Part:BBa_K4719019

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.