Difference between revisions of "Part:BBa K3520004"

Revision as of 16:20, 27 October 2020

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.

bcsC

General Functional Documentation

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 exact function of bcsD still remains undetermined, but it has been suggested that it is involved in the crystallization of cellulose into nanofibrils .

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

@@ Line 1: / Line 1: @@
-PROJECT-GENERAL
+<b>LONG DESCRIPTION</b><br /><br />
-The current part is designed by iGEM Athens 2020 team during the project MORPHÆ. In this project Flavobacteriia were used to produce a non-cellular structurally coloured biomaterial which would require the secretion of a biomolecule that Flavobacteriia 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.
+<b>Project-General</b><br /><br />
+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.<br /><br />
-OPERON RELATED
+<b>Operon related</b><br /><br />
-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.
+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 <i>Komagataeibacter xylinus</i> (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.<br /><br />
-bcsD
+<b>bcsC</b><br /><br />
-GENERAL FUNCTION DOCUMENTATION
+General Functional Documentation<br /><br />
-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.
+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 exact function of bcsD still remains undetermined, but it has been suggested that it is involved in the crystallization of cellulose into nanofibrils .
+<br /><br />
-SOURCE
-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:
+<b>SOURCE OF THIS PART</b><br /><br />
-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
+The nucleotide sequences of the bacterial cellulose operon come from the strain <i>Komagataeibacter xylinus</i> and GenBank database (Acc.No.X54676.1). <i>K.xylinus</i> is a member of the acetic acid bacteria, a group of Gram-negative aerobic bacteria that produce acetic acid during fermentation. <br /><br />
-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
+<b>Useful Links:</b><br /><br />
+NCBI taxonomy:<br /><br />
+https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=28448&lvl=3&lin=f&keep=1&srchmode=1&unlock<br /><br />
+GenBank link:<br /><br />
+https://www.ncbi.nlm.nih.gov/nuccore/X54676.1<br /><br />
+Codon optimisation bank:<br /><br />
+http://genomes.urv.es/OPTIMIZER/?fbclid=IwAR0ALbP_C8UVY4itvYdNX8b5KYYUM5ulQojz8UJAK6Zj5llobNNxE-jYmXQ<br /><br />
+Codon optimization table:<br /><br />
+https://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=376686&fbclid=IwAR0gwwrIarZsiYhWvHPc2BKy-iB_2OM-DPB5I2HYJZwBNiasmlLXWK87PwM<br /><br />
+<b>REFERENCES</b><br /><br />
 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
@@ Line 38: / Line 42: @@
 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
-. Codon optimization via Flavobacteriia 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).
-. 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
-. 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.