Difference between revisions of "Part:BBa K4719024"
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<figcaption><center>Figure 3:Chromatogram of ClCDA-MBP expressed in ArticExpress (DE3) E. coli strain purification using ÄKTA avant chromatography system. Elution stage peak indicates that new expression conditions are optimal for ClCDA-MBP. | <figcaption><center>Figure 3:Chromatogram of ClCDA-MBP expressed in ArticExpress (DE3) E. coli strain purification using ÄKTA avant chromatography system. Elution stage peak indicates that new expression conditions are optimal for ClCDA-MBP. | ||
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| + | <h3>Deacetylation enzymatic activity analysis with fluorescence microscopy</h3> | ||
| + | <p> | ||
| + | For cellulose-chitosan copolymer generation from cellulose-chitin exopolymer we used chitin deacetylase ClCDA. To determine if the deacetylation of our cellulose-chitin copolymer was successful, we used Alexa Fluor™ 405 NHS ester dye that specifically binds to free amino groups. On that account, only deacetylated copolymers should produce any fluorescent signal at this wavelength. To verify that our purified deacetylases are enzymatically active, at first we checked deacetylation activity on enzymes natural substrate - chitin. | ||
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| + | <figure> | ||
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| + | <center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/clcda-flores-1.png" style = "width:600px;"></center> | ||
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| + | <figcaption><center>Figure 4:A - K. xylinus modified with AGM1-NAG5-UAP1 <a href="https://parts.igem.org/Part:BBa_K4719013">BBa_K4719013</a> producing bacterial cellulose-chitin composite grown on 1% glucose and 1% N-acetylglucosamine. B - K. xylinus modified with AGM1-GFA1-GNA1-UAP1 <a href="https://parts.igem.org/Part:BBa_K4719014">BBa_K4719014</a> producing bacterial cellulose-chitin composite grown on 2% sucrose. C - K. xylinus modified with AGM1-GFA1-GNA1-UAP1 <a href="https://parts.igem.org/Part:BBa_K4719014">BBa_K4719014</a> producing bacterial cellulose-chitin composite grown on 2% fructose. D - chitin control. ClCDA was active on native substrate chitin and on the bacterial cellulose-chitin copolymer. | ||
</center></figcaption> | </center></figcaption> | ||
</figure> | </figure> | ||
Revision as of 19:48, 8 October 2023
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.
Deacetylation enzymatic activity analysis with fluorescence microscopy
For cellulose-chitosan copolymer generation from cellulose-chitin exopolymer we used chitin deacetylase ClCDA. To determine if the deacetylation of our cellulose-chitin copolymer was successful, we used Alexa Fluor™ 405 NHS ester dye that specifically binds to free amino groups. On that account, only deacetylated copolymers should produce any fluorescent signal at this wavelength. To verify that our purified deacetylases are enzymatically active, at first we checked deacetylation activity on enzymes natural substrate - chitin.
