Difference between revisions of "Part:BBa K4719019"
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<partinfo>BBa_K4719019 short</partinfo>
 
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4719019 SequenceAndFeatures</partinfo>
  
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We fused a cellulose binding domain connected to a linker to the N-terminus of deacetylase AnCDA to ensure a higher degree of deacetylation. For protein purification 6x his-tag was added to the N-terminus of cellulose binding domain.
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==Introduction==
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Vilnius-Lithuania iGEM 2023 team's goal was to create a universal synthetic biology system for ''Komagataeibacter xylinus'' for ''in vivo'' bacterial cellulose polymer composition modification. Firstly, we chose to produce a cellulose-chitin polymer that would later be deacetylated, creating bacterial cellulose-chitosan. This polymer is an easily modifiable platform when compared to bacterial cellulose. The enhanced chemical reactivity of the bacterial cellulose-chitosan polymer allows for specific functionalizations in the biomedicine field, such as scaffold design. As a second approach, we designed indigo-dyed cellulose that could be used as a green chemistry way to apply cellulose in the textile industry. Lastly, we have achieved a composite of bacterial cellulose and polyhydroxybutyrate (PHB), which is synthesized by ''K. xylinus''.
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Bacterial cellulose-chitin polymer was achieved by increasing the production of UDP-N-acetylglucosamine, which can be recognized as a viable substrate for cellulose synthase and incorporated in the bacterial cellulose polymer. We employed two strategies to produce this material. The first approach was to add N-acetylglucosamine into the growth medium [https://parts.igem.org/Part:BBa_K4719013 BBa_K4719013], and the second one was the production of N-acetylglucosamine by ''K. xylinus'' from simple sugars such as glucose, fructose, and saccharose in the growth medium [https://parts.igem.org/Part:BBa_K4719014 BBa_K4719014]. After achieving bacterial cellulose-chitin copolymer, we had to deacetylase this material to produce bacterial cellulose-chitosan copolymer.  
  
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==Usage and biology==
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This construct contains a fused cellulose binding domain connected to a linker to the N-terminus of deacetylase AnCDA [https://parts.igem.org/Part:BBa_K4719011 BBa_K4719011] to ensure a higher degree of deacetylation. For protein purification, 6x his-tag was added to the N-terminus of the cellulose binding domain. The composite is contained in pBAD/HisB plasmid. For this part to be functional in your bacterial cellulose-chitosan copolymer production system, we had to purify recombinant protein coded by this composite.
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==Experimental characterization==
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===Optimisation of recombinant protein expression===
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<h3>Enzymatic activity</h3>
  
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===Usage and Biology===
 
  
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K4719019 SequenceAndFeatures</partinfo>
 
  
  

Revision as of 17:54, 23 September 2023


CBD-ProThr box-AnCDA chitin deacetylase and cellulose binding domain fusion protein
Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 756
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 769
    Illegal XhoI site found at 1001
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Introduction

Vilnius-Lithuania iGEM 2023 team's goal was to create a universal synthetic biology system for Komagataeibacter xylinus for in vivo bacterial cellulose polymer composition modification. Firstly, we chose to produce a cellulose-chitin polymer that would later be deacetylated, creating bacterial cellulose-chitosan. This polymer is an easily modifiable platform when compared to bacterial cellulose. The enhanced chemical reactivity of the bacterial cellulose-chitosan polymer allows for specific functionalizations in the biomedicine field, such as scaffold design. As a second approach, we designed indigo-dyed cellulose that could be used as a green chemistry way to apply cellulose in the textile industry. Lastly, we have achieved a composite of bacterial cellulose and polyhydroxybutyrate (PHB), which is synthesized by K. xylinus.

Bacterial cellulose-chitin polymer was achieved by increasing the production of UDP-N-acetylglucosamine, which can be recognized as a viable substrate for cellulose synthase and incorporated in the bacterial cellulose polymer. We employed two strategies to produce this material. The first approach was to add N-acetylglucosamine into the growth medium BBa_K4719013, and the second one was the production of N-acetylglucosamine by K. xylinus from simple sugars such as glucose, fructose, and saccharose in the growth medium BBa_K4719014. After achieving bacterial cellulose-chitin copolymer, we had to deacetylase this material to produce bacterial cellulose-chitosan copolymer.

Usage and biology

This construct contains a fused cellulose binding domain connected to a linker to the N-terminus of deacetylase AnCDA BBa_K4719011 to ensure a higher degree of deacetylation. For protein purification, 6x his-tag was added to the N-terminus of the cellulose binding domain. The composite is contained in pBAD/HisB plasmid. For this part to be functional in your bacterial cellulose-chitosan copolymer production system, we had to purify recombinant protein coded by this composite.

Experimental characterization

Optimisation of recombinant protein expression

Enzymatic activity