Difference between revisions of "Part:BBa K4719020"
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<center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/ancda-cbd-arce4a-cbd-opt.png" style = "width:400px;"></center> | <center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/ancda-cbd-arce4a-cbd-opt.png" style = "width:400px;"></center> | ||
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| − | <figcaption><center><b>Figure 1.</b> CBDcenA-ProThr box fused to ArCE4A (39.0 kDa) or AnCDA (<a href="https://parts.igem.org/Part:BBa_K4719019">BBa_K4719019</a>) (39.4 kDa) biosynthesis optimization in ArcticExpress (DE3) <i>E. coli</i> strains. Protein expression was induced with 0.1 mM IPTG for ArCE4A-CBDcenA or 0.01% arabinose for AnCDA-CBDcenA when cell culture optic density at 600 nm reached 0.4 or 0.8. <b>M</b> - PageRuler™ Unstained Protein Ladder (Thermo Fisher Scientific), <b>S</b> – soluble protein fraction, <b>I</b> – insoluble protein fraction. | + | <figcaption><center><b>Figure 1. SDS-PAGE analysis.</b> CBDcenA-ProThr box fused to ArCE4A (39.0 kDa) or AnCDA (<a href="https://parts.igem.org/Part:BBa_K4719019">BBa_K4719019</a>) (39.4 kDa) biosynthesis optimization in ArcticExpress (DE3) <i>E. coli</i> strains. Protein expression was induced with 0.1 mM IPTG for ArCE4A-CBDcenA or 0.01% arabinose for AnCDA-CBDcenA when cell culture optic density at 600 nm reached 0.4 or 0.8. <b>M</b> - PageRuler™ Unstained Protein Ladder (Thermo Fisher Scientific), <b>S</b> – soluble protein fraction, <b>I</b> – insoluble protein fraction. |
</center></figcaption> | </center></figcaption> | ||
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<h3>Deacetylation enzymatic activity analysis with fluorescence microscopy</h3> | <h3>Deacetylation enzymatic activity analysis with fluorescence microscopy</h3> | ||
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| − | Deacetylation was performed in a reaction of final volume of 200 µL: 2 µL 1 mM CoCl2, deacetylase CBD-ProThr box-ArCE4A 100 nM - 2µM and filling the remaining volume with 20mM HEPES-NaOH ph8, 150mM | + | Deacetylation was performed in a reaction of final volume of 200 µL: 2 µL 1 mM CoCl2, deacetylase CBD-ProThr box-ArCE4A 100 nM - 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. |
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<center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/arce4a-cbd-deac.png" style = "width:700px;"></center> | <center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/arce4a-cbd-deac.png" style = "width:700px;"></center> | ||
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| − | <figcaption><center><b>Figure 2.</b> | + | <figcaption><center><b>Figure 2. Florescent Alexa Fluor™ 405 NHS ester dye staining. A</b> - <i>K. xylinus</i> modified with <i>AGM1-NAG5-UAP1</i> <a href="https://parts.igem.org/Part:BBa_K4719013">BBa_K4719013</a> producing bacterial cellulose-chitin copolymer grown on 1% glucose and 1% N-acetylglucosamine. <b>B</b> - chitin control group. <b>The fluorescence signal is low, therefore, we decided that a new linker is needed to achieve a higher degree of deacetylation</b>. |
</center></figcaption></figure> | </center></figcaption></figure> | ||
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<center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/ancda-frf-arce4a-frf-opt.png" style = "width:400px;"></center> | <center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/ancda-frf-arce4a-frf-opt.png" style = "width:400px;"></center> | ||
</div> | </div> | ||
| − | <figcaption><center><b>Figure 3.</b> CBDcenA fused to ArCE4A (38.4 kDa) or AnCDA (38.8 kDa) through FRF linker biosynthesis in ArcticExpress (DE3) <i>E. coli</i> strain. <b>M</b> - PageRuler™ Unstained Protein Ladder (Thermo Fisher Scientific), <b>S</b> – soluble protein fraction, <b>I</b> – insoluble protein fraction. | + | <figcaption><center><b>Figure 3. SDS-PAGE analysis.</b> CBDcenA fused to ArCE4A (38.4 kDa) or AnCDA (38.8 kDa) through FRF linker biosynthesis in ArcticExpress (DE3) <i>E. coli</i> strain. <b>M</b> - PageRuler™ Unstained Protein Ladder (Thermo Fisher Scientific), <b>S</b> – soluble protein fraction, <b>I</b> – insoluble protein fraction. |
</center></figcaption></figure> | </center></figcaption></figure> | ||
<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 CBD-FRF-ArCE4A 50 nM - 2µM and filling the remaining volume with 20mM HEPES-NaOH ph8, 150mM | + | Deacetylation was performed in a reaction with a final volume of 200 µL: 2 µL 1 mM CoCl2, deacetylase CBD-FRF-ArCE4A 50 nM - 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. The fluorescence microscopy was performed under the same conditions as in the first iteration of this construct. |
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<center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/arce4a-frf-flores.png" style = "width:700px;"></center> | <center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/arce4a-frf-flores.png" style = "width:700px;"></center> | ||
</div> | </div> | ||
| − | <figcaption><center><b>Figure 4. A</b> - chitin control. <b>B</b> - <i>K. xylinus</i> modified with <i>AGM1-NAG5-UAP1</i> <a href="https://parts.igem.org/Part:BBa_K4719013">BBa_K4719013</a> producing bacterial cellulose-chitin | + | <figcaption><center><b>Figure 4. Florescent Alexa Fluor™ 405 NHS ester dye staining. A</b> - chitin control. <b>B</b> - <i>K. xylinus</i> modified with <i>AGM1-NAG5-UAP1</i> <a href="https://parts.igem.org/Part:BBa_K4719013">BBa_K4719013</a> producing bacterial cellulose-chitin copolymer grown on 1% glucose and 1% N-acetylglucosamine. C - <i>K. xylinus</i> modified with <i>AGM1-GFA1-GNA1-UAP1</i> <a href="https://parts.igem.org/Part:BBa_K4719014">BBa_K4719014</a> producing bacterial cellulose-chitin copolymer grown on 2% glucose. <b>The new linker improved deacetylase activity</b>. </center></figcaption> |
</figure> | </figure> | ||
</p> | </p> | ||
Latest revision as of 14:44, 12 October 2023
CBD-ProThr box-ArCE4A chitin deacetylase and cellulose binding domain fusion protein
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 432
Illegal NgoMIV site found at 961
Illegal AgeI site found at 417 - 1000COMPATIBLE WITH RFC[1000]
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 copolymer 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 of bacterial cellulose and polyhydroxybutyrate (PHB) composite, which is synthesized by K. xylinus.
Bacterial cellulose-chitin polymer was achieved by increasing the production of UDP-N-acetylglucosamine, which can be recognized as a viable substrate for cellulose synthase and incorporated in the bacterial cellulose polymer.
We employed two strategies to produce this material:
1.The first approach was to add N-acetylglucosamine into the growth medium
BBa_K4719013.
2.The second one was the production of N-acetylglucosamine by K. xylinus from common sugars such as glucose, fructose, and sucrose in the growth medium BBa_K4719014.
This specific part was used of bacterial cellulose-chitin copolymer deacetylation, as deacetylation of this material produces cellulose-chitosan copolymer.
Usage and Biology
We fused a cellulose binding domain connected to a linker to the N-terminus of deacetylase ArCE4A to ensure a higher degree of deacetylation. For protein purification 6x his-tag was added to the N-terminus of cellulose binding domain. The composite is contained in pNIC-CH plasmid. For this part to be functional in our bacterial cellulose-chitosan copolymer production system, we had to purify recombinant protein coded by this composite. This part is used to produce bacterial cellulose-chitosan copolymer from bacterial cellulose-chitin copolymer via deacetylation.Bacterial cellulose-chitosan copolymer has applications in the biomedicine field due to in vivo biodegradability by the lysosomes. Also, this copolymer is a convenient platform for further modifications that would aid in solving the need for the promotion of tissue development. The uncovered amino groups are susceptible to enzymes catalyzing an addition of targeted organic chemistry groups. For instance, after modifying reaction conditions, deacetylase ClCDA BBa_K4719024 can propylate chitosan, which can later be used for click chemistry reactions [1]. In the future, specific targets like drugs or amino acids could be linked to the polymer, promoting the healing properties of the material [2].
Experimental characterization
Protein expression
Fusion with CBDcenA significantly decreased the solubility of deacetylases. To increase protein stability, we employed E. coli ArcticExpress (DE3), which is adapted for protein expression in lower temperatures. Investigating different biosynthesis conditions in ArcticExpress (DE3) revealed that induction at OD600 0.8 and growing the cells overnight at 16°C is optimal for fusion protein expression.
Deacetylation enzymatic activity analysis with fluorescence microscopy
Deacetylation was performed in a reaction of final volume of 200 µL: 2 µL 1 mM CoCl2, deacetylase CBD-ProThr box-ArCE4A 100 nM - 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 CBD-ProThr box-ArCE4A. 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 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.
Protein expression of recombinant deacetylase containing a new linker
A new linker BBa_K4719023 generated by our software was cloned into the pNIC-CH-6His-CBDCenA-ArCE4A backbone by Gibson assembly. Investigating different biosynthesis conditions in ArcticExpress (DE3) revealed that induction at OD600 0.8 and growing the cells overnight at 16°C is optimal for fusion protein production.
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 CBD-FRF-ArCE4A 50 nM - 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. The fluorescence microscopy was performed under the same conditions as in the first iteration of this construct.
Growth burden
In order to work with E. coli for designing constructs and testing synthetic biology parts, the growth burden of said synthetic biology tools has to be measured. We performed growth burden evaluation by measuring OD600 for five hours of modified and unmodified E. coli DH5α. The composite of recombinant deacetylase CBD-ProThr box-ArCE4A did not inhibit the growth of E. coli as seen in Figure 5.
References
1.Lima, R., Fernandes, C. and Pinto, M.M. (2022) ‘Molecular modifications, biological activities, and applications of chitosan and derivatives: A recent update’, Chirality, 34(9), pp. 1166–1190. doi:10.1002/chir.23477.2.Torkaman, S. et al. (2021) ‘Modification of chitosan using amino acids for wound healing purposes: A Review’, Carbohydrate Polymers, 258, p. 117675. doi:10.1016/j.carbpol.2021.117675.