Part:BBa_K3520002

LONG DESCRIPTION Project-General The current part is designed by iGEM Athens 2020 team during the project MORPHÆ. In this project, Flavobacteria were used to produce a non-cellular structurally coloured biomaterial which would require the secretion of a biomolecule that Flavobacteria do not normally secrete. Our hypothesis is that the formed matrix will have a structure similar to that of the biofilm and thus, it will provide the material with macroscopically the same colouration properties as the biofilm.<a href="https://parts.igem.org/Part:BBa_K3520002">a link is here.</a>

Operon related Our initial idea was to use bacterial cellulose, as an appropriate biomaterial, because of its unique properties, robustness, and biodegradability. The genes responsible for its production were selected from the bcs operon of Komagataeibacter xylinus (GenBank Acc. No. X54676.1), the most efficient bacterial cellulose producer, which consists of four genes, bcsA, bcsB, bcsC and bcsD. The bcsABCD operon encodes membrane-associated proteins that allow BC fibres to span through the membrane. Once the bcsABCD operon expression is triggered, BcsA and BcsB proteins form the BcsAB complex, which binds its substrate, UDP-glucose, at an intracellular glycosyltransferase (GT) domain and is the active core of cellulose synthase. This is followed by the secretion of BC fibres through pores and passageways formed by BcsC and BcsD proteins.Cmcax, CcpAx, cellulose synthase, BcsC, and BcsD are the biocatalysts of UDP-glucose transformation to cellulose. Two main applications of cellulose in biosciences are scaffolds for tissue engineering and generally in biomedicine.

bcsB General Functional Documentation bcsB codes for BcsB, the regulatory subunit of the cellulose synthase. BcsB binds to c-di-GMP, which afterwards aids the activation of the cellulose synthesis process. BcsB is a large periplasmic protein that is anchored to the inner membrane via a single C-terminal transmembrane helix. BcsB may guide the polymer across the periplasm toward the outer membrane via two carbohydrate-binding domains (CBDs). BcsB is crucial for the catalytic activity of BcsA and is localized in the region required for cellulose synthesis within BcsB’s C-terminal, a membrane-associated domain that packs against the transmembrane region of BcsA.

SOURCE OF THIS PART The nucleotide sequences of the bacterial cellulose operon come from the strain Komagataeibacter xylinus and GenBank database (Acc.No.X54676.1). K.xylinus is a member of the acetic acid bacteria, a group of Gram-negative aerobic bacteria that produce acetic acid during fermentation.

Useful Links: NCBI taxonomy: https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=28448&lvl=3&lin=f&keep=1&srchmode=1&unlock GenBank link: https://www.ncbi.nlm.nih.gov/nuccore/X54676.1 Codon optimisation bank: http://genomes.urv.es/OPTIMIZER/?fbclid=IwAR0ALbP_C8UVY4itvYdNX8b5KYYUM5ulQojz8UJAK6Zj5llobNNxE-jYmXQ Codon optimization table: https://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=376686&fbclid=IwAR0gwwrIarZsiYhWvHPc2BKy-iB_2OM-DPB5I2HYJZwBNiasmlLXWK87PwM

REFERENCES

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Buldum, G., Bismarck, A., & Mantalaris, A. (2017). Recombinant biosynthesis of bacterial cellulose in genetically modified Escherichia coli. Bioprocess And Biosystems Engineering, 41(2), 265-279. doi: 10.1007/s00449-017-1864-1

Johansen, V., Catón, L., Hamidjaja, R., Oosterink, E., Wilts, B., & Rasmussen, T. et al. (2018). Genetic manipulation of structural color in bacterial colonies. Proceedings Of The National Academy Of Sciences, 115(11), 2652-2657. doi: 10.1073/pnas.1716214115

McBride, M., & Kempf, M. (1996). Development of techniques for the genetic manipulation of the gliding bacterium Cytophaga johnsonae. Journal Of Bacteriology, 178(3), 583-590. doi: 10.1128/jb.178.3.583-590.1996

Nakamura, Y. (2000). Codon usage tabulated from international DNA sequence databases: status for the year 2000. Nucleic Acids Research, 28(1), 292-292. doi: 10.1093/nar/28.1.292

Omadjela, O., Narahari, A., Strumillo, J., Melida, H., Mazur, O., Bulone, V., & Zimmer, J. (2013). BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis. Proceedings Of The National Academy Of Sciences, 110(44), 17856-17861. doi: 10.1073/pnas.1314063110

Römling, U., & Galperin, M. (2015). Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions. Trends In Microbiology, 23(9), 545-557. doi: 10.1016/j.tim.2015.05.005