The following illustration shows the architecture of our Bacterial Cellulose Modification System:

Module I. Bacterial Cellulose Production

Komagataeibacter is a microorganism widely utilized in large-scale bacterial cellulose production[2]. We have optimized the production process, particularly by refining nitrogen source selection and cultivation conditions, thereby significantly improving both the yield and quality of the produced bacterial cellulose.

Module II. Scaffold Protein Design and Production

Through genetic engineering, we successfully constructed an Escherichia coli strain that expresses a scaffold protein, Curlis Fiber-Spytag, capable of binding to bacterial cellulose. In this system, the Curlis Fiber domain provides the bacterial cellulose-binding functionality, while Spytag enables the heterologous linkage via the SpyCatcher system.

Module III. Target Protein Design and Production

We designed a SpyCatcher-POI (protein of interest) fusion with a standardized interface for expression and purification. Using two distinct color proteins as demonstration models, we validated both the expression and purification processes. In future applications, we anticipate that this approach will allow the facile substitution of various POIs through the Golden Gate cloning method.

Composition

Finally, our experimental results confirmed the high-efficiency binding of bacterial cellulose, the scaffold protein Curlis Fiber-Spytag, and the target protein SpyCatcher-POI. These findings demonstrate the effectiveness and feasibility of this modular system, providing a POC and novel strategy for the modification and application of bacterial cellulose.

Work going on

Future teams can focus on identifying more protein pairs with strong binding affinity, like SpyTag-SpyCatcher, and adopt iGEM 2022 Michigan’s molecular chaperone strategy to improve the expression of challenging proteins, such as antimicrobial peptides, in E. coli.

Check the detailed DBTL of each module and the composition, in the following contents.

Cycle 1: Expression Test of Curlis-Spytag

Through reviewing previous iGEM projects[3-6] and related literature[7], we discovered that Curli fibers, composed of the CsgA protein, have excellent protein fiber production characteristics and a strong binding ability to cellulose. Therefore, this year we linked the CsgA protein with the SpyCatcher peptide through a linker, constructing a fusion protein to be used as a scaffold protein in cellulose modification. This fusion protein was cloned into the pET28a+ vector for expression.

Since the CsgA protein, which forms Curli fibers, exists in an insoluble state after self-assembly and cannot be verified by conventional electrophoresis, we used Congo red staining as a preliminary validation method to detect its expression in Escherichia coli.

1. Plasmid Synthesis and Transformation: The plasmid was obtained through full synthesis and transformed into Escherichia coli BL21(DE3). Three single colonies were picked and cultured overnight in LB medium containing antibiotics. Part of the bacterial solution was sent to Anshengda for sequencing confirmation, while the remaining solution was stored in 30% glycerol for future experiments.
2. Congo Red Staining Plate Preparation: Congo red plates with different IPTG concentrations were prepared to detect the expression and self-assembly of Curli fibers. Detailed experimental steps and concentration gradients can be found in the "Experiment" section.
3. Control Experiment: LB agar plates with different IPTG concentrations were also prepared as a control group to compare expression levels under different IPTG induction conditions. Detailed experimental steps and concentration gradients can be found in the "Experiment" section.

1. Bacterial Culture: Three single colonies of Curlis-Spytag were picked from the plate and cultured overnight in LB medium with kanamycin. At the same time, a strain containing the empty pET28a+ plasmid was picked as a control group and cultured overnight in LB-kanamycin medium.
2. Bacterial Solution Dropping and Incubation: 30 µL of the overnight culture was dropped onto Congo red plates with different IPTG concentrations and onto the control plates. The plates were incubated at 30°C for 48 hours to observe colony color changes and self-assembly.

By observing the results of Congo red staining, we can preliminarily validate the expression of the CsgA-SpyCatcher fusion protein in Escherichia coli and the formation of Curlis fibers, providing data support for subsequent experiments.

Through a simple Congo red staining experiment combined with extensive control experiments, we successfully validated that the designed plasmid could induce Escherichia coli to normally express Curlis fibers under the induction conditions. The experimental results showed that the induction effect was optimal at an IPTG concentration of 0.5 mM, which may represent the best induction concentration. Future work will focus on further optimizing Curlis fiber expression in liquid culture environments to collect and purify Curlis fibers, preparing for subsequent binding experiments.

Cycle2: Expression of Curlis-Spytag

After completing the identification of solid-phase expression, we now turn to liquid culture, as it allows for more efficient collection of the target protein. The plasmid design remains the same. To validate our approach, we first designed a small-scale 4 mL liquid culture system to optimize the expression of Curlis fibers, facilitating more efficient collection and subsequent purification.

Samples were taken from the previously sequenced and verified Curlis-Spytag glycerol stock and streaked onto LB plates containing kanamycin to isolate single colonies. At the same time, a strain carrying the empty pET28a+ plasmid was used as a control and similarly streaked onto LB plates containing kanamycin for single colony isolation.

Three single colonies of Curlis-Spytag were picked from the plate and inoculated into LB medium containing kanamycin. The cultures were grown overnight at 37°C and 220 rpm. The next day, the overnight cultures were diluted 1:100 and grown until the OD600 reached 0.3, at which point different concentrations of IPTG were added for induction. The cultures were then incubated at 30°C and 220 rpm for another 24 hours.

After 24 hours, we observed the appearance of obvious filamentous precipitates in the bacterial cultures. This indicates that Curlis fibers were successfully expressed and self-assembled into fibrous structures in the liquid culture system, providing a solid basis for subsequent protein collection and functional validation.

After washing with ddH2O, 1X PBS, and 5X PBS, we found that the filamentous precipitates in the culture medium could not be dissolved. To dissolve the Curlis fibers for detection using electrophoresis, we referred to literature and decided to use 8M guanidine hydrochloride, which successfully dissolved the precipitates. Subsequently, we purified the dissolved protein solution using a GST purification kit from Beyotime to remove non-specific proteins and obtain high-purity Curlis target protein.

Next, we performed Western blot (WB) analysis on the purified Curlis protein samples. Our designed Curlis-Spytag fusion protein carries a GST tag, and the target protein bands were detected using an anti-GST antibody. The results are shown in the following figure:

1. The plasmid we designed can successfully express Curlis-Spytag in a liquid culture system, and the protein can be secreted extracellularly and self-assemble into fibrous structures.
2. The Curlis-Spytag fibers can be effectively dissolved into a soluble state using 8M guanidine hydrochloride, facilitating subsequent experiments such as protein purification, validation, and cellulose binding.

Cycle 1: Construction of the Modified Protein Expression System and Demonstration of Expression and Purification Using Color Proteins

After successfully obtaining Curlis, the next step is to acquire target proteins with Spycatcher and verify their binding with Spytag[8], ultimately validating their feasibility for cellulose binding.

We used the J23119 strong promoter to drive the expression of functional proteins, with a His tag fused at the 5' end for downstream purification, and a Spycatcher fused at the 3' end for binding with Spytag. Additionally, a BsaI restriction site was introduced between the His tag and Spycatcher to simplify the integration of other proteins in the future. This allows for seamless assembly into our system through a simple Golden Gate reaction, eliminating the need for tedious molecular cloning.

As a demonstration to validate this system, we selected two commonly used fluorescent and color proteins, sfGFP and amilCP. These proteins were linked to Spycatcher via a linker to construct fusion proteins, which were then inserted into the pCDuet vector. The T7 expression system on the pCDuet was replaced with the J23119 promoter and BBa_B0034 expression system.

This design allows us to express fluorescent and color proteins with the Spycatcher tag, making it easier to observe the expression of the target sample system and subsequent binding. Additionally, inspired by the blue-white screening experiment, as the target vector for future protein expression vector construction, the colored empty vector provides an effective negative control for the assembly of other target proteins

All plasmids were obtained through full synthesis and transformed into BL21(DE3). Three single colonies were picked and cultured overnight in LB medium with the corresponding antibiotics. Part of the bacterial culture was sent to Anshengda for sequencing to confirm the correct sequence, while the rest was stored in 30% glycerol for future use.

Colonies were streaked from the glycerol stock, and single colonies were picked and cultured in 50 mL LB medium with streptomycin, incubated overnight at 37°C. The bacterial culture was collected at 10,000 rpm in a 50 mL centrifuge tube and washed with 50 mL of 1X PBS solution. Then, using Thermo B-PER reagent, the bacteria were lysed by sonication, and subsequent purification was performed according to the protocol of the Beyotime His-Tag Purification Kit.

Next, we performed Western blot (WB) analysis on the purified target protein samples. Our target protein carries a His tag, and the target protein bands were detected using an anti-His tag antibody. The results are shown in the following figure:

Through our experimental validation, the plasmids we designed successfully expressed the GFP-SpyCatcher and amilCP-SpyCatcher fusion proteins in Escherichia coli. We completed protein purification and obtained purified products. SDS-PAGE results showed that the molecular weights of the fusion proteins GFP-SpyCatcher and amilCP-SpyCatcher were 40.38 kDa and 39.38 kDa, respectively. However, the molecular weight bands for individual GFP (26.8 kDa), amilCP (25.8 kDa), and SpyCatcher (12.58 kDa) did not appear on the gel, indicating that no monomeric proteins were expressed in our experiment, confirming the successful expression and purification of the fusion proteins. Additionally, the presence of the SpyCatcher peptide did not affect the properties of the color proteins, further enhancing the feasibility of our system.

Next, we are preparing to conduct fusion experiments between the two modules.

Cycle 2: Attempt with Antimicrobial Peptide

After successfully engineering the color proteins, we aim to test the assembly and expression of other proteins. We selected a functional antimicrobial peptide as our target protein. Through literature review, we found that antimicrobial peptides can bind well to cellulose, making them suitable for use in antimicrobial dressings for medical applications. After further research, we identified an antimicrobial peptide, HBCM2 (BBa_K4143336), that can be expressed in Escherichia coli[9]. We decided to encode this peptide upstream of Spycatcher, replacing the color proteins used in the previous demonstration experiment.

The HBCM2 gene, containing BsaI restriction sites, was obtained through gene synthesis and cloned into the pUC57 vector. To switch vectors, we amplified and extracted the HBCM2 gene from the plasmid, and sequencing confirmed the accuracy of the sequence. We then used the Golden Gate reaction to integrate this gene into our expression plasmid, replacing the original sfGFP gene.

After unsuccessful transformation attempts using the common competent cells DH5a, DH10B, and T1, we decided to use EPI400 super-competent cells[10]. These cells can reduce plasmid copy number, lowering the metabolic burden on E. coli caused by antimicrobial peptide expression, thus allowing us to obtain single colonies with the correct target plasmid.

We transformed the Golden Gate product into EPI400 cells, obtained clones, and selected three single colonies to grow overnight in LB medium with the appropriate antibiotic. Part of the bacterial solution was sent to Anshengda for sequencing confirmation, and the rest was stored in 30% glycerol for future use.

The HBCM2 (AMP) glycerol stock and pCDuet were streaked onto streptomycin plates, and three single colonies were picked and cultured overnight in LB. The next day, the whole culture from the overnight growth was analyzed using SDS-PAGE.

1. At the molecular level, our standardized interface design works effectively, allowing us to replace the current fluorescent protein gene with other genes.
2. The presence of Spycatcher does not reduce the burden of antimicrobial peptide expression on bacterial growth. For this class of challenging proteins or those that affect E. coli growth, the design of additional molecular chaperones may be needed to assist in their expression.
3. Due to time constraints, we were unable to complete more experiments.

Cycle 1: Improvement of Nitrogen Source for Cellulose Production

A significant challenge in the application of bacterial cellulose (BC) is its high cost. The commonly used HS medium typically consists of 20 g/L glucose, 5 g/L yeast extract, and 5 g/L soytone. These expensive components are difficult to use in large-scale industrial applications. Some studies on alternative carbon sources have discussed replacing glucose with fructose, mannose, and fruit peels. However, since the final product we synthesize is cellulose, substituting carbon sources directly affects the substrate and may significantly impact the quality of the final product[11].

There has been less focus on nitrogen source alternatives in previous work[12-14], primarily centering around corn steep liquor and laundry wastewater. We aim to analyze the nitrogen source preferences of Komagataeibacter xylinus using metabolic modeling and to identify potential alternative nitrogen sources. To this end, we utilized genomic data from the Komagataeibacter xylinus strain CGMCC 17276, obtained from the China General Microbiological Culture Collection Center (CGMCC), submitted by East China Normal University.

Using the genome of Komagataeibacter xylinus strain CGMCC 17276, we generated a corresponding metabolic model through CarveMeand imported the resulting SBML file into cnapy for Flux Balance Analysis (FBA). To understand the optimal conditions for bacterial cellulose (BC) production, we utilized cobrapy to calculate the theoretical optimal medium that maximizes both growth rate and BC production simultaneously.

Based on our metabolic model analysis, the strain shows a higher demand for certain amino acids, specifically arginine (Arg), asparagine (Asn), threonine (Thr), and lysine (Lys). This suggests that these nitrogen sources play a significant role in supporting both growth and bacterial cellulose (BC) production. Interestingly, our model also indicates that the optimal carbon source for maximizing both growth and BC yield is not the commonly used glucose, but rather fructose.

Additionally, we explored the effects of varying concentrations of fructose as the carbon source and different nitrogen source combinations in cnapy to examine trends in BC production and growth. By conducting these simulations, we were able to analyze the relationship between production and growth, providing insight into how different nutrient conditions influence both the yield of BC and the overall biomass formation of K. xylinus. Our approach aims to uncover potential alternative nitrogen sources that can be used to lower the cost of BC production while maintaining high efficiency in industrial applications.

We selected three candidate nitrogen sources from non-food crops: cottonseed meal, soybean meal, and corn steep liquor, and analyzed their specific amino acid compositions. Cottonseed meal is rich in arginine, making it an ideal choice to meet this requirement, but its lysine and threonine contents are low, which cannot fully satisfy the demand for these amino acids. Soybean meal has insufficient levels of arginine and asparagine. While corn steep liquor has a more balanced amino acid profile with moderate lysine content, it is relatively low in arginine and asparagine.

This combination balances the strain’s amino acid needs while being cost-effective. We designed an experimental test using a total nitrogen equivalent of 40 mg/L to calculate the final concentration of each nitrogen source. Three groups were set up: one using only corn steep liquor, one using only cottonseed meal, and one with a cottonseed meal to corn steep liquor ratio of 1:1. Soytone was used as a positive control. The experiment was conducted in a 250 mL, 10x16 cm stainless steel pan culture system, with three replicates per group for experimental validation.

We incubated both plates statically at 30°C for 5 days, during which we monitored the formation of cellulose mats. At the end of the incubation period, we harvested the BC mats, washed them thoroughly with distilled water, and treated them with 4M NaOH at 80°C for 10min to remove residual cells. After neutralizing with water, we dried the cellulose mats in an oven at 80°C until constant weight was achieved.

To calculate the conversion efficiency of bacterial cellulose (BC) production, we used the formula:

Conversion Efficiency (%) = (Dry Weight of BC Produced / Total Sugar Input) x 100

For each experimental condition, we measured the dry weight of the BC mats after drying to constant weight and compared this to the total mass of nitrogen sources used in the culture medium. For example, if 2.5 grams of BC were produced using 5 grams of Sugar, the conversion efficiency would be 50%.

Our findings demonstrated that the original soytone condition provided the highest conversion efficiency for BC production, with the mixture of corn steep liquor and cottonseed meal yielding higher conversion rates than either alternative used alone. This study provides insight into the potential of using cost-effective, alternative nitrogen sources for the production of bacterial cellulose while maintaining high efficiency.

Our experiment shows that, for now, cottonseed meal and soybean meal alone cannot fully replace soytone. However, combining cottonseed meal with soybean meal or corn steep liquor can compensate for the deficiencies of a single nitrogen source. This combination meets the demand for arginine, supplements lysine and threonine, and helps control production costs. This approach may potentially be used in the future to reduce the use of soytone.

Cycle2 : Improvement of Cellulose Cultivation Environment

In the previous experiment, we observed an interesting phenomenon: the thickness of the BC membrane we cultivated was generally around 0.5 cm, but there was still some remaining liquid medium underneath it. We hypothesize that this is because the liquid below is unable to access oxygen, and BC is only produced at the interface between oxygen and the liquid. To optimize bacterial cellulose production efficiency, we designed an experiment to study the effect of culture vessel size on production efficiency while keeping the volume of the medium constant.

We selected two different culture plate sizes: a smaller 10 x 16 cm plate and a larger 15 x 27 cm plate. We hypothesize that the larger plate's greater surface area will enhance gas exchange and nutrient distribution, thereby improving cellulose production efficiency. By comparing the cellulose yield from these two different plate sizes, we aim to determine whether the size of the container can significantly impact conversion rates and overall production efficiency. This experimental design allows us to explore how physical dimensions affect key factors in bacterial cellulose synthesis.

Each plate was filled with 250 mL of HS media, which we prepared by dissolving 20 g of glucose, 5 g of peptone, 5 g of yeast extract, 2.7 g of disodium phosphate, and 1.15 g of citric acid per liter of distilled water, followed by autoclaving. After the media cooled, we inoculated both plates with the bacterial strain Komagataeibacter xylinus, ensuring equal inoculum concentration and uniform distribution across the surface of the media.

We incubated both plates statically at 30°C for 5 days, during which we monitored the formation of cellulose mats. At the end of the incubation period, we harvested the BC mats, washed them thoroughly with distilled water, and treated them with 4M NaOH at 80°C to remove residual cells. After neutralizing with water, we dried the cellulose mats in an oven at 80°C until constant weight was achieved. We then weighed each replicate individually and performed statistical analysis and plotting.

Our results showed that the smaller plate (10 x 16 cm) resulted in a conversion rate of 11.5% on average, while the larger plate (15 x 27 cm) achieved a conversion rate of over 20%. We believe that the larger surface area of the 15 x 27 cm plate allowed for better gas exchange and nutrient distribution, leading to more efficient cellulose production. This experiment demonstrates that the dimensions of the cultivation container play a critical role in bacterial cellulose production efficiency, even when the media volume remains constant.

From the experimental results, we can learn that the surface area of the culture vessel has a significant impact on the efficiency of bacterial cellulose production. The larger culture plate (15 x 27 cm), with its greater surface area, facilitated better gas exchange and nutrient distribution, resulting in a higher cellulose conversion rate. Therefore, future designs should prioritize increasing the culture surface area while optimizing gas exchange and nutrient distribution, or explore different vessel shapes to enhance production efficiency. The experiment also showed that even with the culture medium volume remaining constant, changes in container size significantly affect production outcomes. This provides quantitative guidance for container selection in large-scale production.

Cycle 1: Three-Module Composition Verification

We designed a control experiment to verify the binding of bacterial cellulose, Curlis-Spytag, and color protein-Spycatcher.

1. Cut a piece of BC gel into small blocks of similar size.

2. Take 6 stainless steel bowls (304 grade), and add 50 ml of ddH2O, 50 ml of POI-Spycatcher solution, and 50 ml of POI-Spycatcher-Curlis-Spytag binding solution into each. Place the similarly sized gel blocks into the solutions and incubate overnight at room temperature with shaking at 80 rpm.

3. The next day, remove the corresponding cellulose from the stainless steel bowls and place it in 50 ml ddH2O. Incubate at room temperature with shaking at 80 rpm for 6 hours.

4. Remove the gel from each group, drain, and take photos.

Figure 19 and Figure 20 show the experimental comparison of amilCP and sfGFP binding to bacterial cellulose via scaffold proteins under different conditions. In both experiments, it was observed that the experimental groups with the mixed scaffold protein solution exhibited a deeper color compared to the groups without the scaffold protein solution. This indicates that our scaffold protein design enhances the binding capability between the POI (Protein of Interest) and bacterial cellulose.

1. We aim to identify additional protein pairs with strong binding affinity, similar to the SpyTag-SpyCatcher system, to accommodate a wider range of proteins for our scaffold protein work.
2. For antimicrobial peptides, iGEM22_Michigan has provided an effective strategy by using a molecular chaperone to enhance the expression of antimicrobial peptides in E. coli. We plan to adopt this approach to validate the expression of our antimicrobial peptide-SpyCatcher system, integrating it into our platform. This will allow for future compatibility with proteins that impose a high metabolic burden on bacterial growth.

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2. Cielecka, I., Ryngajłło, M., Maniukiewicz, W. & Bielecki, S., 2021. Highly stretchable bacterial cellulose produced by Komagataeibacter hansenii SI1. Polymers, 13(24), p.4455. Published on 19 December 2021. doi:10.3390/polym13244455.
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