Difference between revisions of "Part:BBa K4719019"
 
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<partinfo>BBa_K4719019 short</partinfo>
 
<partinfo>BBa_K4719019 short</partinfo>
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==Sequence and Features==
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<partinfo>BBa_K4719019 SequenceAndFeatures</partinfo>
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==Introduction==
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<b>Vilnius-Lithuania iGEM 2023</b> team's goal was to create <b> synthetic biology tools for <i>in vivo</i> alterations of <i>Komagataeibacter xylinus</i> bacterial cellulose polymer composition</b>. Firstly, we chose to produce a <b>cellulose-chitin copolymer</b> that would later be deacetylated, creating <b>bacterial cellulose-chitosan</b>. 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 <b>indigo-dyed cellulose</b> that could be used as a green chemistry way to apply cellulose in the textile industry. Lastly, we have achieved a of <b>bacterial cellulose and polyhydroxybutyrate (PHB) composite</b>, which is synthesized by <i>K. xylinus</i>.
 +
<html>
 +
<body>
 +
<br>
 +
<br>
 +
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. <br>We employed two strategies to produce this material: <br>
 +
<b>1.</b>The first approach was to add N-acetylglucosamine into the growth medium
 +
<a href="https://parts.igem.org/Part:BBa_K4719013">BBa_K4719013</a>. <br>
 +
<b>2.</b>The second one was the production of N-acetylglucosamine by <i>K. xylinus</i> from common sugars such as glucose, fructose, and sucrose in the growth medium <a href="https://parts.igem.org/Part:BBa_K4719014">BBa_K4719014</a>. <br>
 +
After achieving bacterial cellulose-chitin copolymer, we had to deacetylase this material to produce bacterial cellulose-chitosan copolymer <a href="https://parts.igem.org/Part:BBa_K4719019">BBa_K4719019</a>, <a href="https://parts.igem.org/Part:BBa_K4719020">BBa_K4719020</a>, <a href="https://parts.igem.org/Part:BBa_K4719024">BBa_K4719024</a>. <b>This specific part was used for bacterial cellulose-chitin deacetylation</b>, as deacetylation of this material produces cellulose-chitosan copolymer.
  
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We fused a cellulose binding domain connected to a linker to the N-terminus of deacetylase AnCDA to ensure a higher degree of deacetylation. For protein purification 6x his-tag was added to the N-terminus of cellulose binding domain.
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<h2>Usage and Biology</h2>
  
 +
<p>
 +
This construct contains a fused cellulose binding domain connected to a linker to the N-terminus of deacetylase AnCDA (<a href="https://parts.igem.org/Part:BBa_K4719011">BBa_K4719011</a>) to ensure a higher degree of deacetylation. For protein purification, 6x his-tag was added to the N-terminus of the cellulose binding domain. The composite is contained in pBAD/HisB plasmid. For this part to be functional in your 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.
 +
<br>
 +
<br>
 +
Bacterial cellulose-chitosan copolymer has applications in the biomedicine field due to <i>in vivo</i> biodegradability by the lysosome. What is more, the bacterial cellulose-chitosan 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 (<a href="https://parts.igem.org/Part:BBa_K4719024">BBa_K4719024</a>) 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].
 +
</p>
 +
<h2>Experimental characterization</h2>
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<h3>Protein expression</h3>
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<p>
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Fusion with CBDcenA significantly decreased the solubility of deacetylases. To increase protein stability, we employed <i>E. coli</i> ArcticExpress (DE3) which is adapted for protein expression in lower temperatures. <b>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</b>.
 +
</p>
 +
<figure>
 +
<div class = "center" >
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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>
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</div>
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<figcaption><center><b>Figure 1. SDS-PAGE analysis.</b> CBDcenA-ProThr box fused to ArCE4A <a href="https://parts.igem.org/Part:BBa_K4719020">BBa_K4719020</a> (39.0 kDa) or AnCDA (39.4 kDa) biosynthesis optimization in ArcticExpress (DE3) E. coli 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.
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</center></figcaption>
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</figure>
  
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<!-- Add more about the biology of this part here
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<h3>Deacetylation enzymatic activity analysis with fluorescence microscopy</h3>
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===Usage and Biology===
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<p>
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Deacetylation was performed in a reaction with a final volume of 200 &#181;L: 2 &#181;L 1 mM CoCl2, deacetylase CBD-ProThr box-AnCDA 100 nM - 2&#181;M and filling the remaining volume with 20mM HEPES-NaOH ph8, 150mM NaCl buffer.  The samples were incubated for 14 h at 37&deg; while shaking at 300 rpm, reaction was stopped by incubating for 3 min at 98&deg;C.
 +
<br>
 +
<br>
 +
For cellulose-chitosan copolymer generation from cellulose-chitin exopolymer we used chitin deacetylase CBD-ProThr box-AnCDA. 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.
 +
<figure>
 +
<div class = "center" >
 +
<center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/deact-ancda-cbd.png" style = "width:700px;"></center>
 +
</div>
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<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>
  
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<!-- -->
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<h3>Protein expression of recombinant deacetylase containing a new linker</h3>
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<span class='h3bb'>Sequence and Features</span>
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<p>
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<partinfo>BBa_K4719019 SequenceAndFeatures</partinfo>
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A new linker <a href="https://parts.igem.org/Part:BBa_K4719023">BBa_K4719023</a> generated by our <a href=https://2023.igem.wiki/vilnius-lithuania/software>software</a> was cloned into the pBAD/HisB-CBDCenA-AnCDA backbone by Gibson assembly. <b>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</b>.
 +
</p>
 +
<figure>
 +
<div class = "center" >
 +
<center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/ancda-frf-arce4a-frf-opt.png" style = "width:400px;"></center>
 +
</div>
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<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) E. coli 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>
 +
 
 +
<h3>Deacetylation enzymatic activity analysis with fluorescence microscopy</h3>
 +
<p>
 +
Deacetylation was performed in a reaction with a final volume of 200 &#181;L: 2 &#181;L 1 mM CoCl2, deacetylase CBD-FRF-AnCDA 100 nM - 2&#181;M and filling the remaining volume with 20mM HEPES-NaOH ph8, 150mM NaCl buffer.  The samples were incubated for 14 h at 37&deg; while shaking at 300 rpm, reaction was stopped by incubating for 3 min at 98&deg;C. The fluorescence microscopy was performed under the same conditions as in the first iteration of this construct.
 +
<br>
 +
<br>
 +
<figure>
 +
<div class = "center" >
 +
<center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/ancda-frf-flores.png" style = "width:700px;"></center>
 +
</div>
 +
<figcaption><center><b>Figure 4. Florescent Alexa Fluor™ 405 NHS ester dye staining. A</b> - <i>K. xylinus</i> modified with <i>AGM1-NAG5-UAP1</i> producing bacterial cellulose-chitin copolymer grown on 1% glucose and 1% N-acetylglucosamine. <b>B</b> - chitin control. <b>The improvement of deacetylase activity by the new linker was satisfactory</b>.
 +
</center></figcaption></figure>
 +
 
 +
<h3>Growth burden</h3>
 +
<p>
 +
 
 +
In order to work with <i>E. coli</i> for designing constructs and testing synthetic biology parts, the growth burden of said synthetic biology constructs has to be measured. We performed growth burden evaluation by measuring OD600 for five hours of modified and unmodified <i>E. coli</i> DH5&alpha;. The composite of recombinant deacetylase CBD-ProThr box-AnCDA did not inhibit the growth of <i>E. coli</i> as seen in Figure 5.
 +
<figure>
 +
<div class = "center" >
 +
<center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/cbd-ancda-growth-burden.png" style = "width:600px;"></center>
 +
</div>
 +
<figcaption><center><b>Figure 5.</b> growth burden of CBD-ProThr box-AnCDA composite. </center></figcaption>
 +
</figure>
 +
</p>
 +
 
 +
<h2>References</h2>
 +
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.
 +
<br>
 +
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.
  
  

Latest revision as of 14:44, 12 October 2023

CBD-ProThr box-AnCDA chitin deacetylase and cellulose binding domain fusion protein

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 756
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 769
    Illegal XhoI site found at 1001
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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.
After achieving bacterial cellulose-chitin copolymer, we had to deacetylase this material to produce bacterial cellulose-chitosan copolymer BBa_K4719019, BBa_K4719020, BBa_K4719024. This specific part was used for bacterial cellulose-chitin deacetylation, as deacetylation of this material produces cellulose-chitosan copolymer.

Usage and Biology

This construct contains a fused cellulose binding domain connected to a linker to the N-terminus of deacetylase AnCDA (BBa_K4719011) to ensure a higher degree of deacetylation. For protein purification, 6x his-tag was added to the N-terminus of the cellulose binding domain. The composite is contained in pBAD/HisB plasmid. For this part to be functional in your 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 lysosome. What is more, the bacterial cellulose-chitosan 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 production.

Figure 1. SDS-PAGE analysis. CBDcenA-ProThr box fused to ArCE4A BBa_K4719020 (39.0 kDa) or AnCDA (39.4 kDa) biosynthesis optimization in ArcticExpress (DE3) E. coli 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. M - PageRuler™ Unstained Protein Ladder (Thermo Fisher Scientific), S – soluble protein fraction, I – insoluble protein fraction.

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-ProThr box-AnCDA 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-AnCDA. 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.

Figure 2. Florescent Alexa Fluor™ 405 NHS ester dye staining. A - K. xylinus modified with AGM1-NAG5-UAP1 BBa_K4719013 producing bacterial cellulose-chitin copolymer grown on 1% glucose and 1% N-acetylglucosamine. B - chitin control group. The fluorescence signal is low, therefore, we decided that a new linker is needed to achieve a higher degree of deacetylation.

Protein expression of recombinant deacetylase containing a new linker

A new linker BBa_K4719023 generated by our software was cloned into the pBAD/HisB-CBDCenA-AnCDA 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.

Figure 3. SDS-PAGE analysis. CBDcenA fused to ArCE4A (38.4 kDa) or AnCDA (38.8 kDa) through FRF linker biosynthesis in ArcticExpress (DE3) E. coli strain. M - PageRuler™ Unstained Protein Ladder (Thermo Fisher Scientific), S – soluble protein fraction, I – insoluble protein fraction.

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-AnCDA 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. The fluorescence microscopy was performed under the same conditions as in the first iteration of this construct.

Figure 4. Florescent Alexa Fluor™ 405 NHS ester dye staining. A - K. xylinus modified with AGM1-NAG5-UAP1 producing bacterial cellulose-chitin copolymer grown on 1% glucose and 1% N-acetylglucosamine. B - chitin control. The improvement of deacetylase activity by the new linker was satisfactory.

Growth burden

In order to work with E. coli for designing constructs and testing synthetic biology parts, the growth burden of said synthetic biology constructs 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-AnCDA did not inhibit the growth of E. coli as seen in Figure 5.

Figure 5. growth burden of CBD-ProThr box-AnCDA composite.

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.