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Introduction

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Bacterial cellulose (BC) is an organic compound with the formula (C_6 H_10 O_5 )_n produced by certain types of bacteria. It is characterized by high purity, strength, moldability and water holding ability. Bacterial cellulose has some potential applications in many areas, including water purification, tissue scaffolds, wound dressings. Bacterial cellulose nano-fibers are one of the stiffest natural organic materials. It is purer than cellulose originated from plants.(Fig.1)

Fig.1. Scanning electron-micrographs of fracture edge of bacterial cellulose membrane

Note: Iguchi, M., Yamanaka, S., & Budhiono, A. (2000). Bacterial cellulose—a masterpiece of nature's arts. Journal of Materials Science, 35(2), 261-270.

Some scientists employed soy protein isolate (SPI) and BC to study high efficiency air filtering materials (Liu, X., 2017). In the experiment, it was proved that the 3D nano-network of BC had the ability of the preliminary physical capturing of particulate matter (PM) particles.

In molecular biology, a carbohydrate-binding module (CBM) is a protein domain found in carbohydrate-active enzymes. The majority of these domains have carbohydrate-binding activity. CBMs were previously known as cellulose-binding domains. In 2014, team Imperial considered a way of functionalizing cellulose. They expressed the protein of interest fused with cellulose binding domains by a linker sequence. Then the cellulose binding domains could attach to the cellulose via hydrophobic interactions. (Fig.2)

Fig.2. A way of functionalizing cellulose

Note: retrieved from https://2014.igem.org/Team:Imperial/Functionalisation

Design

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Inspired by what mentioned above, we decided to develop a cellulose-based membrane to adsorb nicotine in the air.

When culturing Acetobacter xylinum, a membrane appears on the surface of the medium. Immerse the membrane in E. coli expressing the protein of interest and then these proteins are automatically assembled onto the cellulose scaffold. If you remove the membrane directly from the medium, it will lose the specialized nanostructures. Therefore, we designed to retain the nanostructure of the bacterial cellulose membrane by lyophilization with liquid nitrogen. After all the steps, the membrane finally gets functionalized.

We designed a plasmid expressed in E. coli to link CBM to the protein NicA2 for nicotine adsorption. (Fig.3)

Fig.3. CBni plasmid

Result

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We obtained the sequence of gene NicA2 and made the codon optimization for the gene. Then we ordered the gene synthesis from GenScript. We inserted this gene to plasmid PCG and added gene of CBM upstream successfully. The sequence of gene CBni was validated with DNA sequencing by Sangon Biotech. We transformed this plasmid into E.coli BL21. Then we picked some colonies for cultivation and confirmed the transformation result by double digestion after plasmid extraction (shown in Figure 4). From the result of electrophoresis, we confirmed the transformation of CBni was a success.

Figure 4. Electrophoresis result of double digestion of CBni plasmid.

We cultivated the bacteria with 200mL LB medium in 37℃, 150 rpm till OD600 reach 0.8. Then we added IPTG (final concentration=1mM) in it to induce their expression in 37℃ for 3 hours.

Fig.5. result of expression of protein nicA2&cbm

As the Fig.5 shows, the large protein nicA2&cbm is successfully expressed. Since the bacterial cellulose membrane produced by Acetobacter xylinum growed very slow, we failed to complete the whole experiment. The bacterial cellulose membrane used in the freeze-drying experiment was provided by Hainan Yeguo Foods Co.,Ltd..

Application

Our nicotine absoption membrane can be used in smoking room, train, house, etc. We can sale the membranes to those places, then recycle and replace them regularly to process nicotine and absorb some other harmful component.

Because of the gas permeability of this membrane, as well as the ability to absorb other small molecules, such as PM particles, it may eventually be used to make a mask for second-hand smoke in the future.

[1]A. Steinbuhel, "Bacterial Cellulose." Biopolymers. Weinheim: Wiley-VCH, 2001. Print.

[2]Bajaj, I; Chawla, P; Singhal, R; Survase, S. "Microbial cellulose: fermentative production and applications". Food Technology and Biotechnology. 47 (2): 107–124.

[3]Gilkes NR, Claeyssens M, Aebersold R, Henrissat B, Meinke A, Morrison HD, Kilburn DG, Warren RA, Miller RC (December 1991). "Structural and functional relationships in two families of beta-1,4-glycanases". Eur. J. Biochem. 202 (2): 367–77. doi:10.1111/j.1432-1033.1991.tb16384.x. PMID 1761039.

[4]Hestrin, S.; Schramm, M. (1954). "Synthesis of cellulose by Acetobacter xylinum: II. Preparation of freeze-dried cells capable of polymerizing glucose to cellulose". Biochem. J. 58 (2): 345–352.

[5]Hong, J., Ye, X., Wang, Y., & Zhang, Y. H. P. (2008). Bioseparation of recombinant cellulose-binding module-proteins by affinity adsorption on an ultra-high-capacity cellulosic adsorbent. Analytica chimica acta, 621(2), 193-199.

[6]Hu, W., Chen, S., Yang, J., Li, Z., & Wang, H. (2014). Functionalized bacterial cellulose derivatives and nanocomposites. Carbohydrate polymers, 101, 1043-1060.

[7]Iguchi, M., Yamanaka, S., & Budhiono, A. (2000). Bacterial cellulose—a masterpiece of nature's arts. Journal of Materials Science, 35(2), 261-270.

[8]Jonas, R.; Farah, Luiz F. (1998). "Production and application of microbial cellulose". Polymer Degradation and Stability. 59 (1–3): 101–106.

[9]Lee, K.-Y., Buldum, G., Mantalaris, A. and Bismarck, A. (2014), More Than Meets the Eye in Bacterial Cellulose: Biosynthesis, Bioprocessing, and Applications in Advanced Fiber Composites. Macromol. Biosci., 14: 10–32. doi: 10.1002/mabi.201300298

[10]Liu, X., Souzandeh, H., Zheng, Y., Xie, Y., Zhong, W. H., & Wang, C. (2017). Soy protein isolate/bacterial cellulose composite membranes for high efficiency particulate air filtration. Composites Science and Technology, 138, 124-133.

[11]Meinke A, Gilkes NR, Kilburn DG, Miller RC, Warren RA (December 1991). "Bacterial cellulose-binding domain-like sequences in eucaryotic polypeptides". Protein Seq. Data Anal. 4 (6): 349–53. PMID 1812490.

[12]Shah, N., Ul-Islam, M., Khattak, W. A., & Park, J. K. (2013). Overview of bacterial cellulose composites: a multipurpose advanced material. Carbohydrate Polymers, 98(2), 1585-1598.