Difference between revisions of "Part:BBa K4719024"
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<h3>Deacetylation enzymatic activity analysis with fluorescence microscopy</h3> | <h3>Deacetylation enzymatic activity analysis with fluorescence microscopy</h3> | ||
<p> | <p> | ||
| + | Deacetylation was performed in a reaction with a final volume of 200 µL: 2 µL 1 mM CoCl2, deacetylase ClCDA - 2µM and filling the remaining volume with 20mM HEPES-NaOH ph8, 150mM NaCL buffer. The samples were incubated for 14 h at 37° while shaking at 300 rpm, reaction was stopped by incubating for 3 min at 98°C. | ||
| + | <br> | ||
| + | <br> | ||
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. | 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. | ||
</p> | </p> | ||
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</div> | </div> | ||
<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. | <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> | ||
| + | </figure> | ||
| + | |||
| + | <h3>Acetylation enzymatic activity analysis with fluorescence microscopy</h3> | ||
| + | <p> | ||
| + | Acetylation activity was investigated by performing click chemistry reaction on ClCDA acetylated bacterial cellulose-chitosan polymer achieved by deacetylation accomplished by using all of our recombinant deacetylases. Acetylation was performed in 200 µL propiolic acid buffer pH 6.5 (Tris 0.1M, propiolic acid 0.5M) with 2µM ClCDA. The reaction was incubated for 24h at 37 while shaking. | ||
| + | <br> | ||
| + | <br> | ||
| + | Click chemistry reaction was performed on propiolated bacterial cellulose-chitosan copolymer after washing 2 times with 0.1M Tris-NaOH pH 6.5 buffer. Adding copolymer to 100 µL HCl (pH 4) and 2.3 µL 3-azido-7-hydroxyazidocoumarin, 23.5 µL sodium ascorbate and 23.5 µL CuSO4 solution, incubated for 10 min. We evaluated the fluorescence (using DAPI light cube) of our copolymer after different wash times (30min and 90min). | ||
| + | </p> | ||
| + | <figure> | ||
| + | <div class = "center" > | ||
| + | <center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/click-chemistry-clcda.png" style = "width:900px;"></center> | ||
| + | </div> | ||
| + | <figcaption><center>Figure 5:A,E - <i>K. xylinus</i> 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 and deacetylated with ClCDA <a href="https://parts.igem.org/Part:BBa_K4719024">BBa_K4719024</a>. B,F - <i>K. xylinus</i> 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, deacetylated with CBD-FRF-ArCE4A <a href="https://parts.igem.org/Part:BBa_K4719020">BBa_K4719020</a>. C,G - control of bacterial cellulose produced by unmodified K<i>. xylinus</i> grown on 2% glucose. D,H - control of <i>K. xylinus</i> 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, not deacetylated. A,B,C,D - polymers washed for 30 min; E,F,G,H - polymers washed for 90 min. | ||
| + | |||
</center></figcaption> | </center></figcaption> | ||
</figure> | </figure> | ||
Revision as of 20:31, 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
Deacetylation was performed in a reaction with a final volume of 200 µL: 2 µL 1 mM CoCl2, deacetylase ClCDA - 2µM and filling the remaining volume with 20mM HEPES-NaOH ph8, 150mM NaCL buffer. The samples were incubated for 14 h at 37° while shaking at 300 rpm, reaction was stopped by incubating for 3 min at 98°C.
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.
Acetylation enzymatic activity analysis with fluorescence microscopy
Acetylation activity was investigated by performing click chemistry reaction on ClCDA acetylated bacterial cellulose-chitosan polymer achieved by deacetylation accomplished by using all of our recombinant deacetylases. Acetylation was performed in 200 µL propiolic acid buffer pH 6.5 (Tris 0.1M, propiolic acid 0.5M) with 2µM ClCDA. The reaction was incubated for 24h at 37 while shaking.
Click chemistry reaction was performed on propiolated bacterial cellulose-chitosan copolymer after washing 2 times with 0.1M Tris-NaOH pH 6.5 buffer. Adding copolymer to 100 µL HCl (pH 4) and 2.3 µL 3-azido-7-hydroxyazidocoumarin, 23.5 µL sodium ascorbate and 23.5 µL CuSO4 solution, incubated for 10 min. We evaluated the fluorescence (using DAPI light cube) of our copolymer after different wash times (30min and 90min).
