Difference between revisions of "Part:BBa K4719012"

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Latest revision as of 14:54, 12 October 2023

ArCE4A chitin deacetylase
Sequence and Features

Introduction

Vilnius-Lithuania iGEM 2023 team's goal was to create synthetic biology tools for in vivo alterations of Komagataeibacter xylinus bacterial cellulose polymer composition. 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

ArCE4A is a novel chitin deacetylase isolated from marine Arthrobacter sp. AW19M34-1. Sequence of ArCE4A was obtained from metagenomic data. ArCE4A consists of 246 amino acids of which first 31 are predicted extracellular signal peptide. Synthetic ArCE4A gene sequence, optimized for expression in E. coli and cloned into pNIC-CH, is fused with His-tag in N-terminal for protein purification with Ni-NTA chromatography.

ArCE4A belongs to carbohydrate esterase family 4 (CE4) of the CAZy database ([http://www.cazy.org/, www.cazy.org]). This enzyme deacetylates wide variety of substrates but has higher activity for longer chitooligosaccharides. For catalysis ArCE4A requires cofactor Co2+, optimal pH for substrate deacetylation is 8.0.

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_K4719020 to improve the degree of deacetylation.

Experimental characterization

Protein expression optimization

E. coli  BL21 Star (DE3) was the most suitable strain for ArCE4A biosynthesis.

Cultivating conditions:
Medium:TB;
Antibiotics: Ampicillin 100µg/ml;
Strain: BL21 Star (DE3);
Temperature: 28°C;
Time: overnight;
Inducer: IPTG, 0.2 mM.