Part:BBa_K3520009
pHimarEm1
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Illegal EcoRI site found at 6663
Illegal NheI site found at 330
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Illegal NotI site found at 9
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Illegal EcoRI site found at 6663
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Illegal XbaI site found at 6678
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Illegal AgeI site found at 2883 - 1000INCOMPATIBLE WITH RFC[1000]Plasmid lacks a prefix.
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This part codes for the BscD protein, an assisting protein in cellulose biosynthesis in Bacteria. It is codon optimised for Flavobacterium johnsoniae UW101
Description
The exact function of bcsD still remains undetermined, but it has been suggested that it is involved in the crystallization of cellulose into nanofibrils .
Protein Analysis
BcsD seems to be required for the arrangement of the BCS complex along the longitudinal cell axis. Although most biofilm-forming bacteria likely produce amorphous cellulose that is embedded in a 3D matrix of polysaccharides, proteinaceous fibers, and nucleic acids, some bacteria produce cellulose microfibrils resembling those synthesized by eukaryotic cells. In such bacteria, CesA complexes are linearly arranged along the cell axis, and the CesA operons encode at least one additional subunit, BcsD, that might facilitate the linear organization of the synthases.
The Bacterial Bcs Operon
The putative operon consists of four genes, bcsA, bcsB, bcsC and bcsD. It encodes membrane-associated proteins can catalyse extracellular Bacterial Cellulose synthesis in vivo. 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. This complec is the active core of the cellulose synthase. This is followed by the secretion of BC fibres through pores and passageways formed by BcsC and BcsD proteins. Co expression with Cmcax and CcpAx have shown increased production. The cellulose synthase, BcsC, BcsD, Cmcax, and CcpAx 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.
Athens 2020
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.
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
Braun, T., Khubbar, M., Saffarini, D., & McBride, M. (2005). Flavobacterium johnsoniae Gliding Motility Genes Identified by mariner Mutagenesis. Journal Of Bacteriology, 187(20), 6943-6952. doi: 10.1128/jb.187.20.6943-6952.2005
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
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