Difference between revisions of "Part:BBa K3520031"
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<b>LONG DESCRIPTION</b><br /><br />
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__NOTOC__
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<b>Project-General</b><br /><br />
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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 />
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<b>Operon related</b><br /><br />
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<partinfo>BBa_K3520031 short</partinfo>
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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 />
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<br><br>
  
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<b>bcsA</b><br /><br />
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<partinfo>BBa_K3520031 SequenceAndFeatures</partinfo>
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General Functional Documentation<br /><br />
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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.
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BcsA.<br /><br />
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This part codes for the BscA protein, the catalytic subunit which synthesizes cellulose in Bacteria.
  
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<b>SOURCE OF THIS PART</b><br /><br />
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<br/><br/>  
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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 />
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=Description=
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<br>
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BcsA 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 in vitro. This CDS originates from a very productive cellulose synthesising bacterium, <i>Komagataeibacter xylinus</i> (GenBank Acc. No. X54676.1). It is the catalytically acitve subunit of the bacterial Cellulose Synthetase.
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<br><br>
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==Protein Analysis==
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<br>
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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.
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<br><br>
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==The Bacterial Bcs Operon==
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<br>
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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.
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<br><br>
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=SOURCE OF THIS PART=
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<br><br>
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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.
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<br><br>
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=Useful Links:=
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<br><br>
  
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<b>Useful Links:</b><br /><br />
 
 
NCBI taxonomy:<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 />
 
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 />
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Codon optimization table:<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 />
 
https://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=376686&fbclid=IwAR0gwwrIarZsiYhWvHPc2BKy-iB_2OM-DPB5I2HYJZwBNiasmlLXWK87PwM<br /><br />
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<b>REFERENCES</b><br /><br />
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=REFERENCES=
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<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
 
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

Revision as of 23:51, 27 October 2020


bcsA-Bacterial Cellulose Synthase A


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

This part codes for the BscA protein, the catalytic subunit which synthesizes cellulose in Bacteria.



Description


BcsA 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 in vitro. This CDS originates from a very productive cellulose synthesising bacterium, Komagataeibacter xylinus (GenBank Acc. No. X54676.1). It is the catalytically acitve subunit of the bacterial Cellulose Synthetase.

Protein Analysis


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.

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.

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