Difference between revisions of "Part:BBa K4719028"

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<partinfo>BBa_K4719028 short</partinfo>
 
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==Sequence and Features==
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4719028 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4719028 SequenceAndFeatures</partinfo>
 
Possible application as colorful biodegradable plastic.
 
 
 
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<body>
 
<body>
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<h2>Introduction</h2>
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<p>
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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 composite of <b>bacterial cellulose and polyhydroxybutyrate (PHB)</b>, which is synthesized by <i>K. xylinus</i>.
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<br>
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We produced bacterial cellulose - PHB composite by introducing PHB synthesis operon into <i>K. xylinus</i> <a href="https://parts.igem.org/Part:BBa_K4719017">BBa_K4719017</a>. The bacteria simultaneously produce both polymers combined into the same material during the purification process. As an environmentally friendly way of plastic production, we thought of combining PHB synthesis genes with styrene monooxygenase pKARA_RT3 into one operon. <b>This composite allows the synthesis of a self-dyeing plastic-like polymer</b>.
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</p>
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<h2>Usage and Biology</h2>
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<p>
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This construct is a combination of a polyhydroxybutyrate synthesis operon (<i>phaC, phaA, phaB</i>) producing PHB along with bacterial cellulose, together with styrene monooxygenase pKARA_RT3 in <i>K. xylinus</i>. PHB is stored in bacteria intercellularly, while cellulose is secreted outside of the cell. Simultaneously <i>K. xylinus</i> produces indigoid pigments from added indole compounds as a substrate for styrene monooxygenase. To combine all materials into one composite washing procedure at boiling temperatures is required.
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<br>
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<br>
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Ready dyed bacterial cellulose-PHB composite is an alternative to petroleum-based plastics. The advantage of this material is enhanced strength, resistance and accelerated rate of biodegradation [1].
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<br>
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<br>
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Since polymer production occurs in <i>K. xylinus</i> requires a specific plasmid (pSEVA331-Bb) backbone for successful replication. We choose to use <a href="https://parts.igem.org/Part:BBa_K1321313">BBa_K1321313</a> as it was characterized by iGEM14_Imperial team as the most suitable synthetic biology tool for <i>Komagateibacter</i> species. We performed PCR of the plasmid eliminating mRFP in order to preserve Anderson promoter J23104 (<a href="https://parts.igem.org/Part:BBa_J23104">BBa_J23104</a>), ribose binding site (<a href="https://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>) and terminator (<a href="https://parts.igem.org/Part:BBa_B0015">BBa_B0015</a>). The construct was cloned by utilizing (<a href="https://parts.igem.org/Part:BBa_K4719018">BBa_K4719018</a>) as a plasmid backbone containing styrene monooxygenase pKARA_RT3, where PHB synthesis operon was assembled into the backbone by Gibson assembly.
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</p>
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<h2>Experimental characterization</h2>
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<h3>Verification and transformation of the <i>in situ</i> dyed bacterial cellulose-PHB composite</h3>
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<p>
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Sanger sequencing revealed that pSEVA331-Bb-phaC-phaA-phaB-pKARA_RT3</i> did not contain any deleterious mutations and was successfully transformed into electrocompetent <i>K. xylinus</i> cells as seen in Figure 1.
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<div class = "center" >
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<center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/kolonijos-phb-pkara.png" style = "width:400px;"></center>
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<figcaption><center><b>Figure 1:</b> Colony PCR of <i>K. xylinus</i> transformed with pSEVA331-Bb-phaC-phaA-phaB-pKARA_RT3. <b>L</b> -  Invitrogen™ 1 Kb Plus DNA Ladder. <b>1-12</b> - selected colonies. <b>The positive clones (5,9 and 12) had a PCR product of 2798bp as expected</b>.  </center></figcaption> </figure>
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<h3>Growth burden</h3>
 
<h3>Growth burden</h3>
 
<p>  
 
<p>  
 
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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 <i>in situ</i> dyed PHB did not inhibit the growth of <i>E. coli</i> as seen in Figure 2.
In order to work with <i>E. coli</i> for designing constructs and testing synthetic biology systems, 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 <i>E. coli</i> DH5&alpha;. The composite of <i>in situ</i> dyed PHB did not inhibit the growth of <i>E. coli</i> as seen in Figure 1.
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<figure>
 
<figure>
 
<div class = "center" >
 
<div class = "center" >
 
<center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/phb-pkara-growth-burden.png" style = "width:600px;"></center>
 
<center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/phb-pkara-growth-burden.png" style = "width:600px;"></center>
 
</div>
 
</div>
<figcaption><center>Figure 1: growth burden of CBD-ProThr box-AnCDA composite. </center></figcaption>
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<figcaption><center><b>Figure 2:</b> growth burden of <i>phaC-phaA-phaB</i>-pKARA_RT3 composite. </center></figcaption>
 
</figure>
 
</figure>
 
</p>
 
</p>
  
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<h2>References</h2>
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<p>
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1.Ding, R. et al. (2021) ‘The facile and controllable synthesis of a bacterial cellulose/polyhydroxybutyrate composite by co-culturing Gluconacetobacter xylinus and Ralstonia eutropha’, Carbohydrate Polymers, 252, p. 117137. doi:10.1016/j.carbpol.2020.117137.
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</p>
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
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===Usage and Biology===
 
===Usage and Biology===
  

Latest revision as of 15:25, 12 October 2023

phaC-phaA-phaB-pKARA_RT3 operon for in situ dyed bacterial cellulose - polyhydroxybutyrate composite

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 4992
    Illegal SpeI site found at 37
    Illegal PstI site found at 824
    Illegal PstI site found at 1397
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 4992
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal SpeI site found at 37
    Illegal PstI site found at 824
    Illegal PstI site found at 1397
    Illegal NotI site found at 200
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 4992
    Illegal BglII site found at 642
    Illegal BamHI site found at 3039
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 4992
    Illegal SpeI site found at 37
    Illegal PstI site found at 824
    Illegal PstI site found at 1397
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 4992
    Illegal SpeI site found at 37
    Illegal PstI site found at 824
    Illegal PstI site found at 1397
    Illegal NgoMIV site found at 253
    Illegal NgoMIV site found at 368
    Illegal NgoMIV site found at 602
    Illegal NgoMIV site found at 914
    Illegal NgoMIV site found at 1193
    Illegal NgoMIV site found at 1606
    Illegal NgoMIV site found at 1673
    Illegal AgeI site found at 341
    Illegal AgeI site found at 4455
  • 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 composite of bacterial cellulose and polyhydroxybutyrate (PHB), which is synthesized by K. xylinus.
We produced bacterial cellulose - PHB composite by introducing PHB synthesis operon into K. xylinus BBa_K4719017. The bacteria simultaneously produce both polymers combined into the same material during the purification process. As an environmentally friendly way of plastic production, we thought of combining PHB synthesis genes with styrene monooxygenase pKARA_RT3 into one operon. This composite allows the synthesis of a self-dyeing plastic-like polymer.

Usage and Biology

This construct is a combination of a polyhydroxybutyrate synthesis operon (phaC, phaA, phaB) producing PHB along with bacterial cellulose, together with styrene monooxygenase pKARA_RT3 in K. xylinus. PHB is stored in bacteria intercellularly, while cellulose is secreted outside of the cell. Simultaneously K. xylinus produces indigoid pigments from added indole compounds as a substrate for styrene monooxygenase. To combine all materials into one composite washing procedure at boiling temperatures is required.

Ready dyed bacterial cellulose-PHB composite is an alternative to petroleum-based plastics. The advantage of this material is enhanced strength, resistance and accelerated rate of biodegradation [1].

Since polymer production occurs in K. xylinus requires a specific plasmid (pSEVA331-Bb) backbone for successful replication. We choose to use BBa_K1321313 as it was characterized by iGEM14_Imperial team as the most suitable synthetic biology tool for Komagateibacter species. We performed PCR of the plasmid eliminating mRFP in order to preserve Anderson promoter J23104 (BBa_J23104), ribose binding site (BBa_B0034) and terminator (BBa_B0015). The construct was cloned by utilizing (BBa_K4719018) as a plasmid backbone containing styrene monooxygenase pKARA_RT3, where PHB synthesis operon was assembled into the backbone by Gibson assembly.

Experimental characterization

Verification and transformation of the in situ dyed bacterial cellulose-PHB composite

Sanger sequencing revealed that pSEVA331-Bb-phaC-phaA-phaB-pKARA_RT3 did not contain any deleterious mutations and was successfully transformed into electrocompetent K. xylinus cells as seen in Figure 1.

Figure 1: Colony PCR of K. xylinus transformed with pSEVA331-Bb-phaC-phaA-phaB-pKARA_RT3. L - Invitrogen™ 1 Kb Plus DNA Ladder. 1-12 - selected colonies. The positive clones (5,9 and 12) had a PCR product of 2798bp as expected.

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 in situ dyed PHB did not inhibit the growth of E. coli as seen in Figure 2.

Figure 2: growth burden of phaC-phaA-phaB-pKARA_RT3 composite.

References

1.Ding, R. et al. (2021) ‘The facile and controllable synthesis of a bacterial cellulose/polyhydroxybutyrate composite by co-culturing Gluconacetobacter xylinus and Ralstonia eutropha’, Carbohydrate Polymers, 252, p. 117137. doi:10.1016/j.carbpol.2020.117137.