Part:BBa_K4719024
ClCDA chitin deacetylase
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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
ClCDA is chitin deacetylase isolated from fungus Colletotrichum lindemuthianum. It catalyzes hydrolysis of N-acetamido groups in polymers containing N-acetyl-D-glucosamine monomers. ClCDA requires Co2+ for its catalytical activity.
ClCDA gene consists of two exons and encodes 248 amino acids including extracellular localization signal peptide. Coding sequence excluding signal peptide was cloned into pMAL-p5x-CL-StrepII vector containing MBP (maltose binding protein) sequence in N-terminal and Strep-tag II in C-terminal.
Experimental characterization
Protein expression optimization
ClCDA fused with MBP (72.3 kDa) biosynthesis optimization in ArcticExpress (DE3) E. coli strain. Target protein expression was induced with 0.05 or 0.1 mM IPTG when cell culture optic density at 600 nm reached 0.4 or 0.8. After induction, cultures were incubated at 16 or 28 °C overnight. ClCDA-MBP, expressed in E. coli ER2508, was used as control.
Western blot analysis
Western blot for evaluating Strep-tagged ClCDA fused with MBP (72.3 kDa) biosynthesis in ArcticExpress (DE3) E. coli strain. Target protein expression was induced with 0.05 or 0.1 mM IPTG when cell culture optic density at 600 nm reached 0.4 or 0.8. After induction, cultures were incubated at 16 or 28 °C overnight.
Protein purification
Protein purification was performed with 6 grams of biomass using ÄKTA avant chromatography system.
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