Difference between revisions of "Part:BBa K4719028"
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<h2>Introduction</h2>
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Vilnius-Lithuania iGEM 2023 team's goal was to create synthetic biology tools for <i>in vivo</i> alterations of <i>Komagataeibacter xylinus</i> bacterial cellulose polymer composition. 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 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 has potential as a bioplastic and is synthesized by ''K. xylinus''.
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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, allowing the synthesis of a self-dyeing plastic-like polymer.
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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 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. Combining all materials into one composite washing procedure at boiling temperatures is required.
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Ready dyed bacterial cellulose-PHB composite is an alternative to petroleum-based plastics. The advantage of this material is enhanced strenght and resistance, accelerated rate of biodegradation [1].
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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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<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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Colony PCR and restriction digestion analysis allowed us to select several promising constructs. pSEVA331-Bb-phaC-phaA-phaA-pKARA_RT3 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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<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>Figure 1: Results of colony PCR of <i>K. xylinus</i> transformed with pSEVA331-Bb-phaC-phaA-phaA-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.  </center></figcaption>
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<h3>Growth burden</h3>
 
<h3>Growth burden</h3>
 
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<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>
 
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<figcaption><center>Figure 1: growth burden of CBD-ProThr box-AnCDA composite. </center></figcaption>
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<figcaption><center>Figure 1: growth burden of <i>phaC-phaA-phaB</i>-pKARA_RT3 composite. </center></figcaption>
 
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Revision as of 13:49, 8 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]

Possible application as colorful biodegradable plastic.

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 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 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 has potential as a bioplastic and is synthesized by ''K. xylinus''.

We produced bacterial cellulose - PHB composite by introducing PHB synthesis operon into K. xylinusBBa_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, allowing 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 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. Combining 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 strenght and resistance, 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

Colony PCR and restriction digestion analysis allowed us to select several promising constructs. pSEVA331-Bb-phaC-phaA-phaA-pKARA_RT3 did not contain any deleterious mutations and was successfully transformed into electrocompetent K. xylinus cells as seen in Figure 1.

Figure 1: Results of colony PCR of K. xylinus transformed with pSEVA331-Bb-phaC-phaA-phaA-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 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 E. coli DH5α. The composite of in situ dyed PHB did not inhibit the growth of E. coli as seen in Figure 1.

Figure 1: growth burden of phaC-phaA-phaB-pKARA_RT3 composite.