Difference between revisions of "Part:BBa K4719011"
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<strong>Inductor:</strong> 0.02% L-arabinose.
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<strong>Inducer:</strong> 0.02% L-arabinose.
  
 
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Revision as of 18:47, 8 October 2023


AnCDA chitin deacetylase
Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 348
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 361
    Illegal XhoI site found at 593
  • 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.

Usage and biology

AnCDA is chitin deacetylase from Aspergilus nidulans FGSCA4. This deacetylase is a member of carbohydrate esterase family 4 (CE4) of the CAZy database ([http://www.cazy.org/, www.cazy.org]). It has broad substrate specifity – AnCDA hydrolyzes N-acetamido groups in chitin oligomers, crystalline chitin and chitosan, however, it does not deacetylase peptidoglycan. AnCDA reaches its highest level of activity at approximately 50°C and pH 8.0 when Co2+ is used as a cofactor.

AnCDA gene has three exons and encodes the primary product of 237 amino acids, including the N-terminal extracellular signal sequence. Coding regions except leader sequence were cloned into pBAD/HisB vector for recombinant protein expression in E. coli and protein purification using Ni-NTA chromatography with His-tag fused to AnCDA in N-terminal.

This basic part is used for achieving bacterial cellulose-chitosan polymer by enzymatic reaction of deacetylation from bacterial cellulose-chitin. In addition, we have created a construct BBa_K4719019 to improve the degree of deacetylation.

Experimental characterization

Protein expression optimization

AnCDA biosynthesis in E. coli TOP10 (Liu et al., 2017) was not sufficient for purification. After testing three more strains, we found out that arabinose non-metabolizing DH10B is the best fit for AnCDA.

Cultivating conditions:
Medium:2XYT;
Antibiotics: Ampicillin 100 µg/ml and Streptomycin 50 µg/ml;
Strain: DH10B;
Temperature: 16°C;
Time: overnight;
Inducer: 0.02% L-arabinose.

Figure 1:ArCE4A BBa_B0012(25.1 kDa) and AnCDA (25.6 kDa) biosynthesis optimization in BL21 Star (DE3), ArcticExpress (DE3) and DH10B E. coli strains. M - PageRuler™ Unstained Protein Ladder (Thermo Fisher Scientific), S – soluble protein fraction, I – insoluble protein fraction.

References

Liu, Z. et al. (2017) ‘Structure and function of a broad-specificity chitin deacetylase from aspergillus nidulans FGSC A4’, Scientific Reports, 7(1). doi:10.1038/s41598-017-02043-1.