Goal — The Neoleathic age wants to create a suitable leather substitute with bacterial cellulose (BC) produced from a Symbiotic Co-culture Of Bacteria (Komagataeibacter) and Yeast (SCOBY) by modifying BC in three ways:

1. Binding spider silk proteins to improve BC’s properties
2. Dyeing BC with natural dyes
3. Creating fragrance compounds in BC

Major achievements:

1. Characterized and perfected BC growth protocols
2. Successfully bound spider silk proteins in vitro to BC and tested its softness and strength, which showed significant results
3. Proved the viability of in situ spider silk binding
4. Achieved mass scale production of natural dyes
5. Engineered and characterized new bio-bricks relating to dye production
6. Measured the rate and titer of the enzymes in dye production
7. Engineered yeast to produce fragrance from the byproducts of BC production

Future directions:

To achieve our goal of BC becoming a suitable leather substitute, more research needs to be done regarding industrial production and processing of BC. Additionally, an interesting prospect is the direct synthesis of dyes by modifying Komagataeibacter. A simpler, one-celled model for dye synthesis would also greatly improve dye production, as less human resources will be needed (something our modeling team is trying to model!). Overall, while we demonstrate the viability of the Neoleathic age, more research is needed to implement our ideas to reality.

The base material for our project, bacterial cellulose (BC), is produced by a Symbiotic Co-culture Of Bacteria and Yeast (SCOBY) of Komagataeibacter intermedius/rhaeticus and Saccharomyces cerevisiae (1), similar to the health drink kombucha. Before we begin our project, we had to acquire Komagataeibacter strains for our SCOBY.

Isolating and characterizing Komagataeibacter strains

To acquire Komagataeibacter strains, we bought commercially available kombucha and isolated Komagataeibacter from the drink. We performed 16sRNA PCR and sequencing of different strains and constructed a phylogenetic tree (Fig. 1) to identify our strains of Komagataeibacter.

Figure 1. Phylogenetic tree of our strains (25, 12, B2, and 40) constructed using 16sRNA analysis. Due to size constraints, “rh” stands for rhaeticus, “xy" for “xylinus”, "in" for “intermedius”, “su” for “sucrofermentans”, and “ob” for “oboediens”

We then cultured Komagataeibacter rhaeticus and Komagataeibacter intermedius on YPD plates with 0.5% glucose and 1% cellulase and took photos under a microscope (Fig. 2)

Figure 2. A. Pictures of K. rhaeticus and K. intermedius under a microscope. The visible crystals within K. intermedius are likely the result of salt in the medium. B. The edge of a K. rhaeticus culture. The effects of cellulase can be see with the broken cellulose fragments.

After identifying our strains, we grew them, individually, for ten days and measured the dry weight of the BC produced, to figure out which strains can produce the most BC.

Sample BC membranes from Komagataeibacter strain 25 are generally heavier than samples from strain 12, indicating that strain 25 may be a better choice for growing BC membrane due to its higher BC yield. (*还要compare其他的)

Afterwards, we then attempted SCOBY with all four strains 12, 25, 40, and B2. 12 and 25 were extremely unstable in SCOBY, and were incapable of consistently forming a BC membrane in SCOBY. 40 was more stable than 12 and 25, and was able to form BC fairly consistently, but it had a slow growth rate in SCOBY. B2, on the other hand, was able to consistently form BC in SCOBY, and had a fast growth rate in SCOBY compared to 40. Thus, B2 was selected for the rest of the project.

Comparison of samples

Different growth stages of SCOBY and BC

We grew our SCOBY and characterized the BC in different growth environments (Fig. 3a). A side note here is that Komagataeibacter can produce BC on its own with the presence of glucose. All cultures are grown in YPD with 0.5% sucrose. Aerobic conditions are ensured for all cultures.

Figure 3. A. In clockwise order starting from the top left: Komagataeibacter in 14mL culturing tube with 5mL YPD, SCOBY in 50mL centrifuge tube with 15mL YPD, Komagataeibacter in 50mL centrifuge tube with 15mL YPD, SCOBY in 2L glass bowl with 600mL YPD, SCOBY in 4L glass tank with 2L YPD. B. Unprocessed BC from 4L glass tank.

In the 14mL culturing tube, the Komagataeibacter grew into a furry, ball-like structure, with the inner part made of cellulose, and the outer part made of bacteria. After approximately one week, Komagataeibacter culture’s BC will be 2-3 mm thick, while SCOBY’s BC will be 5-6 mm thick, indicating that yeast is extremely helpful in increasing BC yields.

Measuring membrane growth

To gain a better understanding of BC's growth, we measured SCOBY's BC's thickness over time in different growth conditions (Fig. 4), with the same medium as before.

Figure 4. From top to bottom, measurement of the thickness of SCOBY's BC's thickness in 50mL centrifuge tubes, 1.5L glass bowl, and 4L glass tank over time.

We noticed that the BC membrane generally grows faster during the first few days, and its growth rate gradually slows down to zero after seven to ten days, something useful in order to determine when to harvest the BC.

Producing BC on a large scale

To produce BC on a more massive scale for further experimentation and proof of concept, we amplified production from a 50mL test tube to 1.5L glass bowl to finally a 4L glass tank. The biggest barrier during the entire process was contamination, and we had multiple attempts fail because of contamination, especially our first few attempts. This inspired us to visit Classy-Kiss (read more on our visit on our iHPs page), where we gained insight into how they sterilized their equipment. Furthermore, they also provided helpful advice regarding optimal fermentation conditions. As we wanted our BC to be a leather substitute in the future, we designed a “box” (the name of our hardware) capable of growing and processing the leather in one place, without the need for humans. Visit our hardware page for more information!

Dry BC is very brittle, similar to dry tree bark (they are both made of cellulose). This brittleness makes it unsuitable as a leather substitute. Therefore, we hypothesized that by using a synthetic spider silk protein NT2RepCT (first characterized by GBSZ_2019; it was the best basic part of 2019) fused with cellulose binding matrixes (CBMs) to create CBM-NT2RepCT-CBM proteins, which can bind to our BC and improve its physical characteristics. Additionally, by engineering yeast to secrete CBM-NT2RepCT-CBM, we hope to bind spider silk proteins to BC as it is forming.

Structure and properties of spider silk and reinforcing BC

Spider silk proteins in nature are made of multiple repetitive amino acid sequences, up to hundreds of repeats long. These amino acid sequences form beta-pleated sheets, and hydrogen-bonds between the repeats stabilize the structure and form the spider silk we see. Artificial spider silk proteins contains much less repeats, only two repeats in NT2RepCT. However, they still function similarly to natural spider silk. When not in the “string” form we see, spider silk proteins display hydrogel characteristics, forming a net of proteins, similar to our BC. By interlacing cellulose with spider silk, we create a reinforced net-like material, denser than both spider silk or BC alone. Furthermore, by attaching the spider silk to BC, we create a hydrogel made of two connected “nets”, making it stronger and softer than our original BC (Fig. 7).

Structure and properties of CBMs

To bind our spider silk proteins to BC, we used cellulose binding matrixes (CBMs). As implied by its name, CBMs bind to cellulose fibers. It is an artificial protein with its sequence being derived from natural proteins with cellulose-binding functions, such as cellulase, thus its name “matrix”. There are three types are CBMs, CBM1, CBM2, and CBM3, with differ in their size. CBM1 is the smallest, whilst CBM3 is the biggest.The CBMs used throughout our project is CBM3 from Ruminiclostridium thermocellum (Protein Data Bank accession number 1NBC) (2) and CBM2 from Cellulomonas fimi. Figure 8 shows their predicted structures and active sites.

Figure 8. Predicted structure and active site of CBM2 (left) and CBM3 (right)

Constructing CBM2/3-NT2RepCT-CBM2/3 plasmids

We constructed our spider silk fused with CBMs (CBM3-NT2RepCT-CBM3 and CBD-NT2RepCT-CBD) by golden gate assembly. Primers were used to add BsaI restriction sites and short flexible linkers of 5 amino acids (GSGGS and GGGGS) in NT2RepCT (we have access to GBSZ 2019’s strain) to fuse the respective domains together in the pET28a vector with two BsaI sites sandwiched between two CBMs (acquired by DNA synthesis) (Fig. 9a). After construction, the plasmids were transformed successfully into E. coli strain BL21 for inducible expression under the T7 system with IPTG (Fig. 9b).

Figure 9. A. Schematic representing the design and construction of CBM-NT2RepCT-CBM. CBM represents either CBM3 or CBD. B. Colony PCR gel electrophoresis results from transformation into E. coli BL21.

Comparing the solubility of CBM2/3 fused with NT2RepCT

Expression of CBM3, CBM2, CBM3-NT2RepCT-CBM3, and CBD-NT2RepCT-CBD in E. coli BL21 was induced with IPTG and a SDS-PAGE analysis was conducted (Fig. 10).

Figure 10. A. Schematic representing the constructed CBM3-NT2RepCT-CBM3 and CBD-NT2RepCT-CBD plasmids. B. SDS-PAGE gel results of expression of CBM3-NT2RepCT-CBM3 and CBD-NT2RepCT-CBD in E. coli BL21. The black and red arrows indicate CBM3-NT2RepCT-CBM3 and CBD-NT2RepCT-CBD respectively.

As shown by the SDS-PAGE results, CBM2-NT2RepCT-CBM2 was present in the whole cell sample, but absent in the supernatant, whilst CBM3-NT2RepCT-CBM3 was present in both, indicating poor water solubility of CBM2-NT2RepCT-CBM2. Therefore, CBM3-NT2RepCT-CBM3 was chosen for the rest of the project. The difference in size between CBM2 and CBM3 can also be seen.

Purification of CBM3-NT2RepCT-CBM3

To test whether or not NT2RepCT or CBM3-NT2RepCT-CBM3 made a difference after mixing or binding with our BC, we first needed to purify both proteins. We cultured and induced both proteins with IPTG, and used standard his-tag purification methods and a BCA test to determine the concentration of our spider silk proteins (Fig. 10).

There was a higher concentration of NT2RepCT than CBM3-NT2RepCT-CBM3, with their concentrations being 0.38mg/mL and 0.265mg/mL respectively.

Measuring softness and tensile stress of BC mixed with NT2RepCT and CBM3-NT2RepCT-CBM3

After purification of both NT2RepCT and CBM3-NT2RepCT-CBM3, we mixed each with dried BC. We then tested the softness of BC mixed with different amounts of CBM3-NT2RepCT-CBM3 (10-day pellicle with approx. same weight grown in a 50mL tube with 0, 0.5, 1.5, and 3.4mg of CBM3-NT2RepCT-CBM3) (Fig. 11a), and the tensile strength of pure BC, BC with NT2RepCT, and BC with CBM3-NT2RepCT-CBM3 (Fig. 11b). More information about how we did the experiments can be found here.

Figure 11. A. Comparison of the softness of BC mixed with 0, 0.5, 1.5, and 3.4mg of CBM3-NT2RepCT-CBM3. B. Comparison of maximal stress between pure BC, BC with NT2RepCT, and BC with CBM3-NT2RepCT-CBM3. All error bars represent two standard deviations from the mean. */** represents a confidence level of 95/99%.

There is a steady increase of softness as more CBM3-NT2RepCT-CBM3 is added to BC, showing an increase of approximately 50% between pure BC and BC with 3.4mg of CBM3-NT2RepCT-CBM3. More surprising is the one fold increase between pure BC and BC with CBM3-NT2RepCT-CBM3, demonstrating that the combination of BC and spider silk is a viable way to increase BC’s strength. Additionally, the difference in tensile strength is significant between NT2RepCT and CBM3-NT2RepCT-CBM3, showing that the addition of CBM3 is crucial in achieving a stronger material.

Engineering yeast to produce and secrete CBM3-NT2RepCT-CBM3 as BC is forming

As we addition of CBM3-NT2RepCT-CBM3 is shown to increase the softness and tensile strength of BC, making it more like traditional leather, we wanted to engineer yeast to produce and secrete CBM3-NT2RepCT-CBM3 to bind to the BC as it is forming in SCOBY (Fig. 12). This way, the labor-intensive process of protein purification can be skipped.

To enable secretion of production in yeast, we need to attach a short maturation factor alpha (Mα) to our protein. Previous research has characterized Mα and mutated it to become more efficient at protein secretion. For our project, we selected Mα E86T; A87N (referred to as MαMut) as it had one of the highest secretion rates (3). We constructed two plasmids, MαMut-sfGFP-CBM3 and MαMut-CBM3-NT2RepCT-CBM3.

Verifying secretion of protein with MαMut-sfGFP-CBM3 (Fig. 13)

Growing SCOBY with MαMut-sfGFP-CBM3(Fig. 14)

Comparing in situ and in vitro binding of MαMut-CBM3-NT2RepCT-CBM3 (Fig. 15)

Tanning and dyeing animal leather is very polluting process. In areas with lax regulations, such pollution will enter the natural ecosystem, significantly impacting its health. Therefore, we produced three natural dyes — indigo, tyrian purple (6, 6’dibromoindigo), and tyrian red (6, 6’dichloroindigo) in E. coli from tryptophan (trp) to dye our BC. We experimented with different expression systems and protein linkers and quantified the production of our intermediate product and our final dyes.

Production pathway of indigo, tyrian purple, and tyrian red from Trp

Production of indigoid dyes from trp require three enzymes: Trp-6-halogenase, TnaA and FMO (4). Trp-6-halogenase will halogenize trp’s 6th carbon in the presence of halide salts, turning trp into 6-X-trp, where X is a halogen. TnaA will separate the indole sidechain of trp from the amino acid backbone, turning trp or 6-X-trp into indole or 6-X-indole (we only care about indole). FMO will add a hydroxyl group to the 3rd carbon on indole or 6-X-indole to convert it to 3-hydroxy-indole or 3-hydroxy-6-X-indole. Finally, 3-hydroxy-indole or 3-hydroxy-6-X-indole will spontaneously dimerize into indigo or 6, 6’diXindigo (Fig. 16).

Figure 16. Schematic representing conversion of trp into indigo or tyrian purple under the presence of trp-6-halogenase, TnaA, and FMO. The problem arises when trp-6-halogenase, TnaA, and FMO is simultaneously expressed, as trp will trend towards indigo, not tyrian purple.

The problem arises when we simultaneously express all three enzymes (trp-6-halogenase, TnaA, and FMO) under the presence of trp. Trp will, more likely, take the shorter enzymatic pathway and be turned into indigo by TnaA and FMO, without being halogenated first by trp-6-halogenase. To achieve optimal production of halogenated indigoid dyes, we need to separate expression of trp-6-haloganese and TnaA and FMO. To do this, we constructed two strains of E. coli, one expressing trp-6-halogenase, the other TnaA and FMO.

Improving solubility of trp-6-halogenase

Previous research has already characterized the fused enzyme Fre-SttH as a trp-6-halogenase (4). Thus, we chose to use Fre-SttH as our trp-6-halogenase.

Fre-SttH is composed of two separate domains, Fre and SttH. SttH is a trp-6-haloganese that requires FADH2 as a cofactor to convert trp into 6-X-trp, and is highly insoluble in E. coli. Therefore, previous researchers have fused Fre, a highly-soluble flavin reductase which reduces FAD to FADH2 from E. coli, with SttH as a N-terminal soluble tag, enabling the protein to become soluble and eliminating the need for costly FADH2 cofactors to be added (Fig. 17)(4).

Figure 17. Schematic representing the fusion of Fre to SttH to eliminate the need for costly FADH2 cofactors to be added and to make SttH soluble in E. coli (4).

Expressing Fre-SttH in E. coli

To express Fre-SttH, we first attempted the T7 system, commonly used in E. coli BL21. We constructed histag-Fre-SttH in the T7 system with a lacO promoter, but found both its expression and solubility to be fairly low, thus making it unsuitable for our project.

Because we needed a knockout TnaA (TnaA is naturally expressed in E. coli, giving it its characteristic scent) strain to express Fre-SttH so that our 6-X-Trp will not turn into 6-X-indole for measurement purposes, we switched from the T7 system to the ptac system. The ptac system, unlike the T7 system which can only be used in E. coli BL21, can be used in all E. coli strains. Our knockout strain was supplied in E. coli DH5α, courtesy of Sha Zhou, in which we constructed ptac-histag-Fre-SttH and ptac-Fre-SttH.

We then expressed induced expression of both proteins (histag-Fre-SttH and Fre-SttH) and performed SDS-PAGE analysis. Results show that histag-Fre-SttH expression and solubility were poor, but Fre-SttH had extremely high expression and high solubility. Thus, ptac-Fre-SttH in ΔtnaA E. coli DH5α was used for all further experiments.

Measuring Fre-SttH activity and substrate-product concentrations using HPLC

In order to quantify our enzymatic rate of Fre-SttH and measure the substrate-product concentrations of Trp and 6-X-Trp, we induced Fre-SttH expression and added Trp and NaCl/NaBr. We then took samples of the culture every 6 hours for 24 hours for HPLC analysis. The relative concentrations of Trp and 6-X-Trp was calculated for every sample.

The rate of conversion from Trp + NaCl to 6-Cl-Trp was faster than that of its Br counterpart, reaching around 1.4mM after 24 hours for Cl, compared to 1.0mM for Br. To learn more about how we did the experiment, visit our experiment and measurement pages. From this, we acquired several enzymatic constants, which was vital to our modeling team.

Fusing TnaA and FMO together

To improve the reaction speed of TnaA and FMO, we fused these two proteins together into TnaA-FMO. This increases the chance of 6-X-Trp, after being halogenized by FMO, encountering TnaA to form the dyes, rather than being de-halogenated or degraded by the cell.

Designing TnaA-FMO

We designed and engineered three strains of E. coli, 044, 042, and 047, which expresses TnaA-rbs-FMO (TnaA and FMO is expressed separately), TnaA-rigid linker-FMO (TnaA-RL-FMO), and TnaA-Flexible linker-FMO (TnaA-FL-FMO). The sequences for RL and FL are EAAAKEAAAK and GGGGSGGGGS respectively. As TnaA is expressed as a tetramer and FMO a dimer, we speculate that a TnaA tetramer at the center of the fused protein, with FMO forming two dimers to each side of the TnaA tetramer, similar to a CO2 molecule. As such, we speculate that 047 (TnaA-FL-FMO) will have higher efficiency than 044 (TnaA-rbs-FMO) and 042 (TnaA-RL-FMO), as the fused protein can more easily form into its working conformation.

We constructed 044, 042, and 047 plasmids for the ptac system, and we cultured and induced these strains with IPTG. Afterwards, 1mM of either Trp, 6-Cl-Trp, or 6-Br-Trp was added, and the relative titers of each strain was taken by using a standard calibration curve of the three dyes. 缺这里的数据

Drawing inspiration upon GBSZ 2019’s TnaA-rbs-FMO expression system using a constitutive promoter system(TALEsp2) (025), we designed TALEsp2-TnaA-FL-FMO (046), and compared the titers of 025 and 046, to further test whether a TnaA-FL-FMO has higher titres than TnaA-rbs-FMO.缺数据

After confirming the efficacy of Fre-SttH and TnaA-FMO, we wanted to attempt producing dyes from Trp and NaX salts. We cultured and induced Fre-SttH, then took its supernatant and added it to the TnaA-FMO cultures.

The 047 (TnaA-FL-FMO) culture is much more darker and purer in color compared to the rest, showing that 047 probably produced more dyes than the other strains. 025 and 044, on the other hand, whilst still producing a substantial amount of dyes, have colors that are bluer compared to other strains, indicating that apart from tyrian purple/red, these strains also produced indigo. The reason for this is still unknown, but we speculate that it might be because after TnaA acts upon 6-X-Trp to form 6-X-indole, the 6-X-indole might then be de-halogenated before FMO can turn into tyrian purple/red. FMO will then turn the indole into indigo.

We then measured the titers of the above cultures. 缺数据

To dye clothe and our BC, we scaled up production of our dyes. We then dyed silk handkerchiefs and our BC.

The resulting large-scale production was optimal, and the color on the silk and BC is similar to commercial, artificial dyes.

Metabolic pathway of producing ethyl acetate

In SCOBY, yeast will break down sucrose into glucose and fructose, and Komagataeibacter will then oxidize glucose, to gain energy, into acetate. The resulting acetate is a useless byproduct of SCOBY, which will accumulate in the culture. This acetate creates two problems. One, acetate is acidic, and can interfere with secreted yeast proteins, such as MαMut-CBM3-NT2RepCT-CBM3. Second, acetate itself is odorous and smells like vinegar.

In order to utilize the accumulated acetate and to convert it into something useful, we decided on a simple metabolic pathway to turn ethanol (also produced by yeast in SCOBY) and acetate into ethyl acetate. Ethyl acetate contains a fruity smell. This pathway can be completed with two heterologous enzymes: SaACS2 from Salmonella enterica and AeAT9 from Actinidia eriantha (kiwifruit). SaACS2 operates best under anaerobic conditions, and converts acetate into acetyl-CoA. AeAT9 combines acetyl-CoA and ethanol to form ethyl acetate.

We then constructed plasmids containing pTEF1-SaACS2-tADH1 and pRPL8B-AeAT9-tSSA1, through the use of a yeast toolkit first characterized by Lee et al. (5). We synthesized the level 0 plasmids, each containing a basic bio-brick (promoter, terminator, coding region, etc.) flanked by two BsaI restriction sites. Then we constructed the level 1 plasmids (pTEF1-SaACS2-tADH1 and pRPL8B-AeAT9-tSSA1), containing 1 transcriptional unit capable of expression in yeast, using golden gate assembly of 8 level 0 plasmids.

To construct the final large plasmid, we used the previous two level 1 plasmids which contained our target transcriptional units and another level 1 plasmid which acts as the plasmid to perform BsmBI golden gate assembly. During this entire process, we were in close contact and collaboration with AISSU_Union (we share the same lab), where we shared resources, plasmids, and experience with each another. Specifically, their instructor, Tianxiang Wang, was instrumental in guiding our plasmid construction, after we failed multiple times. Read more about our partnership here.

After the final level 2 plasmid (pTEF1-SaACS2-tADH1-pRPL8B-AeAT9-tSSA1) was successfully constructed, we transformed this plasmid into yeast.

After transformation was confirmed, we cultured the yeast for 24 hours in YPD + glucose, then added 0.1% and 0.2% pure ethanol and acetate to different cultures. We took samples at 24h, 48h, and 72h to perform gas chromatography analysis.

We received surprising cultures of yeast, unlike anything we or our instructors have seen before. It was a globulous yellow structure that floated around in the medium, with some similarity to ginseng. This glob continuously grew as time progressed, indicating that it was not dead cells caused by SaACS2 or AeAT9 protein toxicity. Furthermore, this glob was present in all 5 cultures (1 0% culture as control, 2 0.1% acetate & ethanol cultures, and 2 0.2% acetate & ethanol cultures), meaning that the formation of the glob was independent of the addition of acetate and ethanol.

We speculate that the glob is a result of SaACS2’s affinity to be expressed in anaerobic conditions. During shake culturing, the yeast cells with the least contact with air started to express SaACS2. As SaACS2 is highly efficient at synthesizing acetyl-CoA (more than 50 times as efficient as homologous ACS1 in yeast), the cells which expressed SaACS2 grew substantially faster than the other cells, using acetyl-CoA as an energy source. Over time, these cells accumulated to form the glob we see. However, as time is limited (wiki freeze), we did not have time to test our hypothesis.

Despite the weird culture, our GC results show that … 没数据