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.

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.

bcsA General Functional Documentation bcsA is responsible for the expression of BcsA, the catalytic subunit which synthesizes cellulose and forms the transmembrane pore across the inner membrane. BcsA, together with the periplasmic membrane-anchored BcsB subunit, forms a complex that is sufficient for cellulose synthesis and translocation. BcsA is homologous to eukaryotic cellulose synthases and contains eight transmembrane helices and a cytosolic glycotransferase domain between transmembrane helices four and five. BcsA also forms a polysaccharide channel across the membrane, directly above the active site, thereby allowing the coupling of cellulose synthesis and translocation. In addition, BcsA forms a PilZ domain within its C-terminal intracellular extension, which consists of a six-stranded ß-barrel and a preceding linker region. The ß-barrel rests against the intracellular glycotransferase domain and is connected to BcsA’s C-terminal transmembrane helix.

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

DESIGN CONSIDERATIONS 1. Codon optimization via Flavobacteria johnsoniae UW101 as an example organism.

To start with, all the sequences of the genetic construct were codon optimized for the increased expression in Flavobacterium according to the Codon Usage Table UW101 (Nakamura, 2000).

2. Elimination of restriction sites via usage of synonym codons for the illegal restriction enzymes of TYPE IIS and RFC10 Assembly and compatibility with it. The Type IIS Assembly standard was used in order to insert these genes in the pHimarEm1 plasmid, avoiding the presence of illegal sites. Each gene will be assembled with a promoter, RBS and a terminator. Specific prefixes and suffixes are required in order to isolate and assemble the parts. The parts are flanked by fusion sites that ensure proper order assembly and a BsaI restriction enzyme site. RFC 10 assembly standard-compatible BioBrick prefix and suffix sequences were added in the 5’ and 3’ ends to allow for easy amplification of the ordered parts as well as sequencing. The synthesised transcriptional unit will consist of the assembled parts, the fusion site 5’ of the promoter, and the fusion site 3’ of the terminator. Once each transcriptional unit of each gene is synthesised, they will be inserted in the pHimarEm1 plasmid in one step using Type II S assembly, in the designated order due to the 5’ and 3’ fusion sites.

References Chen, S., Bagdasarian, M., Kaufman, M., & Walker, E. (2006). Characterization of Strong Promoters from an Environmental Flavobacterium hibernum Strain by Using a Green Fluorescent Protein-Based Reporter System. Applied And Environmental Microbiology, 73(4), 1089-1100. doi: 10.1128/aem.01577-06

Chen, S., Kaufman, M., Bagdasarian, M., Bates, A., & Walker, E. (2010). Development of an efficient expression system for Flavobacterium strains. Gene, 458(1-2), 1-10. doi: 10.1016/j.gene.2010.02.006

3. Primers design Internal primers for PCR amplification prior to level 0 Golden Gate assembly. Similarly, internal primers were placed between the SapI sites and transcriptional units, in order to perform further amplification prior to performing the level 1 Golden Gate assembly. The primary reason that this was done is to increase the chances of Golden Gate assembly functioning, as the parts that would be ligated, especially at the level 1 stage, are rather large. The RFC10 prefix and suffix already have well-established primers (VW and VW-R) that most iGEM members are familiar with, forgoing the need to order very different primers to amplify each part. thus reducing cost.

We hope all of the above will make it easier for future teams that work with the particular species and gives them higher manipulation capabilities and accuracy.