Team:LINKS China/Proof Of Concept
Proof of concept:
Our project aims to develop a sustainable leather substitute using bacterial cellulose membranes (BCM) and producing natural dyes to dye our BCM. BCM is produced from a symbiotic co-culture of bacteria (Komagateaibacter) and yeast (SCOBY). Our goal is to improve the physical characteristics of BCM by adding spider silk fibroin proteins, and to use engineered E. coli to produce indigo and two types of tyrian purple (6, 6’-di-chloro-indigo and 6, 6’ di-bromo-indigo) from tryptophan (trp). We hope that our new material can be used in the everyday setting, replacing traditional leather.
Figure 1. Komagateaibacter sp. and S. cerevisiae BY4741 will produce bacteria cellulose membrane. E. coli will produce spider silk proteins fused with cellulose binding matrixes and natural pigment dyes. By combining these three components, we can produce NeoLeather.
Production of spider silk BCM composite material
We successfully cultured SCOBY and produced BCM in a 4L glass tank with 2L of growth media, showing that BCM can be grown on a commercial scale. We also developed post-production washing methods to turn the water-saturated BCM into a drier, more leathery material. By layering the dried and washed BCM together, we are able to achieve a material similar to leather.
Figure 2. BCM in 2L glass tank without growth media.
To reduce the possibility of contamination and to wash the BCM automatically, we designed our NeoLeathic Tanner, capable of culturing, washing, and drying BCM without human interference. We used the NeoLeathic Tanner to grow SCOBY, and the BCM produced was better than that produced from an incubator, likely due to the reduced contact with the outside environment. Visit our hardware page to learn more!
Figure 3. Our NeoLeathic Tanner
We designed and engineered E. coli to produce spider silk fibroins fused with cellulose binding matrixes (CBMs) to bind to our BCM. We improved the water solubility of the fused protein by experimenting with different CBMs, ultimately reaching a titer of 133.08mg/L for CBM-spider silk proteins.
Figure 4. BCA test results for the titer of CBM3-2Rep-CBM3.
After purifying the protein and mixing CBM-spider silk with dried BCM, we observed significant increases in both maximal stress and softness of BCM. There was an approximate one-fold increase in maximal stress of the spider silk BCM material compared with raw BCM, and an approximate three fold increase in softness. Visit our results page for more information.
Figure 5. Effect of concentration of CBM-spider silk on the physical properties of BCM. A) Comparison of the effect of CBM3-spider silk concentration on maximal force of BCM. B)
Comparison of the effect of CBM3-spider silk concentration on softness of BCM. Error bars represent two standard deviations from the mean. */** denotes 95/99% certainty.
Production of indigo and tyrian purple from tryptophan
Using two strains of engineered E. coli, we produced all three dyes from tryptophan and halogen salts, and have achieved large-scale production (400mL cultures) for all three dyes. After extracting the dyes, we dyed silk handkerchiefs and our BCM, demonstrating the viability of our dyes. Visit our results page for more information.
Figure 6. Production of indigo and tyrian purple in 400mL cultures.
Final leather product
Our final leather product is a composite material made from spider silk and BCM, improving the physical characters of BCM. It is dyed with natural dyes produced from E. coli, skipping the environmentally damaging steps of traditional tanning and dyeing. We hope that with more research, we will set the basis for a more sustainable leather industry.
Figure 7. BCM dyed with natural indigoid dyes. From left to right: Pure un-dyed BCM, BCM dyed with indigo, dyed with tyrian purple.
