into a 384-well plate alongside pH-adjusted supernatant samples and pH-adjusted cell cultures. Once full, the reaction plate was centrifuged for 10 s to draw liquid to the bottom of the wells before being sealed with a Breath-Easy sealing membrane. Assay plate was run using the same protocol as used in the Eumelanin production assay.

Wettability experiments

K. rhaeticus ptyr1 was inoculated into a 24-well deep well plate, with each well containing 5 ml of HS-glucose media, with 0.5 g l−1 tyrosine, 10 μM CuSO4 and 340 μg ml−1 chloramphenicol. After incubating at 30 °C for 7 days, pellicles were collected. Eumelanin production was initiated by placing the collected pellicles into eumelanin development buffer. A set of pellicles were held back from eumelanin production and placed into an acetate buffer containing 0.5 g l−1 tyrosine and 10 μM CuSO4 at pH 3.6 to act as a negative control. Melanated and unmelanated pellicles were sterilized by placing them in 70% ethanol overnight. Pellicles were then washed in distilled water to remove leftover ethanol and salt. Pellicles were then dried flat using a heated press set to 120 °C and 1 ton of pressure. This process on average leads to a 98% reduction in mass of the pellicle. To facilitate this drying and prevent the pellicles from sticking to the press, pellicles were sandwiched between three layers of filter paper. Wettability tests were conducted using a KRUSS EasyDrop with 1 μl of water. Each contact angle measurement was derived from the average contact angle from ten back-to-back water drop images taken within 10 s of drop contact with the pellicle surface.

Tensile strength experiments

K. rhaeticus ptyr1 was inoculated into 15-cm square Petri dishes containing 50 ml of HS-glucose media, with 0.5 g l−1 tyrosine, 10 μM CuSO4 and 340 μg ml−1 chloramphenicol. After incubating at 30 °C for 7 days, pellicles were collected and cut into half. One half was placed into an eumelanin development buffer to initiate eumelanin production and the other half into an acetate buffer containing 0.5 g l−1 tyrosine and 10 μM CuSO4 at pH 3.6 to prevent eumelanin production. After 24 h of shaking at 30 °C, melanated and unmelanated pellicles were removed from their respective buffers and sterilized in a 70% ethanol solution overnight. Pellicles were then washed in distilled water to remove ethanol and salts left over from the eumelanin development processes. Pellicles were then dried flat using a heated press set to 120 °C and 1 ton of pressure. This process on average leads to a 98% reduction in mass of the pellicle. The 35 -mm-long dog-bone test specimens were cut out of the dried cellulose using a Zwick ZCP 020 manual cutting press. Pellicle specimen ends reinforced with a card using Everbuild Stick 2 superglue. Dots were marked on the surface of each specimen for the optical measurement of displacement. A silver pen was used to dot melanated cellulose to generate the necessary contrast for optical measurement of displacement. Tensile tests were conducted with a Deben Microtest Tensile Stage using a load cell of 200N and cross-head speed of 0.5 mm min−1.

Scanning electron microscopy

The unmelanated pellicle was prepared by placing it into an acidic acetate buffer at pH 3.6, which prevented eumelanin synthesis and incubated in identical conditions to the melanated pellicle in the eumelanin development buffer bath. Melanated and unmelanated pellicles were prepared for SEM through the following steps. Unsterilized pellicles were placed in a 20% ethanol solution and shaken gently for 1 h before being removed and placed into a 40% ethanol solution and shaken gently. This process was repeated for 60%, 80% and 100% ethanol solutions to ensure the maximum replacement of water with ethanol from the cellulose matrix. Pellicles were then flash-frozen in liquid nitrogen and freeze-dried until completely dry. The fully dried pellicles were then fixed on aluminum studs, sputter coated with gold and imaged at 5 kV with a Zeiss Auriga Gemini FEG FIB-SEM.

Light microscopy

K. rhaeticus ptyr1 and K. rhaeticus ctyr1 were separately inoculated into 3 ml of HS-glucose media containing 2% (vol/vol) cellulase and 340 μg ml−1 chloramphenicol and grown shaking at 30 °C until turbid. The turbid cultures were then pelleted by centrifugation, washed with 1 ml PBS and split into two separate 1.5 ml centrifuge tubes. The cells were then pelleted again. One pellet was resuspended with 500 μl eumelanin development buffer to initiate eumelanin production and the other pellet was resuspended with 500 μl PBS to keep the cells unmelantated. The cells were incubated over 24 h at 30 °C by which point the tube containing the cells in eumelanin development buffer had turned black. To prepare the microscope slides, 1 μl of melanated and unmelanated cells were placed on separate 1% agarose pads and imaged on a Nikon Ti-EX1 invert microscope with a ×40 objective lens. Cells were imaged in bright field with no phase contrast to accurately represent the shade of the cells.

Pellicle cross-sections

K. rhaeticus WT, K. rhaeticus ptyr1 and K. rhaeticus ctyr1 were inoculated into two-well deep well plates containing 50 ml of HS-glucose media, with 0.5 g l−1 tyrosine, 10 μM CuSO4 and 340 μg ml−1 chloramphenicol. After 10 days of incubation at 30 °C, pellicles were collected and placed into eumelanin development buffer. After 24 h, pellicles were sterilized through autoclaving. Pellicles were then placed in a −20 °C freezer for 24 h to minimize compression during sectioning. The frozen pellicles were sectioned by hand using a Leica DB80LX blade and imaged using a macro lens (Leica) on an RS Pro lightbox.

Color resistance to water spotting

K. rhaeticus ptyr1 and K. rhaeticus ctyr1 were inoculated into 12.5 × 16.5 cm2 two-well glass container with 200 ml of HS-glucose media with 0.5 g l−1 tyrosine, 10 μΜ CuSO4 and 340 μg ml−1 chloramphenicol. After incubation for 7 days at 30 °C, pellicles were collected. Eumelanin production was initiated by placing the pellicles into eumelanin development buffer. After 24 h of shaking at 30 °C, pellicles were removed from the buffer and sterilized in 70% ethanol solution overnight. Pellicles were then washed in distilled water to remove ethanol and leftover salts. To make the material pliable after drying, replicate pellicles were placed in 0% or 5% glycerol solution overnight. Pellicles were then dried flat using a heated press set to 120 °C and 1 ton of pressure. To facilitate this drying and prevent the pellicles from sticking to the press, pellicles were sandwiched between three layers of filter paper. Water spotting tests were adapted from ISO 105-E07:2010 standard. Eumelanated pellicles were secured onto an RS Pro lightbox, and 50 μl of distilled water was spotted onto each sample in triplicate. Pellicles were imaged before, immediately after and 16 h after water spotting and assessed for color change.

Patterning mCherry expression in a K. rhaeticus pOpto-T7RNAP*(563-F2)-mCherry pellicle

A custom projection rig was built to project light onto the growing pellicle (Extended Data Fig. 7b). This held an acetate transparency that contained various components that would test the quality of the patterning in the pellicle. The image transparency was designed in Adobe Illustrator and printed on an HP LaserJet 500 MFP M570. Four acetate transparencies were stacked atop each other to form the final transparency. This was then sealed between glass slides and secured to the upper laboratory loop clamp. The pellicle container was sterilized and filled with 500 ml of HS-glucose media, containing 0.1% (wt/vol) arabinose, 1% (vol/vol) ethanol and 170 μg ml−1 chloramphenicol. The media was then inoculated with a 1-ml K. rhaeticus pOpto-T7RNAP*(563-F2)-mCherry glycerol aliquot, and a glass lid was placed on top of the container. This glass lid was warmed before placement to prevent condensation forming on it and distorting the projection. The LED lamp was then turned on, and the lens shuttered with a piece of black card. After 3 days at ~30 °C, a thin pellicle had formed. The lens was uncovered and the image from the transparency focused on the pellicle. Once the pellicle had been exposed to the projected image for 3 days, it was collected and scanned using a FLA-5000 fluorescence scanner (Fujifilm). Image analysis was conducted using the OpenCV Python library.

Patterning tyr1 expression in a K. rhaeticus Opto-T7RNAP(563-F1)-tyr1 pellicle

A custom rig using a commercial LED projector (ViewSonic M1) was built to project light onto the growing pellicle (Extended Data Fig. 7e). The rig was draped with blackout fabric to remove outside light. A time-lapse image was designed in Adobe Illustrator to test how long a given pellicle would need to be exposed to light before an identifiable change in pigmentation could be observed. In this image, blue is represented by an RGB value of (0, 0, 255), cyan by (0, 255, 255), white by (255, 255, 255) and black by (0, 0, 0) (Fig. 4h). The pellicle container was sterilized and filled with 1 l of coconut water media, containing 1% (wt/vol) arabinose, 0.5 g l−1l-tyrosine, 10 μM CuSO4, 1% (vol/vol) ethanol and 200 μg ml−1 spectinomycin. The media was then inoculated with a 1-ml K. rhaeticus Opto-T7RNAP(563-F1)-tyr1 glycerol aliquot and the culture container was covered with foil. While this version of the optogenetic rig did contain a heater, in practice, we found this was only effective at heating the growth area by 1–2 °C above room temperature. After 8 days at near room temperature (~20 °C), a thin pellicle had formed. The foil was then removed, the projector focused on the surface of the pellicle and the 80-h video started. After 80 h, the pellicle was collected and placed into a 300 × 400 mm Eurobox containing 2 l of concentrated eumelanin development buffer and left to develop in stationary conditions at 30 °C until a discernible pattern could be identified. The pellicle was then washed in dH2O to remove eumelanin that had not accumulated within the pellicle. Densitometry scans of the pellicle were taken using an Amersham Typhoon scanner (GE) and set to the digi-blue digitalization setting.

Characterizing mCherry expressing optogenetic strains

K. rhaeticus Opto-T7RNAP strains carrying the pT7-mCherry plasmid and K. rhaeticus pOpto-T7RNAP*(563-F2)-mCherry were cultured, in darkness, shaking in 3 ml of HS-glucose media with 2% cellulase, containing either spectinomycin at 200 μg ml−1 or chloramphenicol at 340 μg ml−1 depending on the plasmid. When all cultures had become turbid, the OD600 was measured and cultures were all either diluted or concentrated to an OD600 of 1, before being inoculated (a ratio of 1:10) into a 96-well deep well plate containing 270 μl HS-glucose media with 2% cellulase and either 0, 1, 10 or 100 mg ml−1 of arabinose. Where appropriate, spectinomycin at 200 μg ml−1 and chloramphenicol at 340 μg ml−1 were added to the wells. After 18 h of shaking growth at 30 °C in darkness, cells were split across two clear 96-well plates, diluted 1:2 into fresh media with a matching arabinose concentration. One plate was placed onto a shaker under a blue LED flood light and the other plate was wrapped in foil and placed on the same shaker. Both plates were sealed with a Breath-Easy sealing membrane. After 6 h in the two lighting conditions at 30 °C and fast shaking, the cells were placed into a plate reader, and red fluorescence in each well was measured using ex of 590 nm and em of 645 nm as well as cell density at OD600.

Characterizing tyr1 expressing optogenetic strains

The Opto-T7RNAP K. rhaeticus strains carrying the pT7-tyr1 plasmid and K. rhaeticus pOpto-T7RNAP(563-F1)-tyr were cultured in the same manner as the mCherry strains—with the exception that the HS-glucose was supplemented with 0.5 g l−1 tyrosine and 10 μM CuSO4. The approach to exposing the cells to blue light was also the same as the mCherry strains, except, after 6 h of exposure time, the two plates were entered into the eumelanin production assay procedure. The two plates were placed onto the OT-2 deck and samples from both plates were mixed with eumelanin development buffer in a 384-well reaction plate. Each well in the two 96-well plates was sampled twice in the 384 reaction plate to give two technical replicates for each well. These two replicates were then averaged during analysis.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.